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Anonymous e 6 April 2013

Created By: Daylen Gargalis

Golden Rice: What it is, what it does and how good it is at doing it

Updated 6 April 2013. For articles on Golden Rice in scientific journals, please scroll down to a list of publications!

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Golden Rice is rice enriched with beta-carotene, a provitamin. It was developed to help prevent vitamin A deficiency and its often severe and sometimes deadly consequences in rice-eating populations in developing countries: in these countries many people are too poor to be able afford a balanced diet with greens, fruits and animal products. Unfortunately it is only realistic to assume that large parts of these populations will remain poor and malnourished for the foreseeable future.

Contents: Rice and vitamin A deficiency | Vitamin A interventions to date | Golden Rice as a new vitamin A intervention | Golden Rice as a humanitarian project | Timeline of Golden Rice | The burden of vitamin A deficiency in India | Impact and cost-effectiveness of Golden Rice | Publications

Rice and vitamin A deficiency

[1] Rice is a staple crop for half of humanity. In particular in Asia it is the main source of dietary energy for many people. Yet, rice is a poor source of some vitamins and minerals, e.g. unlike certain other crops it does not contain any beta-carotene (provitamin A). Therefore people who rely on rice as their main food source are at risk of vitamin A deficiency. This risk is biggest for pregnant and lactating women as well as for young children. The main consequences of vitamin A deficiency are (i) eye problems that can lead to complete blindness and (ii) a higher susceptibility to infectious diseases that are often deadly. In fact, the World Health Organization (WHO) estimates that each year 125,000-250,000 children die due to VAD, with as many becoming blind. In the poorest countries the WHO considers vitamin A deficiency to be one of the major health risk factors. And according to data of the latest study on the "Global Burden of Disease", in 2010 more than 11.5 million so-called "Disability-Adjusted Life Years" (DALYs) – i.e. person-years lost in a population owing to disability and shortened life – were lost globally due to VAD.

Vitamin A interventions to date

So far, efforts to address VAD rely mainly on the distribution of medical doses of synthetic vitamin A. Usually these supplementation programmes are targeted at pre-school children, who have to receive a vitamin A mega dose twice a year. While such interventions are considered to be very cost-effective, it represents a considerable cost to cover millions and millions of children two times year on year. Apart from these recurrent costs, which reduce the funds that are available for other humanitarian efforts, in developing countries there are additional problems that limit the coverage and success of such programmes (infrastructure, logistics, qualified health personnel). Children in remote rural areas or in urban slums may not be reached and older children and adults are not covered at all. Programmes for the industrial fortification (e.g. of sugar) face similar obstacles. And the promotion of nutrition knowledge and dietary diversification, while the most desirable option, is also the most long-term and resource-intensive intervention (for instance on the supply side such projects have high staff requirements and their geographic coverage is limited, while on the side of the beneficiaries there can be opportunity costs – especially in form of the time and costs it takes to cultivate or procure the required produce and to prepare the meals – that prevent an uptake).

[2] Golden Rice as a new vitamin A intervention

Therefore, despite what current efforts have already achieved, developing additional tools to help address VAD is a good idea per se – as long as these alternative interventions can make a difference in terms of impact and cost-effectiveness. This is where Golden Rice comes into play: While beta-carotene is produced in the green parts of the rice plant, none of it gets into the kernels. And if there is nothing, nothing can be used for cross-breeding, i.e. conventional breeding was not an option. Instead, rice was genetically engineered (with the help of a maize gene) to produce kernels that are enriched with beta-carotene. Like in other carotene-rich plants (e.g. carrots, mangoes or orange-fleshed sweet potatoes), this gives the Golden Rice its characteristic yellow hue from which its very name is derived.

Golden Rice as a humanitarian project

Golden Rice was originally developed by a team of researchers led by Ingo Potrykus of the Swiss Federal Institute of Technology and by Peter Beyer of the University of Freiburg in Germany. Later on the project was also supported by a group of seed companies, coordinated by Syngenta, who donated royalty-free intellectual property (materials and patented processes and technologies) for the development and humanitarian use of Golden Rice. For this reason – and contrary to often repeated claims by activists – smallholder farmers in developing countries will be able to get Golden Rice without additional charges and they are free to save the seeds for replanting. The work on Golden Rice is being continued and coordinated by the International Rice Research Institute (IRRI). Target countries for the introduction of Golden Rice are the Philippines and Bangladesh, but also India, Indonesia and Vietnam.

Timeline of Golden Rice

According to an NPR interview with Gary Toenniessen of the Rockefeller Foundation, the story of Golden Rice began in 1984 with an after-work brainstorming of a group of breeders at a meeting at IRRI. The idea that was discussed there – that regular white rice does not provide enough beta-carotene to protect children from vitamin A deficiency and that they can be harmed for the rest of their lives – persuaded Toenniessen to start a Rockefeller programme to develop "yellow rice". Other donors followed, such as the European Commission, but Rockefeller is still funding work on Golden Rice, such as field tests and bio-safety assessments.

Then, in 1999 a press release of the European Commission confirmed that "a project funded by the European Union – Carotene plus – has successfully incorporated the production of ß-carotene into rice. This major scientific achievement, which incidentally turns the rice grains yellow, will [sic!] help prevent severe vitamin A deficiency in countries relying on rice as a staple food." A year later, in 2000, a first proof-of-concept study on the feasibility of rice biofortification with beta-carotene was published, and in subsequent work the beta-carotene content in the rice was increased substantially: By 2005 a "second generation" of Golden Rice had been developed that could provide enough beta-carotene (even in in absolute terms) to prevent VAD in rice-eating populations. Not least, this advance served to disprove the "Golden Rice Hoax" (which is discussed in more detail in the references below).

By 2009 a feeding study had been conducted that showed a high bioavailability of the beta-carotene in Golden Rice, and in 2012 the results of another, larger feeding study showed that the beta-carotene in Golden Rice is as effective as pure beta-carotene and better than spinach at providing vitamin A to children. As IRRI – while itself not involved – pointed out, a statement in this latter study confirmed that the processes and protocol of the study were approved in China and the United States and the study was conducted with the consent of those involved.

Meanwhile not least the strict regulatory frameworks for the approval of GMOs slowed down the development process of Golden Rice, which was moreover met with scepticism by a public that was unsettled because of contradicting but unsubstantiated disinformation campaigns of interested third parties and activist groups that benefit from keeping the public in the dark – and frightened – about GM crops. (One reason why it is important to bring light into the discussion about GMOs.)

Continuing its previous funding, in April 2011 the Gates Foundation announced a US$ 10 million grant to IRRI to fund the development and evaluation of Golden Rice varieties for the Philippines and Bangladesh. The grant is also meant to help generating the data needed for Golden Rice to comply with food safety and environmental regulations. Thus the grant will also be used to compile the regulatory dossier to confirm that Golden Rice is indeed safe to eat; as the coordinator of the Golden Rice Network stresses: "These crops will not be used by farmers or consumers until they pass tests for biosafety in each country." In addition, this new initiative includes a collaboration of IRRI with Hellen Keller International (HKI) to evaluate to what extent the consumption of Golden Rice improves vitamin A status. Then, if Golden Rice is deployed, HKI will help ensure that it reaches those most in need.

In January 2013, two seasons of field trials were concluded in the Philippines. These trials were part of the safety assessment of Golden Rice and the generated data will be evaluated by the national regulatory authority in the Philippines for biotechnology research and development as part of their biosafety regulatory process. As Golden Rice will only be made available broadly to farmers and consumers in the Philippines if it is approved and shown to reduce vitamin A deficiency, this process may take another two years or more.

The burden of vitamin A deficiency

As explained above, and despite some shortcomings, current approaches to address VAD are cost-effective public health interventions. Therefore any alternative or additional vitamin A intervention should be less costly than these remedies, have a discernible impact, and possibly be complementary in scope to cover those people who are neglected so far. Together with an inter-disciplinary group of researchers I carried out a comprehensive case study for India to assess impact and cost-effectiveness of other biofortified crops and Golden Rice. (The work on Golden Rice was done with Dr. Sachdev and Prof. Qaim.) In a first step we measured the burden of disease of VAD by counting the number of "disability-adjusted life years" (DALYs) lost, i.e. the number of years of life lost due to ill-health, disability or early death because of VAD. In India the burden of VAD amounts to an annual loss of 2.3 million DALYs; as reported above, the global figure is 11.5 million DALYs lost due to VAD.

Impact and cost-effectiveness of Golden Rice

To determine the potential impact of Golden Rice on this burden on public health, we simulated the consumption of Golden Rice based on real food expenditure data from a representative sample of 120,000 households in India. We found that in a high impact scenario the widespread consumption of Golden Rice in the target groups could reduce the disease burden of VAD in India by almost 60 percent. But even under pessimistic assumptions the burden could still be reduced by almost 10 percent – i.e. over 200,000 "healthy life years" (DALYs) could be saved. Setting off these gains (in terms of saved lives and improved health) against all the costs needed to make Golden Rice a success (i.e. expenditures for research, breeding, dissemination, public awareness, etc.) showed that Golden Rice could prevent the loss of one DALY for less than $20, even under pessimistic assumptions. In contrast, other vitamin A interventions cost between $80-$600 per DALY saved. Hence, while this was only a computation, it was a very thorough one. (We worked on the overall project for three years and used all available information.) Therefore our conclusion was that pursuing the development of Golden Rice further is justified. The finer details of this study can be found in the peer-reviewed literature listed below, where common arguments against Golden Rice – which are based on double standards or twisted logic and poor data – are refuted point-by-point.

- Alexander Stein

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Publications on the impact and cost-effectiveness of Golden Rice

Please feel free to contact me if you cannot obtain the papers elsewhere.

Stein A.J., Sachdev H.P.S., Qaim M. (2008). "Genetic engineering for the poor: Golden Rice and public health in India." World Development 36(1): 144-158. doi:10.1016/j.worlddev.2007.02.013.

Stein (2008), Golden Rice, World Development Abstract: Vitamin A deficiency (VAD) affects millions of people, causing serious health problems. Golden Rice (GR), which has been genetically engineered to produce beta-carotene, is being proposed as a remedy. While this new technology has aroused controversial debates, its actual impact remains unclear. We develop a methodology for ex ante evaluation, taking into account health and nutrition details, as well as socioeconomic and policy factors. The framework is used for empirical analyses in India. Given broad public support, GR could more than halve the disease burden of VAD. Juxtaposing health benefits and overall costs suggests that GR could be very cost-effective. (Keywords: vitamin A deficiency, biofortification, Golden Rice, disability-adjusted life years, cost-effectiveness, India.)

Qaim M., Stein A.J. (2008). "Economic consequences of Golden Rice." Invited presentation at the 4th Conference of the European Plant Science Organisation (EPSO), 22-26 June, Toulon, France.

Qaim (2008), Golden Rice, EPSO Abstract: Golden Rice (GR), which has been genetically modified to produce beta-carotene in the endosperm of grain, has been proposed to control vitamin A deficiency (VAD), especially among the poor in developing countries. However, the usefulness of GR is questioned by some, and the technology has become one of the centerpieces in the public controversy over genetically modified crops [...] we show that VAD is a serious public health problem in India, causing a sizeable disease burden, especially in terms of increased child mortality [...] if GR were to be consumed widely, the disease burden of VAD could be reduced by 60% [...] Regardless of the underlying assumptions, GR is likely to be more cost-effective than alternative vitamin A interventions, such as food supplementation or fortification. Therefore, it should be considered seriously as a complementary intervention to fight VAD in rice-eating populations.

Stein A.J., Sachdev H.P.S., Qaim M. (2007). "What we know and don't know about Golden Rice." Nature Biotechnology 25(6): 624. doi:10.1038/nbt0607-624a, incl. a point-by-point refutation of arguments made against Golden Rice in response to our earlier study, and incl. an illustration of how such arguments are inconsistent and based on double standards.

Stein (2007), Golden Rice, Nature Biotechnology Abstract: Michael Krawinkel raises three issues in his comment to our economic analysis of Golden Rice. First, he questions the scientific basis of the assumptions that we have used in our impact assessment. Second, he claims that the development of Golden Rice costs “a lot of money” and would mainly benefit “agrochemistry” companies. And third, he states that biofortification in general and Golden Rice in particular cannot replace any of the established micronutrient interventions for the forseeable future. [... In response, we highlight the biomedical foundation and the available evidence for our assumptions; we put the costs of Golden Rice into perspective by citing the costs for alternative interventions, which are two orders of magnitudes bigger; we clarify the misconception that private companies would benefit from Golden Rice, which is a humanitarian undertaking; and more generally we illustrate how Krawinkel uses double standards in his criticism e.g. by stressing the costs of Golden Rice while ignoring the costs of other interventions, or by stressing the limitations of Golden Rice without acknowledging the shortcomings of alternative interventions (which Golden Rice could complement in a sensible way. Finally, we explain how our cost-effectiveness analysis, which uses uniform standards that create a level playing field and allow comparisons across interventions in a transparent and consistent way, represent a more objective and science-based approach.]

Stein A.J., Sachdev H.P.S., Qaim M. (2006). "Potential impact and cost-effectiveness of Golden Rice." Nature Biotechnology 24(10): 1200-1201. doi:10.1038/nbt1006-1200b. With supplementary information.

Stein (2006), Golden Rice, Nature Biotechnology Abstract: A News & Views article by Michael Grusak in last year’s April issue (Nat. Biotechnol. 23, 429-430, 2005) highlighted the unresolved debate concerning the efficacy of Golden Rice in addressing the problem of vitamin A deficiency (VAD). He pointed out that an assessment of the potential impact of Golden Rice on this type of malnutrition requires the consideration of multiple variables, including the target individuals’ life stages, the average amount of rice consumed daily by these individuals and the percentage of β-carotene that would be absorbed from rice. He further explains how early critics of the original Golden Rice technology had used simple estimates of these variables to suggest that unrealistic amounts of the transgenic rice would need to be consumed to satisfy the recommended dietary intakes of vitamin A equivalents (exclusively) through rice consumption. [...]

Stein A.J., Sachdev H.P.S., Qaim M. (2006). "Potential impact and cost-effectiveness of Golden Rice." Nature Biotechnology 24(10): online supplement, on p. 27 incl. a detailed point-by-point refutation of the populistic arguments made against Golden Rice in "The Golden Rice hoax."

Stein (2006), Golden Rice, Nature Biotechnology Abstract: Genetic engineering (GE) in agriculture is a controversial topic in science and society at large. While some oppose genetically modified crops as proxy of an agricultural system they consider unsustainable and inequitable, the question remains whether GE can benefit the poor within the existing system and what needs to be done to deliver these benefits? Golden Rice has been genetically engineered to produce provitamin A. The technology is still in the testing phase, but, once released, it is expected to address one consequence of poverty – vitamin A deficiency (VAD) – and its health implications. Current interventions to combat VAD rely mainly on pharmaceutical supplementation, which is costly in the long run and only partially successful. We develop a methodology for ex-ante evaluation, taking into account the whole sequence of effects between the cultivation of the crop and its ultimate health impacts. In doing so we build on a comprehensive, nationally representative data set of household food consumption in India. Using a refined disability-adjusted life year (DALY) framework and detailed health data, this study shows for India that under optimistic assumptions this country's annual burden of VAD of 2.3 million DALYs lost can be reduced by 59.4% hence 1.4 million healthy life years could be saved each year if Golden Rice would be consumed widely. In a low impact scenario, where Golden Rice is consumed less frequently and produces less provitamin A, the burden of VAD could be reduced by 8.8%. However, in both scenarios the cost per DALY saved through Golden Rice (US$3.06-19.40) is lower than the cost of current supplementation efforts, and it outperforms international cost-effectiveness thresholds. Golden Rice should therefore be considered seriously as a complementary intervention to fight VAD in rice-eating populations in the medium term. Plus, on p. 27, incl. a detailed point-by-point refutation of the populistic arguments made against Golden Rice in "The Golden Rice hoax." (Keywords: genetic engineering, beta-carotene biofortification, vitamin A deficiency, Golden Rice, health benefits, DALYs, cost-effectiveness, cost-benefit analysis, India.)

Stein A.J., Sachdev H.P.S., Qaim M. (2006). "Can genetic engineering for the poor pay off? An ex-ante evaluation of Golden Rice in India." Research in Development Economics and Policy 5, University of Hohenheim.

Stein (2006), Golden Rice, University of Hohenheim Abstract: Genetic engineering (GE) in agriculture is a controversial topic in science and society at large. While some oppose genetically modified crops as proxy of an agricultural system they consider unsustainable and inequitable, the question remains whether GE can benefit the poor within the existing system and what needs to be done to deliver these benefits? Golden Rice has been genetically engineered to produce provitamin A. The technology is still in the testing phase, but, once released, it is expected to address one consequence of poverty – vitamin A deficiency (VAD) – and its health implications. Current interventions to combat VAD rely mainly on pharmaceutical supplementation, which is costly in the long run and only partially successful. We develop a methodology for ex-ante evaluation, taking into account the whole sequence of effects between the cultivation of the crop and its ultimate health impacts. In doing so we build on a comprehensive, nationally representative data set of household food consumption in India. Using a refined disability-adjusted life year (DALY) framework and detailed health data, this study shows for India that under optimistic assumptions this country’s annual burden of VAD of 2.3 million DALYs lost can be reduced by 59.4% hence 1.4 million healthy life years could be saved each year if Golden Rice would be consumed widely. In a low impact scenario, where Golden Rice is consumed less frequently and produces less provitamin A, the burden of VAD could be reduced by 8.8%. However, in both scenarios the cost per DALY saved through Golden Rice (US$ 3.06-19.40) is lower than the cost of current supplementation efforts, and it outperforms international cost-effectiveness thresholds. Golden Rice should therefore be considered seriously as a complementary intervention to fight VAD in rice-eating populations in the medium term. (Inclusive of a scientific appraisal of the populistic arguments gathered by Vandana Shiva in "The 'Golden Rice' hoax" - which are, for instance, more widely circulated and exploited by Greenpeace.) (Keywords: genetic engineering, beta-carotene biofortification, vitamin A deficiency, Golden Rice, health benefits, DALYs, cost-effectiveness, cost-benefit analysis, India.)

Stein A.J., Sachdev H.P.S., Qaim M. (2006). "Potential impacts of Golden Rice on public health in India." Contributed paper presented at the 26th Conference of the International Association of Agricultural Economists (IAAE), August 12-18, Broadbeach, Australia.
Presentation on Golden Rice [PDF | 54KB].

Stein (2006), Golden Rice, IAAE Abstract: Vitamin A deficiency (VAD) affects millions of people world-wide, causing serious health problems. Golden Rice (GR), which has been genetically engineered to produce beta-carotene, is being proposed as a remedy. While this new technology has aroused controversial debates, its nutritional impact and cost-effectiveness remain unclear. We determine the current burden of VAD in India from a public health perspective, and simulate the potential alleviating impact of GR using representative household food consumption data. Given broad public support, GR could more than halve the overall burden of VAD. Juxtaposing health benefits and overall costs suggests that GR is very costeffective. (Keywords: Golden Rice, vitamin A deficiency, biofortification, genetic engineering, DALYs, cost-effectiveness analysis, India.)

Stein A.J. (2006). "Potential impact and cost-effectiveness of Golden Rice in India: an ex-ante study." Invited presentation at the Meeting of the Golden Rice Humanitarian Board, May 2, Freiburg i.Br., Germany.

Presentation on Golden Rice [PDF | 113KB].

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Mestel 12 July 2012

Created By: Daylen Gargalis

Bananas and genetic engineering: Past, present and future
July 12, 2012|By Rosie Mestel, Los Angeles Times | This post has been corrected. See the note below for details.



The Pahang banana -- good for research but not so good for eating because its fruits are full of seeds. Scientists have sequenced its genome. It will help them in their banana biotech and breeding efforts going forward.

The Pahang banana -- good for research but not so good for eating because… (Angelique D'Hont / CIRAD )

Scientists are fighting to protect the hundreds of bananas and plantains people eat around the world from a blizzard of pests: insects, fungi, worms, bacteria and viruses.

They're using old methods and new ones in their fight, as noted in our news story on the successful sequencing of the banana genome by French scientists.

[1] In Uganda, for example, scientists have been using conventional breeding, crossing fertile wild bananas to local bananas that are eaten. They’re trying to develop resistances to banana blights such as black leaf streak disease, a.k.a. black sigatoka, said Andrew Kiggundu, plant biotechnology research officer at the country’s National Agricultural Research Laboratories Institute in Kawanda. The Black Sigatoka fungus attacks the leaves and can cause production losses of up to 50%.

A hybrid banana resistant to black sigatoka is now being scaled up for distribution for farmers, Kiggundu said.

Uganda is also where the first African field trials for genetically modified bananas took place, starting in 2007. (People in Uganda eat almost a kilo of bananas a day, so it’s a very important food crop.)

The trials were done under carefully controlled field conditions, Kiggundu said, and the bananas showed some limited resistance to black sigatoka in the field, with slower disease progression than regular bananas. But the scientists need the plants to do better. They are going back to the drawing board and rejiggering their technology in the hope that they’ll see improved resistance.

In collaboration with a variety of other researchers around the world, the Uganda team is also working on genetically modified bananas with resistance to nematodes, weevils, bacterial wilt and a mold called Panama disease, as well as higher levels of vitamin A and iron, and delayed ripening.

What is a “resistance gene” anyway? How does a plant ward off pests?

In the case of the black sigatoka field trial, bananas were engineered with rice genes that carry instructions for proteins called chitinases. Chitinases break up molecules called chitin – found in insect skeletons and also the cell walls of fungi.

Bananas genetically modified to fight bacterial wilt make two proteins from sweet pepper. One of them is plant ferrodoxin-like protein. It triggers a strong response when plants are attacked by pathogens. The plant, in this response, essentially kills off its own tissue around the site of the infection. At the same time, lots of highly reactive oxygen species are produced, which can attack the invader. And the plant starts making antimicrobial chemicals.

To name just two examples.

[2] Plants naturally have genetic resistances because they are constantly fighting off pests in the wild. And that includes many wild bananas. Since the genome of one wild banana, a subspecies of Musa acuminata (Pahang), has now been sequenced, researchers hope in the future that some of the genes that they use for engineering resistance and other qualities will come from bananas themselves. Variability exists aplenty in bananas -- it's just hellish to breed traits into the ones people like to eat because the edible ones are not fertile.

Bananas were once explored for another biotech use: creating edible vaccines against various human diseases. Charles Arntzen, a plant biotechnologist at Arizona State University, had in mind producing bananas engineered to make a protein from the bacterium that causes cholera.

This protein doesn't cause disease "but it's a very good vaccine," Arntzen said. The bananas would be harvested and made into chips and powder and these would be given to people in developing countries who don't have good access to modern vaccines. The banana chips, once eaten, would expose the body to the cholera protein and offer resistance later on to encounters with the cholera bacterium.

Arntzen once had high hopes for the concept (he worked on something similar in tomatoes), but the regulatory hurdles for an edible vaccine plus the money needed for safety trials made the whole endeavor too complicated, he said. He and most others have abandoned the effort and moved on to growing non-edible vaccines in tobacco plants.

The remaining edible vaccine efforts are mostly for delivery to fish and other animals, Arntzen said.

You can read lots more about bananas at the following websites:

The banana website for the nonprofit group Bioversity International; the International Magazine on Banana and Plantain (if it’s not already on your coffee table); Uganda’s National Banana Research Program; the book “Banana: The Fate of the Fruit that Changed the World.”

And a review at the state of the banana by Pat Heslop-Harrison and Trude Schwarzacher, "Domestication, Genomics and the Future for Banana."

[For the record, 12:30 p.m. July 13: An earlier version of this post incorrectly said that Charles Arntzen works at Northern Arizona University. Arntzen works at Arizona State University.]
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Nilsson 31 August 2006

Created By: Daylen Gargalis

Scientists Genetically Engineer Human Cells to Fight Cancer

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Aug. 31, 2006

Seven years ago, Mark Origer was diagnosed with a malignant melanoma, a sometimes curable skin cancer that can be deadly if it spreads to other parts of the body.

Watch the full report tonight on "World News With Charles Gibson"

By 2004, his cancer had spread to his liver, lung and lymph nodes.

Origer, 53, was optimistic about a cure, but conventional treatments failed him.

"I was hopeful every time I tried a new treatment. I hoped it would be the end of my disease," Origer said.

But nothing worked.

"It felt defeating," he said.

Desperate for a cure, Origer enrolled in a clinical trial at the National Cancer Institute in Bethesda, Md. The trial tested a very experimental therapy that had never before been used in people.

First Human Gene Therapy for Cancer

What attracted Origer, of Waterville, Wis., to the cancer institute was a unique process where genetic engineering is applied to humans.

The process is usually associated with hybrid animals and super foods, but is being tested to fight diseases in people.

The cancer institute's researchers are using genetically engineered immune cells to shrink tumors in cancer patients like Origer.

"This is the first gene therapy for cancer. … That is why it is so important," said Dr. Steven A. Rosenberg, who headed the trial as chief of surgery at the National Cancer Institute.

[1] Researchers took immune system cells from the blood of 17 advanced melanoma patients who, like Origer, had not been helped by conventional treatments. Origer had only three months to four months left to live when the experimental treatment began.

These ordinary blood cells, called T cells, were genetically engineered to become cancer-fighting cells that could recognize and attack the life-threatening melanoma.

The cancer-fighting cells were then injected back into each patient. Researchers hoped that the new T cells would multiply and fight off the cancers.

Scientific Advance, a Lifesaver for Some

The experimental therapy could be a major medical and scientific advance.

The therapy is also important because, for Origer, it worked.

After he was injected with his own engineered cancer-fighting T cells, he was dismissed from the hospital.

Doctors told him they would know within a month whether the therapy had worked.

"There was a lot of anxiety and apprehension that month," Origer said. "I felt like a gladiator in the arena when I was waiting to see that doctor. I was waiting for 'thumbs up' or 'thumbs down.'"

He received the thumbs up.

"Oh, it was euphoric," Origer said. "It really was."

[2] Doctors said that Origer's tumors had shrunk by 50 percent after one month, including those that had spread to his liver and other parts of his body.

He was one of two patients on the trial to be declared disease free, even 18 months after the experimental therapy had started.

Not all of the patients were helped by the therapy -- the other 15 died from their disease.

Some Success, Questions Still Linger

Researchers are not sure exactly why the therapy did not work for everyone. They speculate that the cancer cells may have mutated so the cancer-fighting T cells could not recognize them.

The therapy could also have potential side effects, though Origer said he had not had any unusual side effects.

Dr. Patrick Hwu, chairman of melanoma medical oncology at the University of Texas' M.D. Anderson Cancer Center, expressed concerns that these tumor-fighting T cells might turn around and attack the patients' own tissues.

[3] "T cells are being genetically modified to recognize receptors on the specific cancer cells and to attack those receptors. One must remember that cancer arises from normal tissue," he said. "The side effect could potentially be that the T cells will attack normal tissue that has those same receptors."

Obviously, scientists still have a lot to learn about this approach.

Hwu said, "It is not ready to be used in common practice."

Researchers will keep working on it, though.

"In the future, we plan to perform further trials with patients who have breast, lung or ovarian cancer, but these trials have not begun," Rosenberg said.

This experimental treatment may not be ready for common practice, but it gives science a potential, new approach to cancer therapy.
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Yang et.al. 22 March 2010

Created By: Daylen Gargalis

Narrowing Down the Targets: Towards Successful Genetic Engineering of Drought-Tolerant Crops
Shujun Yang, Barbara Vanderbeld, Jiangxin Wan and Yafan Huang1
+ Author Affiliations

Performance Plants Inc., 700 Gardiners Road, Kingston, Ontario, K7M 3X9, Canada
1To whom correspondence should be addressed. E-mail yafanhuang@gmail.com.
Received March 12, 2010.
Accepted March 22, 2010.

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[1] Drought is the most important environmental stress affecting agriculture worldwide. Exploiting yield potential and maintaining yield stability of crops in water-limited environments are urgent tasks that must be undertaken in order to guarantee food supply for the increasing world population. Tremendous efforts have been devoted to identifying key regulators in plant drought response through genetic, molecular, and biochemical studies using, in most cases, the model species Arabidopsis thaliana. However, only a small portion of these regulators have been explored as potential candidate genes for their application in the improvement of drought tolerance in crops. Based on biological functions, these genes can be classified into the following three categories: (1) stress-responsive transcriptional regulation (e.g. DREB1, AREB, NF-YB); (2) post-transcriptional RNA or protein modifications such as phosphorylation/dephosphorylation (e.g. SnRK2, ABI1) and farnesylation (e.g. ERA1); and (3) osomoprotectant metabolism or molecular chaperones (e.g. CspB). While continuing down the path to discovery of new target genes, serious efforts are also focused on fine-tuning the expression of the known candidate genes for stress tolerance in specific temporal and spatial patterns to avoid negative effects in plant growth and development. These efforts are starting to bear fruit by showing yield improvements in several crops under a variety of water-deprivation conditions. As most such evaluations have been performed under controlled growth environments, a gap still remains between early success in the laboratory and the application of these techniques to the elite cultivars of staple crops in the field. Nevertheless, significant progress has been made in the identification of signaling pathways and master regulators for drought tolerance. The knowledge acquired will facilitate the genetic engineering of single or multiple targets and quantitative trait loci in key crops to create commercial-grade cultivars with high-yielding potential under both optimal and suboptimal conditions.

Key words
Abiotic stress drought tolerance gene expression genetic engineering crop yield potential field trials
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One of the major challenges facing agriculture today is the global water shortage caused by the increasing world population and worldwide climate change. The Intergovernmental Panel on Climate Change (IPCC) has concluded that elevated greenhouse gas concentrations are likely to lead to the general drying of the subtropics by the end of this century, creating widespread drought stress in agriculture (IPCC, 2007). This global water scarcity threatens sustainable crop farming, as agricultural activities account for about 75% of global water consumption, and, in particular, irrigation represents over 90% of water used in many developing countries (UNEP, 2009). Stresses such as drought and salinity affect the productivity of most field crops to variable degrees, depending on the onset time, duration, and intensity of the stress. Rice (Oryza sativa), one of the most important food crops in the world, is very sensitive to drought stress because of its limited adaptation to water-deficit conditions. It is predicted that the depletion of ground water resources and rising soil salinity are pushing some rice-cropping systems away from traditional paddy land to aerobic fields (Delmer, 2005; Bernier et al., 2008). Maize (Zea mays), another staple crop, is also very sensitive to water-deficit stress (Boyer and Westgate, 2004), as its pollination and embryo development during and post flowering are greatly affected by soil water supply (Grant et al., 1989; Bolaños and Edmeades, 1996).

Plants, sessile in their environments, have evolved to adapt to environmental stresses via morphological and physiological changes. These include the efficiency with which the plant draws water from the surrounding soil, the water-retaining capacity within plant tissues or cells, control of water loss from transpiration through stomatal pores, and developmental adaptations to avoid seasonal water shortage during flowering (Zhu, 2002; Shinozaki et al., 2003; Umezawa et al., 2006; Neumann, 2008). At the molecular level, plants utilize multiple chains of signaling molecules (e.g. abscisic acid; ABA), which regulate different sets of stress-responsive genes to initiate the synthesis of various classes of proteins, including transcription factors (TFs), enzymes, and molecular chaperones (Valliyodan and Nguyen, 2006). These proteins function accordingly to enhance plants’ tolerance via processes such as vegetative growth attenuation, osmoprotectant accumulation, and transpiration reduction. Hundreds of genes and their related signaling pathways that control the key processes of plants’ responses to abiotic stresses have been identified (Umezawa et al., 2006; Yamaguchi-Shinozaki and Shinozaki, 2006; Tran et al., 2007; Wan et al., 2009). In general, the primary response of plants to water deficit is the inhibition of shoot growth, allowing cellular essential solutes to be diverted from growth requirements to stress-related functions. However, since this growth arrest decreases plant size and hence limits yield potential, the development of crop varieties able to maintain near-normal growth under moderate water stress is critical for yield stability.

Traditional breeding for yield stability under water-deficit conditions is labor-intensive and time-consuming, primarily because of the complicated genetics that control yield and the difficulty in controlling moisture levels in the field. Furthermore, the long-time practice of breeding for higher yield under well watered conditions has narrowed the genetic spectrum of current cultivars with respect to their ability to tolerate drought stress. Genetic engineering is thus being intensively explored to enhance plants’ tolerance to abiotic stresses, and many engineered plants have manifested improved stress-resistance phenotypes (Bartels and Sunkar, 2005; Vinocur and Altman, 2005; Umezawa et al., 2006; Pennisi, 2008; Wan et al., 2009). Although such genetic engineering has been successfully applied to some crop species, such as rice, canola (Brassica napus), and maize, the majority of published results were based on the analysis of transgenic Arabidopsis, and stress tolerance was often evaluated in potted soil with rapid stress imposition under controlled growth conditions.

Many excellent reviews regarding plant drought response and resistance focus on either particular molecules (e.g. ABA, Wasilewska et al., 2008; aquaporin, Johansson et al., 2000), cis-acting regulatory elements (Yamaguchi-Shinozaki and Shinozaki, 2005), long-distance signals (Schachtman and Goodger, 2008), signaling pathways (Xiong et al., 2002; Chinnusamy et al., 2004; Yamaguchi-Shinozaki and Shinozaki, 2006), crop engineering (Zhang et al., 2004a; Umezawa et al., 2006; Valliyodan and Nguyen, 2006), particular crop species (e.g. maize, Bruce et al., 2002; rice, Leung, 2008; canola, Wan et al., 2009), soybean (Glycine max, Manavalan et al., 2009), or offer an overall retrospective (Ingram and Bartels, 1996; Bartels and Sunkar, 2005; Maggio et al., 2006; Bressan et al., 2009; Charron and Quatrano, 2009). This review, on the other hand, focuses mainly on the recent studies of genes that are involved in molecular or biochemical processes affecting drought tolerance and that have been used successfully in the genetic engineering of staple crop species such as rice, maize, wheat (Triticum aestivum), soybean, and canola for improvement of drought tolerance (summarized in Tables 1–3 and Figure 1). Particular attention is given to those gene candidates that were assessed in a crop species under field conditions for successful yield protection against the stress. Hyperosmotic, high salinity, and cold stress responses are discussed minimally, regardless of their close cross-talk and interaction with drought-resistance responses (Seki, 2002).

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Figure 1.
Schematic Presentation of the Signaling Relationships among the Genes Described in this Review.

Drought induces the accumulation of ABA, which is affected by PARP and LOS5. ABA is perceived by the ABI1/PYR1 receptor and induces downstream TFs such as NF-YA, SNAC, and AREBs through, in some cases, SnRK2 kinase activity. SnRK2 also physically interacts with and activates the S-type anion channel protein SLAC1 to promote stomatal closure. Drought also induces the expression of CBF/DREB1, DREB2, ZFP252, NF-YB, and ZAT10 independently of ABA. DREBs and AREBs regulate, through the interaction with DRE and ABRE cis-elements, the expression of a series of downstream genes to produce osmoprotectants such as LEA, HSP, proline, GB, sugars, and polyamines, whereas farnesyltransferase α-/β-subunit (FTA/B), SNAC, NF-YA5, DST, and NPK1 are involved in the regulation of stomatal movements via, in the cases of DST and NPK1, H2O2 signaling. Drought- or ABA-induced Ca2+ fluxes are affected by PLC and perceived by CDPK and CIPK for downstream drought-tolerance responses.

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Environmental stresses on plants often induce the expression of numerous transcriptional regulators, which, in turn, up-regulate a series of downstream genes for self-protection or stress adaptation. Typical de novo synthesized regulatory proteins include TFs (Yamaguchi-Shinozaki and Shinozaki, 2006). In Arabidopsis, over 1700 genes encoding TFs in more than 50 families have been identified (Xiong et al., 2005; Palaniswamy et al., 2006; Riaño-Pachón et al., 2007; Guo et al., 2008), and some of them have been implicated in plants’ response to drought (Bartels and Sunkar, 2005; Umezawa et al., 2006). In particular, TFs from basic leucine zipper (bZIP) (e.g. ABA responsive element binding protein/ABRE binding factor (AREB/ABF)), AP2/EREBP (e.g. DRE binding protein/CRT binding factor (DREB/CBF)), NAM (no apical meristem), ATAF1-2, CUC2 (cup-shaped cotyledon) (NAC) (e.g. stress-responsive NAC (SNAC)), CCAAT-binding (e.g. nuclear factor Y (NF-Y)), and zinc-finger (e.g. C2H2 zinc finger protein (ZFP)) families have been characterized in detail with regard to their roles in the regulation of drought stress responses (Zhu, 2002; Shinozaki et al., 2003), and the ectopic expression of several of these TFs resulted in improved crop tolerance to drought (Hu et al., 2006; Nelson et al., 2007; Xiao et al., 2009; Table 1).

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Table 1.
Genes Studied in Transcriptional Regulation of Drought Response and Tolerance in Crops.

Drought and ABA-Inducible AREB/ABF
ABA responsive element binding proteins/factors (AREB/ABF) belong to a family of basic leucine zipper (bZIP) plant TFs known to function in ABA signaling during dehydration and seed maturation. In the Arabidopsis genome, 75 AREB/ABF homologs have been identified (Jakoby et al., 2002). In response to ABA, an activated AREB/ABF binds to the conserved regulatory cis-element sequence (ACGTGT/GC) called the ABA-responsive element (ABRE, Mundy et al., 1990) to trigger gene expression. ABRE is found in the promoters of the genes that are induced by ABA (Baker et al., 1994; Yamaguchi-Shinozaki and Shinozaki, 2005, 2006).

Overexpression of AREB1/ABF2, ABF3, or AREB2/ABF4 in Arabidopsis resulted in enhanced ABA response, greater guard cell closure, and reduced transpiration (Kang et al., 2002). The transgenic plants appeared to be tolerant to drought (Fujita et al., 2005), cold, heat, and oxidative stresses (Kim et al., 2004). Rice seedlings overexpressing Arabidopsis ABF3 showed less leaf rolling, wilting, and higher Fv/Fm values after withholding water for 4 d (Oh et al., 2005). However, this ABF3-mediated rice drought tolerance was outperformed by DREB1A/CBF3-mediated stress tolerance (discussed in the ABA-independent DREB/CBF section, below) when the two transgenic events were compared side by side (Oh et al., 2005).

Stress and ABA-Inducible NAC (SNAC)
NAC (NAM, ATAF1-2, CUC2) is a plant-specific TF family consisting of about 100 members that contain a highly conserved DNA-binding NAC domain (Palaniswamy et al., 2006; Guo et al., 2008). Three Arabidopsis NAC genes (ANAC019, ANAC055, and ANAC072) were isolated based on their binding to the NACRS (NAC recognition sequence, CATGTG) element in the promoter of the early responsive to dehydration 1 (ERD1) gene (Tran et al., 2004), suggesting that at least some of the NAC TFs play an important role in plant response to dehydration and drought. These three NAC genes are inducible by drought, high salinity, and ABA. In rice, 140 putative NAC or NAC-like genes are predicted according to sequence analysis, of which 40 are likely to be responsive to drought and/or salt stresses (Kikuchi et al., 2000; Fang et al., 2008). Several rice NACs such as SNAC1 (Hu et al., 2006), SNAC2 (Hu et al., 2008), OsNAC6 (Nakashima et al., 2007), ONAC045 (Zheng et al., 2009), and soybean GmNACs (Tran et al., 2009) are induced by drought, salt, cold, and ABA; canola BnNAC5-1 is slightly induced in seedlings by dehydration (Hegedus et al., 2003).

SNAC1 is also specifically induced in guard cells by drought. SNAC1-overexpressing rice showed significant reduction in stomatal aperture (Hu et al., 2006). In moderate drought or severe drought (sheltered) field conditions, SNAC1-transgenic plants exhibited 17–22% higher spikelet fertility and 22–34% higher seed setting rate than non-transgenic plants, whereas in well irrigated field conditions, transgenic plants performed similarly to wild-type plants. The enhanced drought resistance appeared to be partially due to the reduction in stomatal aperture as a result of increased ABA sensitivity in the guard cells (Hu et al., 2006). In contrast, SNAC2-overexpressing plants did not demonstrate any improvement with respect to drought tolerance but showed improved tolerance to cold, salinity, and osmotic stresses (Hu et al., 2008). Transgenic rice seedlings expressing OsNAC6 under the control of the maize ubiquitin (ZmUbi) promoter had 42–57% higher recovery rate after the hydroponically grown seedlings were removed from their growth medium and exposed to air for 12 h. However, the OsNAC6-transgenic rice suffered from growth retardation with yield penalty (Nakashima et al., 2007). In order to circumvent these problems, the ubiquitin promoter was replaced with the endogenous stress-inducible OsNAC6 promoter (Rabbani et al., 2003). The resulting transgenic rice seedlings showed increased salt tolerance without any growth and yield penalty (Nakashima et al., 2007); however, no data were given regarding the dehydration tolerance of these transgenic rice seedlings.

ABA-Independent DREB/CBF
Unlike AREB/ABF and SNAC described above, some TFs are responsive to dehydration but not to ABA, and are thus called ABA-independent dehydration-responsive TFs. These TFs typically bind to a specific and conserved cis-element (A/GCCGAC) in the promoters of target genes to activate their expression. The signature cis-element is termed the dehydration-responsive element (DRE), first discovered in the promoter of the Arabidopsis RD29A gene (Yamaguchi-Shinozaki and Shinozaki, 1994). Interestingly, DRE was simultaneously characterized as a C-repeat (CRT) element in the promoters of several cold-responsive genes (Baker et al., 1994). The DRE-binding (DREB, Liu et al., 1998) or CRT-binding factors (CBF, Stockinger et al., 1997) were then isolated and found to be the major players in driving dehydration- or cold-induced gene expression (Yamaguchi-Shinozaki and Shinozaki, 1994, 2005, 2006; Stockinger et al., 1997; Gong et al., 2008). All DREB/CBFs belong to the plant-specific AP2/ERF TF superfamily, which includes 147 members (Nakano et al., 2006). Besides their involvement in regulation of dehydration and cold responses, these TFs play a wide range of roles in seed, leaf, and flower development (Riechmann and Meyerowitz, 1998; Fujimoto et al., 2000; Lin et al., 2008).

Two classes of DREBs (i.e. DREB1 and DREB2) were isolated from Arabidopsis using yeast one-hybrid screening for proteins that bind to DRE elements (Stockinger et al., 1997; Liu et al., 1998). DREB1 expression is strongly up-regulated by cold whereas DREB2 is more responsive to drought, salt (Liu et al., 1998), and heat stresses (Sakuma et al., 2006b). DREB1s and CBFs correspond to identical loci in the Arabidopsis genome; for instance, DREB1A is identical to CBF3; DREB1B to CBF1; DREB1C to CBF2; and DREB1D to CBF4. DREB1/CBF TFs belong to Group III of the DREB/CBF subfamily (Nakano et al., 2006), which are expressed in response to various stresses in Arabidopsis, rice, wheat, barley, corn, soybean, and canola (Qin et al., 2004). To date, the DREB1/CBF signaling pathway has been one of the most explored for improving crop stress tolerance (Stockinger et al., 1997; Liu et al., 1998; Nakashima et al., 2009). When rice OsDREB1A (Dubouzet et al., 2003) or corn ZmDREB1A (Qin et al., 2004) were constitutively overexpressed in Arabidopsis, the downstream target genes regulated by the Arabidopsis DREB1 (e.g. RD29A) were induced, resulting in desiccation tolerance under 15% humidity (Qin et al., 2004) as well as salt and cold tolerance (Dubouzet et al., 2003). This suggests that the DREB1/CBF pathway and the function of DREB1 proteins are conserved in both dicot and monocot species.

To employ the DREB1 regulon for the purpose of improving rice drought stress tolerance, OsDREB1A, OsDREB1B, AtDREB1A, AtDREB1B, and AtDREB1C were ectopically overexpressed in rice under the control of either the constitutive 35S, ZmUbi, OsActin1, or the drought-inducible OsHVA22p promoter (Oh et al., 2005; Ito et al., 2006; Xiao et al., 2009). Among the transformants that overexpressed either of these proteins, the majority of the transgenic plants could survive the treatment of a 9-d water withholding while all wild-type plants died from water deprivation in pot trials (Ito et al., 2006). In rain-free and water-stressed fields, AtDREB1A/CBF3 driven by the OsHVA22p promoter significantly improved rice spikelet fertility (42% higher) and yield per plant (11% higher), whereas AtDREB1A/CBF3 driven by the constitutive OsActin1 promoter had only marginal effects (Xiao et al., 2009). It is worthwhile to note that the field drought treatment was quite severe, which resulted in a yield loss in wild-type rice plants of 82%. Under normal field conditions, however, the spikelet fertility and grain yield per plant were comparable between the wild-type control and the AtDREB1A/CBF3-transgenic rice (Xiao et al., 2009), indicating that no growth defect was derived from DREB1A transgene. This result is in agreement with an earlier report that showed the normal growth under greenhouse conditions of AtDREB1A-transgenic rice under the control of the ZmUbi promoter (Oh et al., 2005) but differs from the observation that all ZmUbi-driven DREB1-transgenic rice plants suffer from growth retardation (Ito et al., 2006). This phenotypic discrepancy is possibly due to the different rice cultivars used in these reports. Growth retardation was also observed in 35S:AtDREB1B-transgenic tomato plants, which, similarly to rice, were more resistant to water-deficit stress by showing less plant wilting and leaf curling than wild-type controls after a 21-d water withdrawal in the same pot (Hsieh et al., 2002). Unlike OsDREB1A and OsDREB1B (Ito et al., 2006), OsDREB1F caused no negative growth effects when overexpressed in transgenic rice or Arabidopsis (Wang et al., 2008b) but resulted in enhanced tolerance to drought, salt, and cold stresses in laboratory tests. By activating the expression of both ABA-dependent and ABA-independent downstream genes, OsDREB1F-transgenic rice seedlings grown in liquid medium showed less leaf coil and withering after a 5-h air dry; and transgenic Arabidopsis grown in pots survived an 8-d water withdrawal while controls failed (Wang et al., 2008b). Since OsDREB1F is also induced by ABA application but does not directly bind to the ABRE element, OsDREB1F might be a molecular bridge between ABA-independent DREB and ABA-dependent AREB pathways. Moreover, ICE1, inducer of CBF/DREB1 expression 1, a TF upstream of DREB1, was recently characterized as regulator of stomatal cell differentiation in Arabidopsis (Kanaoka et al., 2008). Therefore, it could be another link between the DREB1/CBF regulon and stomatal function/formation.

One of the main drawbacks for DREB1/CBF application for the enhancement of drought tolerance is the growth defects caused by their constitutive overexpression. In order to minimize this side effect, AtDREB1A, driven by the osmotic stress-inducible RD29A promoter, was transformed into Arabidopsis (Kasuga et al., 1999), bread wheat (Triticum aestivum, Pellegrineschi et al., 2004), tall fescue (Festuca arundinacea Schreb., Zhao et al., 2007b), and peanut (Arachis hypogaea, Bhatnagar-Mathur et al., 2007). In these cases, the transgenic plants demonstrated substantial resistance to water stress in comparison with the non-transgenic controls without displaying obvious defects in growth. Transgenic wheat seedlings in pots developed leaf wilting and bleaching symptoms 5 d later than controls after water withdrawal (Pellegrineschi et al., 2004). Transgenic peanut plants had relatively higher transpiration efficiencies in both well watered and water-limited conditions, likely explained by lower stomatal conductance (Bhatnagar-Mathur et al., 2007), while tall fescue had higher proline contents (Zhao et al., 2007b).

HARDY, another Group III member of the DREB/CBF subfamily (Nakano et al., 2006), is not induced by drought but is normally expressed in inflorescence tissues of Arabidopsis. When constitutively overexpressed in Arabidopsis, HARDY caused pleiotropic effects on vegetative growth, forming small, thick green leaves and an abnormally dense root system (Karaba et al., 2007). In rice, ectopically expressed HARDY improved the plants’ water use efficiency (WUE) in greenhouse tests (Karaba et al., 2007). The transgenic rice did not show any reduction in growth and seed yield, but had dark green leaves with more bundle sheath cells and a significant increase in leaf canopy size and number of tillers. The significant increase (50%) in rice WUE was explained by lower transpiration and higher photosynthetic carbon assimilation under both drought and well watered conditions (Karaba et al., 2007). HARDY and DREB1/CBF may share some common steps in stress signal transduction, since both of them induce the expression of a subset of genes known to respond to water deprivation and osmotic stress.

Arabidopsis DREB2A, a Group IV member of the DREB/CBF subfamily (Liu et al., 1998; Nakano et al., 2006), only weakly induced downstream genes when overexpressed, and had little effect on stress tolerance enhancement because of its possible post-translational modification or quick degradation by the 26S proteasome (Qin et al., 2008). However, as the DREB2A protein can be made constitutively active (DREB2A-CA) by specific residue deletion (Sakuma et al., 2006a), rendering DREB2A constitutively active or blocking DREB2 degradation through ubiquitination could be possibilities for the application of DREB2 in enhancing crop drought tolerance. Indeed, Sakuma et al. (2006a) showed that overexpression of DREB2A-CA significantly enhanced the drought tolerance of Arabidopsis but, as has been the case with the constitutive expression of other DREBs, this was also associated with growth retardation. On the other hand, stress-inducible or constitutive ectopic expression of maize ZmDREB2A (Qin et al., 2007) and soybean GmDREB2 (Chen et al., 2007) in Arabidopsis resulted in the improved tolerance to drought, heat, and salt stress of transgenic plants without growth defects, suggesting also that maize ZmDREB2A and soybean GmDREB2 may not require modification to be active. It has yet to be determined, however, whether the overexpression of ZmDREB2A and GmDREB2 would have similar effects on maize or soybean plants, respectively. Importantly, DREB2A regulates the expression of not only dehydration-inducible genes, but also heat-shock-related genes (Sakuma et al., 2006b). Therefore, the active form of DREB2A could be applicable to crop engineering for both drought and heat tolerance.

Zinc Finger Protein (ZFP) TFs
There are about 134 C2H2-type ZFPs in the Arabidopsis genome, and they play a critical role in many cellular functions through DNA or RNA binding and protein–protein interactions (Ciftci-Yilmaz and Mittler, 2008). The strong induction of some Arabidopsis C2H2 ZFPs by dehydration, salinity, cold, and ABA treatments suggests that these ZFPs may also be actively involved in the stress response by acting as transcriptional activators or repressors (Sakamoto et al., 2000, 2004; Sugano et al., 2003).

Recent reports on rice ZFPs, such as OsZFP252 (Xu et al., 2008), DST (drought and salt tolerance, Huang et al., 2009), and ZAT10 (Xiao et al., 2009), have shed some light on the possible roles of rice ZFP TFs in the regulation of drought responses. Overexpression of OsZFP252, a C2H2-type ZFP, enhanced the tolerance of rice seedlings to drought by showing 74–79% higher survival rate after a 14-d water withdrawal (Xu et al., 2008). The tolerance was correlated with the induced expression of OsDREB1A, suggesting that OsZFP252 might be an upstream regulator of OsDREB1A, and with a higher accumulation of free proline and soluble sugars. On the other hand, DST, another C2H2-type ZFP, was shown to function as a negative regulator in stomatal closing via the direct modulation of H2O2 homeostasis in guard cells (Huang et al., 2009). The rice DST knockdown mutant (dst) acquired a significant improvement in drought and salt tolerance without any yield penalty. In addition, an EAR (ERF-associated amphiphilic repression) C2H2 ZFP, ZAT10 (Xiao et al., 2009), and a cytoplasmic (non-TF) ZFP, OsiSAP8 (Kanneganti and Gupta, 2008), were reported to affect rice drought tolerance. ZAT10 is induced by DREB1A/CBF3 (Maruyama et al., 2004), and its overexpression in rice conferred drought tolerance and, importantly, 17–36% yield increase under rain-free field conditions (Xiao et al., 2009). OsiSAP8 is induced by multiple abiotic stresses (dehydration, salinity, heat, cold, and ABA treatment), and its overexpression in rice resulted in 50% yield penalty even under normal water conditions, but these plants survived water withdrawal for 23 d, while the non-transgenic plants did not (Kanneganti and Gupta, 2008). As the C2H2 zinc finger TFs appear to function in association with either the DREB1 pathway or H2O2-mediated stomatal response, they may be candidates that could be tailored for crop engineering for stress tolerance.

Nuclear Factor (NF-Y)
NF-Ys are ubiquitous CCAAT-binding TFs composed of A, B, and C subunits. The Arabidopsis genome encodes for 10, 13, and 13 A, B, and C subunits, respectively (Gusmaroli et al., 2002), whereas there is only a single gene for each subunit in mammals and yeast. This suggests that plants may have evolved additional functions for the NF-Y heterotrimeric complexes. Using a functional genomics strategy, a B subunit of the Arabidopsis NF-Y complex (named AtNF-YB1, Nelson et al., 2007) and, subsequently, an A subunit named AtNF-YA5 (Li et al. 2008) were isolated in searches for TFs able to improve drought tolerance (Nelson et al., 2007) or for small RNAs involved in stress responses (Li et al., 2008), respectively. In the latter case, the AtNF-YA5 gene in the Arabidopsis genome partially overlaps with its neighbor gene in reverse orientation, resulting in the production of natural antisense transcript (nat)-derived siRNA (natsiRNA, Borsani et al., 2005). AtNF-YB1 and AtNF-YA5 were both able to increase drought tolerance in Arabidopsis when constitutively overexpressed. Whereas AtNF-YB1 regulates genes not known to respond to ABA or to the DREB/CBF regulon, AtNF-YA5 is induced by both drought and ABA and is prominently expressed in the vascular system and in guard cells, where it functions to reduce stomatal aperture. AtNF-YA5 regulates a number of known stress-responsive genes. Interestingly, AtNY-YA5 is post-transcriptionally regulated by microRNA 169 (miR169), suggesting that there might be a complex mechanism controlling the functions of AtNF-YA5 (Li et al., 2008).

When ZmNF-YB2, the maize ortholog of AtNF-YB1, was constitutively expressed in an elite maize inbred line, the transgenic lines showed improved drought tolerance compared to wild-type plants under water-stressed conditions in the field, suggesting the existence of a common regulator for stress response in maize and Arabidopsis. Under such conditions, the transgenic maize had less leaf rolling, a higher photosynthetic rate, cooler leaf temperature, and higher stomatal conductance. Although earlier flowering (1–3 d) and slightly compressed internodes were observed in ZmNF-YB2 transgenic plants under normal water supply, an increase of ∼50% in yield from a best-performing line in relatively severe drought conditions highlights the potential of the application of this gene target for genetic engineering in suboptimal, drought-prone maize production systems (Nelson et al., 2007).

TFs containing conserved WRKY domain (WRKY) TFs (more than 70 in Arabidopsis) are best known for their involvement in the regulation of plant growth, development, and in response to biotic stresses (Ulker and Somssich, 2004). While many studies on WRKYs focused on disease-induced responses, relatively little is known about their roles in abiotic response. One exception is OsWRKY11, which, when driven by the heat-inducible heat shock protein (HSP)101 promoter, rendered transgenic rice significantly heat and drought-tolerant compared to controls, as indicated by slower leaf wilting in potted soil (Wu et al., 2009). It is very interesting to note that the overexpression of soybean GmWRKY54 in Arabidopsis (Zhou et al., 2008) significantly induced the expression of DREB2A, which is known to have dual functions in both drought and heat tolerance (Sakuma et al., 2006b). Unfortunately, the tolerance of GmWRKY54-transgenic Arabidopsis to drought, salt, and cold but not to heat was evaluated by the authors (Zhou et al., 2008). Transgenic plants showed significantly enhanced tolerance to drought and salt, but only slightly improved tolerance to freezing.

Challenges with Transcriptional Manipulation for the Purpose of Enhancing Drought Tolerance
Using TF regulons to improve tolerance of crops to abiotic stresses is a promising strategy because of the ability of a TF to regulate an entire set of genes in a stress-response pathway. Of all the TFs discussed above, only SNAC1 (Hu et al., 2006), NF-YB1 (Nelson et al., 2007), and DREB1A/CBF3 (Xiao et al., 2009) were confirmed as being able to enhance drought tolerance in crops under field conditions. The effects of other TFs were only assessed in potted soil under greenhouse conditions or by air-drying over a rather short period of time. In addition, the physiological assessments were often carried out with transgenic seedlings rather than with flowering plants, which are most sensitive to water deprivation with respect to yield output. According to the literature published so far, the manipulation of DREB regulons appears to be quite effective for improving plants’ tolerance to drought at their seedling stage. Their value for increasing grain yield in crops in the field has only been demonstrated in rice under rather severe drought conditions (Xiao et al., 2009); therefore, further evaluation of this promising gene target is required to determine its impact on economically important crops at the flowering stage under suboptimal field conditions.

Constitutive overexpression of some stress-responsive TFs such as DREBs (controlled by the 35S promoter) frequently caused unwanted side effects, such as growth retardation and yield penalty (Kasuga et al., 1999; Hsieh et al., 2002; Zhang et al., 2004a). In order to minimize the negative effects, DREB1A was expressed under the control of the osmotic stress-inducible RD29A promoter (Kasuga et al., 1999, 2004). One of the unique features of the RD29A:DREB1A expression cassette is the forward feedback between the RD29A promoter and the DREB1A protein. Under drought stress conditions, the drought-induced and de novo synthesized DREB1A binds to the DRE cis-element of the RD29A promoter, which drives the expression of DREB1A for rapid production of this TF. Caution should be taken, however, when using an Arabidopsis-inducible promoter in crops. For example, it was reported that the Arabidopsis RD29A promoter functions in rice root but, unexpectedly, not in leaves (Ito et al., 2006). In the case of OsNAC6 expression, the endogenous OsNAC6 promoter was used in order to minimize growth defects in transgenic rice plants (Nakashima et al., 2007), although the resulting level of OsNAC6 transcripts would be expected to be only double the normal level.

So far, most candidate genes for drought tolerance were first unearthed from studies in the Arabidopsis system. Although most stress-response and signaling pathways are thought to be ubiquitously present throughout the plant kingdom, it will be crucial to examine the involvement and contribution of additional and related gene candidates from crop species in drought tolerance.

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Protein Farnesylation
Farnesylation is a post-translational protein modification that adds a farnesyl group to a target protein. Farnesylated proteins function to regulate many different growth and developmental processes (Nambara and McCourt, 1999; Galichet and Gruissem, 2003). The plant farnesyltransferase consists of an α- (FTA) and a β- (FTB) subunit. Loss of function of FTB in Arabidopsis resulted in mutants that display an enhanced response to ABA (era1, Cutler et al., 1996). ERA1, a negative regulator of the ABA response, is involved in the regulation of stomatal aperture (Pei et al., 1998), suggesting a potential role for protein farnesylation in drought response.

The effectiveness of down-regulation of FTB using antisense technology (Wang et al., 2005) and FTA via RNAi techniques (Wang et al., 2009) for improvement of drought tolerance and yield protection has been demonstrated in transgenic canola. As the era1 mutants exhibit pleiotropic developmental abnormalities, the suppression of endogenous FTA or FTB was fine-tuned to be dependent upon water availability by using the drought stress-inducible RD29A, or the shoot-specific peroxisomal hydroxypyruvate reductase promoters. Under drought stress, transgenic canola showed reduced stomatal conductance and transpiration, likely as a result of being hypersensitive to ABA. Field trials over the course of three consecutive years indicated that under moderate drought stress at flowering stage, transgenic canola had yields significantly higher than controls, while with adequate water supplies, their yields were equal (Wang et al., 2005, 2009).

Protein Phosphorylation
Some TFs such as AREB1 need to be phosphorylated by protein kinases in order to become active (Furihata et al., 2006). Several protein kinases, especially a subset of 604 Arabidopsis receptor-like kinases, have been implicated in osmotic stress responses based on their transcriptional responses to different environmental stimuli (Boudsocq and Laurière, 2005; Chae et al., 2009). Mitogen-activated protein kinases (MAPKs) are often the focus of studies in plant response to environmental stimuli because of the well known yeast HOG1 (high-osmolarity glycerol) MAPK cascade in hyperosmotic signaling. Other protein kinases related to osmotic adaptation include calcium-dependent protein kinases (CDPKs), CBL (calcineurin B-like) interacting protein kinase (CIPK/sucrose non-fermenting protein (SNF1)-related kinase 2 (SnRK3) and SNF1-related kinase 2 (SnRK2).

MAPK cascades, consisting of three interlinked protein kinases (MAPKKK, MAPKK, and MAPK), function in many signal transduction pathways, including those activated by dehydration, cold, and high salt conditions (Nakagami et al., 2005). Based on the results of comparative sequence analysis, about 90 genes have been identified and assigned to the Arabidopsis MAPK cascades, and multiple homologs or orthologs have been reported in crop species such as maize and rice (Ning et al., 2008). The overexpression of a tobacco (Nicotiana tobacum) MAPKKK (designated Nicotiana protein kinase 1 (NPK1) activated an oxidative stress-response signaling cascade and led to enhanced tolerance to freezing, heat, and salt stress in transgenic tobacco (no drought test was performed, Kovtun et al., 2000). When the kinase domain of NPK1 was constitutively expressed in maize, the overexpression lines showed enhanced drought tolerance and maintained significantly higher photosynthetic rates under greenhouse conditions (Shou et al., 2004). The drought-stressed transgenic plants produced kernels comparable to those under well watered conditions. The mechanism behind this drought tolerance is likely that the active NPK1 switches on the oxidative stress-responsive signaling cascade at the onset of drought stress, thus protecting the photosynthetic machinery from oxidative damage. Overexpression of NPK1 had no negative effect on maize growth. In rice, four NPK1-like genes (OsNPKLs) are strongly induced by drought and salt stress, and are genetically co-localized with a drought resistance quantitative trait loci (QTL) on chromosome 1 (Ning et al., 2008). NPK1-transgenic rice under control of the drought-inducible OsHVA22P promoter also showed significantly increased spikelet fertility (23% higher) and grain production (28% higher) under rain-free field conditions, while its constitutive expression under OsActin1 promoter had a less significant effect with spikelet fertility of only 1% higher and grain yield of 11% higher (Xiao et al., 2009).

In plants, CDPKs are important sensors of environmentally induced Ca2+ fluxes. The majority of Ca2+-stimulated protein phosphorylation is performed by CDPKs (Ludwig et al., 2004). Rice OsCDPK7 is induced by salt and cold in roots and shoots. When OsCDPK7 was constitutively overexpressed in transgenic rice, the extent of the tolerance of rice seedlings to drought, salt, or cold stresses correlated well with the expression level of the transgene (Saijo et al., 2000), with 50% fewer transgenic seedlings showing wilting after a 3-d water withdrawal when compared with non-transgenic controls. Three rice late embryogenesis abundant (LEA) genes were found to be induced in the overexpression lines. It was proposed that OsCDPK7 is likely kept in an inactive form under normal conditions, but is then quickly activated by Ca2+ once stress occurs. As a consequence, no negative effects on plant growth were observed as a result of OsCDPK7’s overexpression.

Another group of Ca2+-sensing protein kinases involved in stress signaling are CIPKs. CIPKs are activated by Ca2+ through interaction with Ca2+-binding CBL proteins during the ABA response (Hirayama and Shinozaki, 2007). Activated CIPKs transduce Ca2+ signals by phosphorylating downstream protein components such as the Na+ transporter SOS1 (Quintero et al., 2002). Arabidopsis has at least 10 CBLs and 25 CIPKs, which provide a high level of diversity and flexibility with respect to CBL–CIPK interactions. The CIPK-mediated signaling pathways are also found in rice (Kim et al., 2003) and maize (Zhao et al., 2009). Three rice CIPKs (OsCIPK03, OsCIPK12, and OsCIPK15) and a maize ZmCIPK16 are all responsive to osmotic stress (Xiang et al., 2007; Zhao et al., 2009). Interestingly, when overexpressed in rice seedlings, OsCIPK12 enhanced tolerance to drought, OsCIPK15 to salt, and OsCIPK03 to cold stress (Xiang et al., 2007). This suggests that each OsCIPK may have a distinctive role in the response to different stresses. The observed drought tolerance conferred by OsCIPK12 expression was observed to be mediated by a significant increase in the proline and soluble sugar content after the onset of drought. As a result, the transgenic plants displayed less leaf rolling and a 33–57% higher growth recovery rate after a week-long water withdrawal and re-watering. No negative growth effects were observed in plants overexpressing the OsCIPK genes. Since CIPK's kinase activity also depends on its interaction with CBLs, the manipulation of CIPK alone may not always result in drought enhancement, as expected. However, constitutively active forms of CIPKs generated by mutations in the catalytic kinase domains or by deletion of the auto-inhibitory FISL motif in the kinases (Gong et al., 2002; Pandey et al., 2008) may prove useful for the application of these kinases in crop genetic engineering.

Unlike CDPKs and CIPKs, the activity of plant SnRK2 (also called SRK2 for the Arabidopsis ortholog) does not require direct Ca2+ binding, but can be induced by ABA or osmotic stress. SnRK2s belong to a relatively small plant-specific kinase family of about 10 members. SnRK2.6/SRK2E/OST (open stomata) in Arabidopsis was originally characterized as an ABA-activated protein kinase involved in the regulation of stomatal movement (Li and Assmann, 1996; Mustilli et al., 2002). ABA is known to induce stomatal closure by activating specific anion channels, such as the slow-type anion channel (SLAC1). It has recently been demonstrated that SnRK2.6 physically interacts with SLAC1 (Lee et al., 2009; Geiger et al., 2009). Furthermore, the interaction leads to SLAC1 activation through phosphorylation (Lee et al., 2009). Conversely, SLAC1 activity appears to be negatively regulated by selected members of the protein phosphatase 2C (PP2C) family such as ABA-insensitive (ABI)1 via dephosphorylation (Lee et al., 2009; Geiger et al., 2009). Therefore, the movement of guard cells might be controlled through the actions of activation/inactivation by a specific pair of kinase–phosphatase (Figure 1). It would be interesting to see whether activation of SLAC1 constitutively or conditionally would lead to drought stress tolerance.

SnRK2 also phosphorylates a motif in the Constant (C) subdomains in bZIP TFs including AREB1, AREB2, and ABI5 to make them active for transcription (Furihata et al., 2006). SRK2E (SnRK2.6) was identified as a key regulator of stomatal closure in Arabidopsis leaves (Mustilli et al., 2002), while SRK2C (SnRK2.8), abundant in roots tips, mediates drought stress signaling in roots. When overexpressed in Arabidopsis, SRK2C (SnRK2.8) improved drought tolerance by up-regulating stress-responsive genes such as DREB1 (Umezawa et al., 2004). Since the signal transduction pathways of stress-activated SnRK2s are highly conserved in higher plants, the manipulation of SnRK2 expression in Arabidopsis could be developed into a valuable tool for enhancing drought stress tolerance in important crops.

Two types of proteins are critical to the process of ABA perception: pyrabactin resistance (PYR)1/PYL and protein phosphatase 2C (e.g. ABI1 and ABI2) (Park et al., 2009). ABI1, a negative regulator of the ABA response, is repressed upon ABA binding to ABI1 and PYR1. Inactivation of multiple ABI1 functional homologs was shown to enhance ABA sensitivity and reduce water consumption in Arabidopsis (Saez et al., 2006). Certainly, the ABA receptor proteins are of immediate interest as targets for improvement of drought tolerance in crops. However, due to the high redundancy of these receptors in each species as well as in their interacting components downstream in the ABA signaling pathways, a crop-specific, precise engineering approach would be required in order to utilize these components to achieve satisfactory effects in protecting crop productivity against stresses.

Protein Poly(ADP-ribosyl)ation
Many abiotic stresses cause the accumulation of reactive oxygen species (ROS) in plant cells, which can, among other things, cause damage to genetic material and subsequently interfere with cellular energy homeostasis through the activation of Poly(ADP-ribose) polymerase (PARP, Amor et al., 1998; Doucet-Chabeaud et al., 2001), which catalyzes the synthesis of long branching poly(ADP-ribose) polymers covalently attached to proteins by consuming NAD+ pools (Bakondi et al., 2002). Reducing the activity of PARP, thus limiting its consumption of NAD+, could conceivably reduce the impact of environmental stresses on cellular energy homeostasis, resulting in the enhanced ability of the plants to tolerate stress. Both Arabidopsis and canola with reduced PARP activity following transformation with a PARP hairpin (hpPARP) construct were more resistant to various abiotic stresses such as drought, heat, and high light without accompanying growth defects (de Block et al., 2005). This stress tolerance was initially attributed to the maintenance of energy homeostasis due to reduced NAD+ consumption (de Block et al., 2005); however, another explanation for stress tolerance conferred by PARP deficiency is the apparent induction of specific ABA signaling pathways (Vanderauwera et al., 2007). PARP-deficient plants had elevated ABA levels and an over-representation of transcripts of ABA-responsive genes such as ABF3 and RD29A in addition to transcripts for genes involved in starch metabolism and flavonoid biosynthesis. Field trials with canola and corn hpPARP-transgenic plants showed a significant difference in yield (20–40% increase for best lines) under drought (Vanderauwera et al., 2007). Since PARP is able to modify some chromatin-associated proteins and function in transcriptional regulation, it will be of great interest to dissect the chromosomal control of ABA-related gene expression by PARP activity.

Challenges with Post-Translational Manipulation for the Purpose of Enhancing Drought Tolerance
The majority of known regulatory genes (e.g. TFs and protein kinases) were originally isolated and characterized as important stress regulators based on their transcriptional induction by various stresses. However, as discussed, many proteins undergo some form of post-translational modification in order to be biologically active in vivo (e.g. DREB2A, Sakuma et al., 2006a; AREB1/ABF2, Furihata et al., 2006; CIPK, Gong et al., 2002; see Table 2). The expression of such genes may be constitutive in terms of transcript accumulation as revealed by transcript profiling, so the challenge in this regard is to identify components whose activities are controlled by post-translational modification or intracellular translocation rather than by transcription during stress signaling. Direct manipulation of the active form of a regulator could bypass the control from its upstream modifier, and thus may be a better approach for genetic engineering for drought-resistant plants. For instance, modifying the phosphorylation site of the bZIP TF TRAB1 (Kagaya et al., 2002) or AREB1/ABF2 (Furihata et al., 2006) to enhance protein activity resulted in the induction of many ABA-responsive genes without exogenous ABA application.

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Table 2.
Genes Studied in Post-Translational Regulation of Drought Tolerance in Crops.

The successful application of manipulating genes involved in protein modification was well demonstrated in drought-tolerant canola suppressing protein farnesylation (Wang et al., 2005, 2009) and rice with elevated levels of CDPK or CIPK (Saijo et al., 2000; Xiang et al., 2007). Many regulatory proteins and enzymes can be switched on and off by phosphorylation and dephosphorylation so as to control a wide range of cellular processes or signal relays. However, the in vivo substrates of many stress-responsive protein kinases remain unknown because of the low substrate specificity of kinases in in vitro activity assays. Therefore, one of the great challenges ahead is the identification of the in vivo substrates of these protein kinases or other post-translational modifiers in order to be able to genetically fine-tune the effectors for stress tolerance.

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ABA Metabolism
One of the quick responses of plants to drought stress is the accumulation of ABA in plant cells (Hsiao, 1973), which triggers ABA-inducible gene expression (Yamaguchi-Shinozaki and Shinozaki, 2006) and stomatal closure to reduce transpirational water loss (Schroeder et al., 2001). ABA de novo biosynthesis occurs in leaves, stems, and roots of almost all plant species. The initial four catalytic steps of ABA biosynthesis, which involves the conversion of β-carotene to xanthoxin, primarily take place in plastids (e.g. chloroplasts in the leaf) via ABA1/low expression of osmotically responsive gene 6 (LOS6), ABA4, and 9-cis-epoxycarotenoid dioxygenase (NCED) enzymatic activities, while the last two steps occur in the cytoplasm to produce bioactive ABA via ABA2 and ABA3/LOS5 (Wasilewska et al., 2008; Wan et al., 2009). In the cytoplasm, ABA is readily perceived by the recently identified ABA receptors (PYR1/ABI1, Park et al., 2009), which then relay the signal to the AREB TFs to regulate the ABA-dependent expression of numerous downstream target genes for drought adaptation. ABA can also be directly converted from its inactive glucose ester conjugate storage form in vacuoles to bioactive ABA via β-glucosidase (AtBG1, Lee et al., 2006) and vice versa. After rehydration or drought release, ABA is catabolized to phaseic acid or dehydrophaseic acid by Cyp707A-mediated hydroxylation to maintain its homeostasis (Kushiro et al., 2004).

Although the genes for the metabolism of ABA in Arabidopsis have all been characterized, and those for ABA perception and the downstream signal relay are being described at rapid pace, the manipulation of only one gene (LOS5/ABA3) has been reported so far to have an effect on crop (rice) drought tolerance enhancement (Xiao et al., 2009). LOS5/ABA3 is a key enzyme working in the last step of ABA biosynthesis, and its loss-of-function mutant los5 had reduced tolerance to drought, salt, and cold stresses (Xiong et al., 2001). The constitutive or drought-inducible overexpression of LOS5 in rice significantly increased the spikelet fertility and yield of transgenic plants under field conditions (Xiao et al., 2009). NCED is drought-inducible and a rate-limiting enzyme for ABA biosynthesis. Thus, it may be the best candidate for gene manipulation to regulate ABA homeostasis in plants. However, although overexpression of LeNCED1 in tomato (Solanum lycopersicum L.) did increase ABA accumulation and WUE in transgenic tomato, it had no effect on dehydration tolerance (Thompson et al., 2007) while causing severe growth defects such as stunted plants and increased seed dormancy (Thompson et al., 2000; Tung et al., 2008).

Phospholipid Signaling
Membrane-component phospholipids and their metabolites are important second messengers in plant development and in response to environmental stimuli (Testerink and Munnik, 2005; Xue et al., 2009). Phosphatidylinositol-specific phospholipase C2 (PtdIns-PLC2) hydrolyzes PtdIns-4,5-biphosphate (PtdIns(4,5)P2) to produce Inositol 1,4,5,-triphosphate (Ins(1,4,5)P3), which acts as a second messenger for Ca2+ release from internal storages. The Ca2+ oscillation mediated by PtdIns(4,5)P2 and Ins(1,4,5)P3 is crucial for guard cell movement as well as for other plant growth processes. In addition, phospholipase D (PLD) hydrolyses phospholipids at the terminal phosphodiester bond to generate phosphatidic acid (PA), which interacts with and represses the activity of ABI1, resulting in enhanced ABA response and stomatal closure (Zhang et al., 2004b). PA is likely the functional link between the phospholipid and ABA signaling pathways with respect to stomatal movement and drought response.

Arabidopsis PLC (Tasma et al., 2008) and PLD (Katagiri et al., 2001) genes are both induced by dehydration and involved in drought tolerance (Sang et al., 2001; Hong et al., 2008). Maize overexpressing ZmPLC1 (Wang et al., 2008a) exhibited a higher cellular solute content and increased rate of photosynthesis, whereas canola overexpressing BnPtdIns-PL2 (Georges et al., 2009) exhibited a lower rate of stomatal transpiration and a smaller stomatal aperture. Transgenic maize were more productive, as indicated by showing 14% higher kernel weight per ear under drought, while transgenic canola were able to survive progressive water deprivation for 24 d, under which conditions the control plants died.

Heat or Cold Shock Protein (HSP or CSP) Chaperones
Drought, salt, and high temperature can cause the misfolding and dysfunction of many RNAs and proteins. Some stress-induced genes encode proteins to protect the conformation of other proteins, RNAs, or cell structures. These include numerous HSPs and CSPs, which are required not only for quick adaptation to temperature changes, but also for rapid recovery after heat or cold release. Small HSPs (sHSPs) represent the major family of HSPs induced by heat in plants. It has been observed that the exposure of rice seedlings to high temperature (42°C for 24 h) resulted in a significant increase in drought tolerance, and rice seedlings overexpressing rice sHSP17.7 also had enhanced tolerance to drought stress, suggesting that the observed drought-tolerance acquisition after heat shock was associated with the accumulation of sHSP proteins (Sato and Yokoya, 2008). Endoplasmic reticulum (ER) luminal binding protein (BiP), a member of the HSP70 family of protein chaperones, is induced by abiotic stresses and overexpression of soyBiPD conferred drought resistance to soybean plants and resulted in a delay in drought-induced leaf senescence through an unknown mechanism (Valente et al., 2009).

CSPs, which rapidly accumulate in some bacteria under low-temperature stress, function as RNA chaperones to stimulate growth following stress acclimation. Bacterial CspA and CspB, when expressed in Arabidopsis, rice, and maize, conferred enhanced tolerance to drought, cold, and heat by protecting and improving vegetative growth, photosynthesis, and reproductive development (Castiglioni et al., 2008). Multiple-location, multiple-year, and multiple-hybrid field trials with CspA or CspB-expressing maize showed yield increases of 11–21% for maize plants under limited water supply during or after vegetative-to-reproductive transition. No yield drag was observed for CspB-transgenic maize under normal water supply; however, the significant effect of CspB on corn yield protection was achieved only under severe drought that often caused about 50% overall yield losses when compared with yields from unstressed transgenic plants (Castiglioni et al., 2008). Nevertheless, commercial grade hybrid corn modified with this technology is on its way to the marketplace, intended for sub-optimal corn farming systems, and the regulatory approval for CspB transgenic corn is underway (Edmeades, 2008).

Late Embryogenesis Abundant (LEA) Proteins
LEA proteins accumulate in embryos during seed desiccation and are also induced in vegetative tissues by dehydration, cold, salt, and ABA treatment. LEAs are extremely hydrophilic and are involved in adaptive responses to hyperosmotic conditions through the maintenance of protein or membrane structure, sequestration of ions, binding of water, and their operation as molecular chaperones (Bray, 1997). ABA-inducible HVA1, a group 3 LEA protein from barley, was reported to sequester ions (e.g. Na+) during cellular dehydration (Hong et al., 1988). The idea of overexpressing LEA genes for drought tolerance has been tested. These include rice expressing barley HVA1 (Xu et al., 1996; Rohila et al., 2002; Babu et al., 2004) or rice OsLEA3-1 (Xiao et al., 2007); spring wheat (Triticum aestivum) expressing barley HVA1 (Sivamani et al., 2000); and Chinese cabbage (Brassica chinensis) expressing a canola LEA (Park et al., 2005). The transgenic wheat had significantly higher dry mass accumulation and WUE under moderate water-deficit conditions in greenhouse tests (Sivamani et al., 2000); however, no consistent results were obtained in multiple-season/location field trials (Bahieldin et al., 2005). The HVA1-transgenic rice and LEA-transgenic Chinese cabbage exhibited improved tolerance at the vegetative stage to water deprivation and to salt stress in potted soil, as indicated by higher growth rates and faster recovery from the stresses (Xu et al., 1996; Park et al., 2005). The dehydration and salt tolerance likely resulted from better cell membrane protection, as indicated by low membrane electrolyte leakage, but was not likely due to osmotic adjustment (the ratio of leaf osmotic potential before and after dehydration stress), which was found to be quite similar among transgenic and control plants (Rohila et al., 2002; Babu et al., 2004). Under rain-free field drought conditions, the transgenic rice expressing OsLEA3-1 under either its own drought-inducible promoter or the 35S promoter had significantly higher spikelet fertility and grain yield (∼45%) than wild-type (Xiao et al., 2007). Even more promisingly, no significant yield penalty was detected between the transgenic and wild-type plants under normal field conditions. However, the significant yield protection attributed to OsLEA3-1 expression was observed only under severe stress that still led to about 45% yield loss when compared with wild-type plants under normal field condition (Xiao et al., 2007).

Amino Acids (Proline)
Drought resistance could also be improved by increasing in plant cells the accumulation of metabolites that function as adaptive osmolytes or antioxidants. These metabolites function via either osmotic adjustment (OA) to increase water retention or as osmoprotectants to stabilize other molecules under stress conditions.

Proline is one of the most common compatible solutes (small hydrophilic organic molecules that accumulate to high concentrations) in water-stressed plants and in many other organisms (Delauney and Verma, 1993). The accumulation of proline in dehydrated plants is caused by both the promotion of its biosynthesis and by the prevention of its degradation. Water stress-induced overexpression of 1-pyrroline-5-carboxylate synthase (P5CS), the rate-limiting enzyme for proline biosynthesis in rice (Zhu et al., 1998), or 1-pyrroline-5-carboxylate reductase (P5CR) in soybean (de Ronde et al., 2004a, 2004b) increases proline content by about 2.5-fold after drought induction, resulting in increased relative water content (RWC) and better growth under water-deficit conditions, whereas the suppression of P5CR in soybean resulted in an increased sensitivity to osmotic, drought, and heat stresses (de Ronde et al., 2000). Other evidence suggests a role for proline in drought adaptation through the DREB1 pathway. For example, tall fescue and Arabidopsis overexpressing AtDREB1A/CBF3 (Gilmour et al., 2000; Zhao et al., 2007b), tomato overexpressing AtDREB1B/CBF1 (Hsieh et al., 2002), and rice overexpressing OsDREB1 or AtDREB1 (Ito et al., 2006) have been shown to accumulate higher levels of proline than wild-type plants under both normal and water-deficit conditions. Correspondingly, the transgenic plants were more resistant to water-deficit stress than the wild-type controls.

Glycine Betaine (GB)
Plants from several families (e.g. Gramineae, Chenopodiaceae, Compositae) produce GB in response to salt or water stress. GB accumulates in the chloroplasts and plastids of these and many other halotolerant plants, presumably to stabilize photosynthetic and translational machineries via ROS scavenging and protein protection (Chen and Murata, 2008). When applied exogenously, GB translocates actively to all parts of plants, whereas GB produced in vivo accumulates five times more in flowers and siliques than in leaves of transgenic Arabidopsis, indicating its potential role in protection in reproductive machinery (Sulpice et al., 2003).

Genetic engineering to increase GB biosynthesis (by dehydrogenation or oxidation of choline) had only a marginal effect on GB levels (Bartels and Sunkar, 2005), and tolerance enhancement by this method has been more successful in salt and chilling stresses than in drought stress (Chen and Murata, 2008). Maize plants transformed with betA, which encodes a choline dehydrogenase, accumulated more GB than controls when exposed to drought stress in the field (Quan et al., 2004). After a 3-week drought stress, reproductive development of transgenic plants was less inhibited, as evidenced by a 10–23% higher grain yield per plant than that of wild-type. betA-transgenic cotton were tolerant to drought stress, as they displayed a higher RWC, increased photosynthesis, better OA, a lower percentage of ion leakage, and less lipid membrane peroxidation than the wild-type controls (Lv et al., 2007).

A novel GB biosynthesis pathway via glycine was reported recently in halophilic microorganisms (Nyyssola et al., 2000; Waditee et al., 2003). Arabidopsis expressing genes from this pathway accumulated more GB than plants expressing the choline monooxygenase gene from the conventional choline-mediated pathway (Waditee et al., 2005). Whether commercially important crops can be engineered to take advantage of this novel pathway remains to be tested.

Sugars (Trehalose, Mannitol)
Sugar osmolytes include simple sugars (e.g. glucose), sugar alcohols (e.g. mannitol), and complex sugars (e.g. trehalose). Trehalose is synthesized in response to dehydration stress in various organisms, such as insects and lower plants, to stabilize dehydrated proteins, lipid membranes, and biological structures. But, among higher plants, most species, except for highly desiccation-tolerant resurrection plants, accumulate only trace amounts of trehalose. To increase the biosynthesis of trehalose in rice (Garg et al., 2002; Jang et al., 2003) and tomato (Cortina et al., 2005), bacterial and yeast trehalose-6-phosphate synthase (TPS) and phosphatase (TPP), key enzymes in trehalose biosynthesis, were engineered into the plant genomes under the control of either tissue-specific, stress-responsive, or constitutive promoters. Transgenic rice plants could accumulate two to nine times more trehalose than non-transgenic controls (Garg et al., 2002) or even more when TPS and TPP were expressed as bifunctional fusion proteins in rice (Jang et al., 2003). After 8 or 12 d of water withholding, non-transgenic rice exhibited leaf rolling and wilting, while transgenic rice showed vigorous shoot growth with considerably fewer visual stress symptoms (Garg et al., 2002; Jang et al., 2003). In addition, transgenic rice exhibited less photo-oxidative damage under salt and low-temperature stresses.

Wheat does not naturally synthesize mannitol; however, expressing the E. coli mtlD gene caused the accumulation of mannitol in transgenic wheat cells (Abebe et al., 2003). A high accumulation of mannitol generally causes severe abnormalities such as sterility and stunted growth; however, a moderate accumulation of mannitol was shown to improve the tolerance of transgenic wheat to water and salt stresses by facilitating increased biomass and plant height under stressed conditions compared to wild-type (Abebe et al., 2003).

Polyamines are small nitrogenous compounds involved in plant responses to various stresses. The physiological significance of polyamines in stress response is not yet thoroughly understood (Groppa and Benavides, 2008). In plants, polyamines (spermidine and spermine) are derived from arginine via enzymes such as arginine decarboxylase (adc) and S-adenosylmethionine decarboxylase (samdc). Transgenic rice expressing oat adc (Capell et al., 1998) or jimsonweed (Datura stramonium) adc had a significant increase in putrescine, a precursor of spermidine and spermine (Capell et al., 2004). The increase surpassed the threshold required to trigger the conversion of putrescine to spermidine and spermine; however, only 1.5-fold increases of each compound were observed in transgenic rice subjected to drought stress, suggesting a tight control of polyamine homeostasis in plants. Nevertheless, adc-transgenic plants showed no chlorophyll loss after 8 d of drought stress (Capell et al., 1998) and developed no stress symptoms, such as rolling leaves or wilting, as controls did under drought (Capell et al., 2004). Since the spermidine and spermine content in rice was not correlated with mRNA levels of adc but was with that of samdc, the latter, which actually catalyzes the formation of the polyamines from putrescine precursors, should be the primary candidate for plant engineering. An alternative explanation for the low polyamine accumulation in transgenic rice is that the over-accumulation of polyamines is toxic to plants (Capell et al., 1998), in which case constitutive overexpression of adc or samdc should be avoided for genetic engineering.

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There have been very few successful genetic engineering examples to improve plant tolerance against dehydration stress by increasing cellular content of compatible solutes (see Table 3). This is likely due to two main reasons: (1) the transgene-induced increase in compatible solutes, in most cases, represented only a small portion of the total OA of plant cells under osmotic stress; and (2) the induced increase often resulted in impaired plant growth, even in the absence of stress (Holmström et al., 1996; Abebe et al., 2003; Karim et al., 2007). Osmolyte-mediated OA responsible for maintaining cell turgor, when it occurs in the root or root tips, could allow continued root development into deeper soil and thus give plants access to a deep water reservoir (Serraj and Sinclair, 2002; Sinclair et al., 2004). In contrast, OA-mediated cell turgor maintenance in leaves may prolong stomatal opening and thus lead to more water transpiration during drought stress. Taking into account the growth retardation of plants overproducing osmolytes, investigations that seek to improve crop performance by enhancing OA need to focus on the root or root tip by using tissue-specific expression systems.

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Table 3.
Metabolites and Osmoprotectants Studied in Drought Tolerance in Crops.

The functional mechanisms of some compatible solutes in drought tolerance enhancement are, however, still unclear or controversial. Experiments expressing tomato P5CS in yeast cells, for example, showed that yeast growth is inversely correlated with proline accumulation under osmotic stress conditions (Maggio et al., 2002), suggesting that proline or other osmolytes may not act as osmoprotectants.

The successful application of betA gene expression (GB biosynthesis) in drought-tolerance enhancement of maize and cotton propelled the search for the mechanism behind GB function. GB, like plant hormones, can be exogenously applied to, quickly taken up by, and actively spread to all parts of the plant. Moreover, GB has an in vivo concentration in the millimolar range, which is the effective concentration range of most plant hormones. As such, it was postulated that, like phytohormones, GB's effects might be manifested through the induction of specific downstream genes. However, no drought-related genes have been confirmed so far to be directly induced by GB.

In the case of sugar solutes, it was proposed that the primary effect of trehalose is not actually as a compatible solute (Garg et al., 2002). Rather, increased trehalose accumulation correlates with higher soluble carbohydrate levels and an elevated capacity for photosynthesis under both stress and non-stress conditions. This suggests that the role of trehalose in stress tolerance is actually through the modulation of sugar sensing and carbohydrate metabolism. In transgenic wheat that accumulated mannitol, it was found that the concentration of mannitol was too low to function as an osmolyte, but may have functioned as a ROS scavenger (Abebe et al., 2003). It should also be noted that the high level of osmolyte accumulation in plants necessary for drought tolerance improvement is biologically expensive; therefore, the effect on plant biomass production or on yield should be evaluated.

Single genes are more amenable to manipulation but less sustainable under various field conditions. To compensate for built-in feedback control mechanisms or to avoid the over-accumulation of intermediate products, it is sometimes required that multiple genes be manipulated. For instance, stress-tolerant TPS-expressing tobacco plants had a higher trehalose content but also presented with developmental irregularities such as stunted growth and altered leaf shape (Holmström et al., 1996; Karim et al., 2007), possibly due to the over-accumulation of trehalose-6-phosphate (T6P, Schluepmann et al., 2004). To avoid growth defects while maintaining drought tolerance, three strategies were successfully applied: (1) co-expression of TPS and TPP to reduce the over-accumulation of harmful T6P intermediates; (2) stress-induced expression of TPS using an appropriate promoter; and (3) the targeting of TPS to chloroplasts where it functions naturally (Karim et al., 2007). In addition, a fusion-gene strategy was successfully applied in rice to allow the overexpression of two trehalose biosynthesis genes (otsA and otsB), requiring only a single transformation event and resulting in a higher net catalytic efficiency for trehalose formation (Garg et al., 2002).

Nevertheless, very encouraging results for enhanced drought tolerance of commercial crops is coming from, for instance, the overexpression of some molecular chaperones in maize or rice (Castiglioni et al., 2008). Constitutive overexpression of ubiquitous RNA chaperones (e.g. CSP) of bacterial origin made transgenic plants more tolerant to multiple stresses, but caused no negative pleiotropic effects, suggesting that the small chaperone molecules might be good candidates for drought tolerance enhancement.

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This review focused on the recent progress in genetic manipulation of important crops for the enhancement of drought tolerance, which is a very complex plant trait. It should be borne in mind that tolerance to a given stress such as drought should ultimately be evaluated in association with other major stresses such as high temperature, salt, and cold because of the cross-talk between these stress-response pathways. The majority of the genes in the literature reported to play a role in drought tolerance also, to some extent, confer some degree of tolerance to salt and cold stresses. However, crops tolerant to drought as well as heat will be in immediate demand for a world that is generally hotter and drier as a result of global climate change (Battisti and Naylor, 2009). Since our knowledge about stress signaling networks is still limited, the challenge in the near future will be to identify the signaling elements missing in our current models of pathways and increasing our understanding of the cross-talk between pathways.

Outside of the scope of this review due to the absence of literature available on the subject as it pertains to drought tolerance in crop species are non-coding regulatory RNAs. Regulatory RNAs including siRNAs and miRNAs have been emerging as important regulators in abiotic stress response and tolerance (Borsani et al., 2005; Zhao et al., 2007a; Li et al., 2008). It is very likely that regulatory proteins work in concert with regulatory RNAs to fine-tune the temporal and spatial expression of genes in stress response (Li et al., 2008; Alexandre et al., 2009).

The identification of commercial grade transgenes that enhance crop performance under both drought and optimal conditions is a lengthy, tedious, and expensive process. Nevertheless, the successful genetic engineering of canola (Wang et al., 2005, 2009), rice (Hu et al., 2006; Xiao et al., 2007, 2009), and maize (Nelson et al., 2007; Castiglioni et al., 2008) for improved drought tolerance as reviewed herein confirms that the approach is feasible (Tables 1–3 and Figure 1). However, among the reports with promising drought-tolerance enhancement under field conditions, there were only a few showing significant net crop yield benefit (Wang et al., 2005, 2009), while the majority of the reports showing a yield benefit are based on experiments in which the drought conditions were too severe and yields were usually too low to be acceptable for agricultural practice, especially in developed countries. So far, none of the reported technologies has been tested in actual farmers’ fields, although some commercial plans have been proposed for within the next couple of years by some biotech companies. Ultimately, with the use of the powerful molecular and genetic tools available, such as stress-metabolite profiling (Urano et al., 2009), functional genomics, and proteomics, more and more key regulators of plant stress responses will be identified and utilized as target genes or molecular markers for crop genetic engineering or marker assistant selection to create cultivars with yield stability in drought-prone environments.

No conflict of interest declared.

© The Author 2010. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS.
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Radakovits 10 April 2013

Created By: Daylen Gargalis

Genetic Engineering of Algae for Enhanced Biofuel Production ▿
Randor Radakovits1, Robert E. Jinkerson1, Al Darzins2 and Matthew C. Posewitz1,*
+ Author Affiliations

1Department of Chemistry and Geochemistry, Colorado School of Mines, 1500 Illinois St., Golden, Colorado 80401, and
2National Renewable Energy Laboratory, 1617 Cole Blvd., Golden, Colorado 80401

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There are currently intensive global research efforts aimed at increasing and modifying the accumulation of lipids, alcohols, hydrocarbons, polysaccharides, and other energy storage compounds in photosynthetic organisms, yeast, and bacteria through genetic engineering. Many improvements have been realized, including increased lipid and carbohydrate production, improved H2 yields, and the diversion of central metabolic intermediates into fungible biofuels. Photosynthetic microorganisms are attracting considerable interest within these efforts due to their relatively high photosynthetic conversion efficiencies, diverse metabolic capabilities, superior growth rates, and ability to store or secrete energy-rich hydrocarbons. Relative to cyanobacteria, eukaryotic microalgae possess several unique metabolic attributes of relevance to biofuel production, including the accumulation of significant quantities of triacylglycerol; the synthesis of storage starch (amylopectin and amylose), which is similar to that found in higher plants; and the ability to efficiently couple photosynthetic electron transport to H2 production. Although the application of genetic engineering to improve energy production phenotypes in eukaryotic microalgae is in its infancy, significant advances in the development of genetic manipulation tools have recently been achieved with microalgal model systems and are being used to manipulate central carbon metabolism in these organisms. It is likely that many of these advances can be extended to industrially relevant organisms. This review is focused on potential avenues of genetic engineering that may be undertaken in order to improve microalgae as a biofuel platform for the production of biohydrogen, starch-derived alcohols, diesel fuel surrogates, and/or alkanes.

[2] Interest in a variety of renewable biofuels has been rejuvenated due to the instability of petroleum fuel costs, the reality of peak oil in the near future, a reliance on unstable foreign petroleum resources, and the dangers of increasing atmospheric CO2 levels. Photosynthetic algae, both microalgae and macroalgae (i.e., seaweeds), have been of considerable interest as a possible biofuel resource for decades (165). Several species have biomass production rates that can surpass those of terrestrial plants (41), and many eukaryotic microalgae have the ability to store significant amounts of energy-rich compounds, such as triacylglycerol (TAG) and starch, that can be utilized for the production of several distinct biofuels, including biodiesel and ethanol. It is believed that a large portion of crude oil is of microalgal origin, with diatoms being especially likely candidates, considering their lipid profiles and productivity (153). If ancient algae are responsible for creating substantial crude oil deposits, it is clear that investigation of the potential of living microalgae to produce biofuels should be a priority. Microalgae are especially attractive as a source of fuel from an environmental standpoint because they consume carbon dioxide and can be grown on marginal land, using waste or salt water (41). In addition, it may be possible to leverage the metabolic pathways of microalgae to produce a wide variety of biofuels (Fig. 1). In contrast to corn-based ethanol or soy/palm-based biodiesel, biofuels derived from microalgal feedstocks will not directly compete with the resources necessary for agricultural food production if inorganic constituents can be recycled and saltwater-based cultivation systems are developed.

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FIG. 1.
Microalgal metabolic pathways that can be leveraged for biofuel production. ER, endoplasmic reticulum.

However, several technical barriers need to be overcome before microalgae can be used as an economically viable biofuel feedstock (139). These include developing low-energy methods to harvest microalgal cells, difficulties in consistently producing biomass at a large scale in highly variable outdoor conditions, the presence of invasive species in large-scale ponds, low light penetrance in dense microalgal cultures, the lack of cost-effective bioenergy carrier extraction techniques, and the potentially poor cold flow properties of most microalga-derived biodiesel. To advance the utilization of microalgae in biofuel production, it is important to engineer solutions to optimize the productivity of any microalgal cultivation system and undertake bioprospecting efforts to identify strains with as many desirable biofuel traits as possible. Over 40,000 species of algae have been described, and this is likely only a small fraction of the total number of available species (75). The U.S. Department of Energy's Aquatic Species Program analyzed approximately 3,000 different microalgae for their potential to produce biofuels, and numerous additional species have subsequently been investigated (165). Although these efforts demonstrated that many species of microalgae have properties that are desirable for biofuel production, most have drawbacks that have prevented the emergence of an economically viable algal biofuel industry. It is postulated that a light-harvesting footprint of at least 20,000 square miles will be required to satisfy most of the current U.S. transportation fuel demand (41). Therefore, even modest improvements in photon conversion efficiencies will dramatically reduce the land area and cost required to produce biofuels. Consequently, continued bioprospecting efforts and the development and engineering of select microalgal strains are required to improve the yields of bioenergy carriers. Current commercial agriculture crops have been cultivated for thousands of years, with desired traits selected over time. It stands to reason that with microalgae, it would be beneficial to use genetic engineering in an attempt to bypass such a lengthy selection process. However, despite the recent advances in biotechnological approaches, the full potential of genetic engineering in some microalgal species, particularly diploid diatoms, can be fully realized only if conventional breeding methods become firmly established, thereby allowing useful traits or mutations to be easily combined (5, 24, 25). Since the topic of microalgal sexual breeding is beyond the scope of this review, we will instead focus on genetic engineering approaches that could be utilized in the industry's efforts to improve microalgae as a source of biofuels.

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Significant advances in microalgal genomics have been achieved during the last decade. Expressed sequence tag (EST) databases have been established; nuclear, mitochondrial, and chloroplast genomes from several microalgae have been sequenced; and several more are being sequenced. Historically, the green alga Chlamydomonas reinhardtii has been the focus of most molecular and genetic phycological research. Therefore, most of the tools for the expression of transgenes and gene knockdown have been developed for and are specific for this species. However, tools are now also being rapidly developed for diatoms and other algae that are of greater interest for industrial applications.

Microalgal genomes.Access to microalgal genome sequences that are of interest for academic or industrial applications greatly facilitates genetic manipulation, and the availability of rapid large-scale sequencing technology represents a revolution in microalga research. Several nuclear genome sequencing projects have now been completed, including those for C. reinhardtii (116, 171), Phaeodactylum tricornutum (15), Thalassiosira pseudonana (6), Cyanidioschyzon merolae (109), Ostreococcus lucimarinus (135), Ostreococcus tauri (36), and Micromonas pusilla (201). Currently, ongoing microalgal genome sequencing projects include those for Fragilariopsis cylindrus, Pseudo-nitzschia, Thalassiosira rotula, Botryococcus braunii, Chlorella vulgaris, Dunaliella salina, Micromonas pusilla, Galdieria sulphuraria, Porphyra purpurea, Volvox carteri, and Aureococcus anophageferrens (100). In addition, there are several completed and ongoing efforts to sequence plastid and mitochondrial genomes, as well as dynamic transcriptomes from many different microalgae (4, 9, 29, 66, 73, 101, 105, 130, 133, 149, 161, 169, 171, 196, 198).

Methods for transformation and expression.Successful genetic transformation has been reported for the green (Chlorophyta), red (Rhodophyta), and brown (Phaeophyta) algae; diatoms; euglenids; and dinoflagellates (2, 3, 21,–,23, 26, 30, 37, 42, 44, 45, 49, 54,–,56, 58, 65, 69, 76, 83, 85, 87, 88, 92,–,95, 108, 119, 121, 123, 147, 148, 150, 151, 163, 170, 180, 181, 183, 187, 210, 211, 214, 217). More than 30 different strains of microalgae have been transformed successfully to date. In many cases, transformation resulted in stable expression of transgenes, from either the nucleus or the plastid, but in some cases only transient expression was observed. Methods developed primarily with C. reinhardtii (for a recent review by Eichler-Stahlberg et al., see reference 48) demonstrate that the stability of expression can be improved through proper codon usage, the use of strong endogenous promoters, and inclusion of species-specific 5′, 3′, and intron sequences. The efficiency of transformation seems to be strongly species dependent, and the method of transformation has to be carefully selected and optimized for each microalga. A variety of transformation methods have been used to transfer DNA into microalgal cells, including agitation in the presence of glass beads or silicon carbide whiskers (44, 87, 119), electroporation (21, 22, 26, 108, 170, 181, 184), biolistic microparticle bombardment (2, 45, 49, 51, 52, 81, 83, 88, 183, 187, 210, 211), and Agrobacterium tumefaciens-mediated gene transfer (23, 93).

Efficient isolation of genetic transformants is greatly facilitated by the use of selection markers, including antibiotic resistance and/or fluorescent/biochemical markers. Several different antibiotic resistance genes have been used successfully for microalgal transformant selection, including bleomycin (2, 52, 56, 104, 210), spectinomycin (19, 42), streptomycin (42), paromomycin (81, 173), nourseothricin (210), G418 (45, 148, 210), hygromycin (12), chloramphenicol (184), and others. Due to the fact that many microalgae are resistant to a wide range of antibiotics, the actual number of antibiotics that work with a specific strain may be much more limited. In addition, antibiotics like nourseothricin and G418 are much less effective in salt-containing media and are not ideal for use with marine algae (210). Other markers that have been used include luciferase (51, 55, 83), β-glucuronidase (22, 23, 26, 49, 51, 92), β-galactosidase (58, 85, 151), and green fluorescent protein (GFP) (23, 50, 54, 56, 148, 210).

Transgene expression and protein localization in the chloroplast is needed for the proper function of many metabolic genes of interest for biofuel production. In C. reinhardtii, it is possible to achieve transformation of the chloroplast through homologous recombination (for a review by Marín-Navarro et al., see reference 106). While chloroplast transformation has not been demonstrated with diatoms, several publications have used plastid targeting sequences to translocate proteins to the chloroplast (3, 65).

Nuclear transformation of microalgae generally results in the random integration of transgenes. While this may be suitable for transgene expression or for random mutagenesis screens, it makes it difficult to delete specific target genes. Some progress in homologous recombination has been made with the nuclear genome of C. reinhardtii, but the efficiency remains low (217). Homologous recombination has also been reported for the red microalga C. merolae (121). Another option for gene inactivation is the use of RNA silencing to knock down gene expression; the mechanisms for RNA silencing have been studied with microalgae, and RNA silencing has been used successfully with both C. reinhardtii and P. tricornutum (16, 37, 122, 123, 214). Recent improvements in gene knockdown strategies include the development of high-throughput artificial-micro-RNA (armiRNA) techniques for C. reinhardtii that are reportedly more specific and stable than traditional RNA interference (RNAi) approaches (123, 214).

Members of the chlorophyte group that have been transformed include C. reinhardtii, which has been transformed using a variety of methods (44, 87, 88, 93, 170); Chlorella ellipsoidea (22, 83); Chlorella saccharophila (108); C. vulgaris (30, 69); Haematococcus pluvialis (177, 187); V. carteri (81, 163); Chlorella sorokiniana (30); Chlorella kessleri (49); Ulva lactuca (76); Dunaliella viridis (180); and D. salina (181, 183). Heterokontophytes that have reportedly been transformed include Nannochloropsis oculata (21); diatoms such as T. pseudonana (147), P. tricornutum (2, 210, 211), Navicula saprophila (45), Cylindrotheca fusiformis (52, 148), Cyclotella cryptica (45), and Thalassiosira weissflogii (51); and phaeophytes, such as Laminaria japonica (150) and Undaria pinnatifada (151). Rhodophytes, such as C. merolae (121), Porphyra yezoensis (23), Porphyra miniata (92), Kappaphycus alvarezii (94), Gracilaria changii (58), and Porphyridium sp. (95), have also been transformed. Dinoflagellates that have been transformed include Amphidinium sp. and Symbiodinium microadriaticum (119). The only euglenid that has been transformed to date is Euglena gracilis (42).

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Understanding microalgal lipid metabolism is of great interest for the ultimate production of diesel fuel surrogates. Both the quantity and the quality of diesel precursors from a specific strain are closely linked to how lipid metabolism is controlled. Lipid biosynthesis and catabolism, as well as pathways that modify the length and saturation of fatty acids, have not been as thoroughly investigated for algae as they have for terrestrial plants. However, many of the genes involved in lipid metabolism in terrestrial plants have homologs in the sequenced microalgal genomes. Therefore, it is probable that at least some of the transgenic strategies that have been used to modify the lipid content in higher plants will also be effective with microalgae.

Lipid biosynthesis.In recent years, many of the genes involved in lipid synthesis have been subjected to both knockout and overexpression in order to clarify their importance in lipid accumulation and to establish strategies to increase the lipid content in the oleaginous seeds of higher plants, such as Arabidopsis thaliana, soy bean (Glycine max), and rapeseed (Brassica napus). See Fig. 2 for a simplified overview of lipid biosynthesis pathways. Several of these transgenic overexpression strategies have resulted in the increased production of triacylglycerols in seeds and in other plant tissues. Ohlrogge and Jaworski have proposed that the fatty acid supply helps determine the regulation of oil synthesis (134); therefore, some efforts have been made to increase the expression of enzymes that are involved in the pathways of fatty acid synthesis. One early committing step in fatty acid synthesis is the conversion of acetyl-coenzyme A (CoA) to malonyl-CoA, catalyzed by acetyl-CoA carboxylase (ACCase), which is considered the first committed step in fatty acid biosynthesis in many organisms. However, several attempts to utilize ACCase overexpression to increase lipid content in various systems have been somewhat disappointing. Dunahay et al. overexpressed native ACCase in the diatom C. cryptica (45). Despite a 2- or 3-fold increase in ACCase activity, no increased lipid production could be observed (165). ACCase from A. thaliana has been overexpressed in B. napus and Solanum tuberosum (potato) (89, 157). Overexpression of ACCase in the oleaginous seeds of B. napus resulted in a minor increase in seed lipid content of about 6% (384 mg g−1 and 408 mg g−1 dry weight for wild-type [WT] and transgenic ACCase rapeseed lines, respectively). Interestingly, the effect of ACCase overexpression in potato tubers, a tissue that normally is very starch rich and lipid poor, resulted in a 5-fold increase in TAG content (from 0.0116 to 0.0580 mg g−1 fresh weight). It may be that ACCase levels are a limiting step in lipid biosynthesis mainly in cells that normally do not store large amounts of lipid. Another attempt to increase expression of a protein involved in fatty acid synthesis, 3-ketoacyl-acyl-carrier protein synthase III (KASIII), was not successful in increasing lipid production. KASIII from spinach (Spinacia oleracea) or Cuphea hookeriana was expressed in tobacco (Nicotiana tabacum), A. thaliana, and B. napus, resulting in either no change or reduced seed oil content (33).

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FIG. 2.
Simplified overview of the metabolites and representative pathways in microalgal lipid biosynthesis shown in black and enzymes shown in red. Free fatty acids are synthesized in the chloroplast, while TAGs may be assembled at the ER. ACCase, acetyl-CoA carboxylase; ACP, acyl carrier protein; CoA, coenzyme A; DAGAT, diacylglycerol acyltransferase; DHAP, dihydroxyacetone phosphate; ENR, enoyl-ACP reductase; FAT, fatty acyl-ACP thioesterase; G3PDH, gycerol-3-phosphate dehydrogenase; GPAT, glycerol-3-phosphate acyltransferase; HD, 3-hydroxyacyl-ACP dehydratase; KAR, 3-ketoacyl-ACP reductase; KAS, 3-ketoacyl-ACP synthase; LPAAT, lyso-phosphatidic acid acyltransferase; LPAT, lyso-phosphatidylcholine acyltransferase; MAT, malonyl-CoA:ACP transacylase; PDH, pyruvate dehydrogenase complex; TAG, triacylglycerols.

While increasing the expression of genes involved in fatty acid synthesis has had small successes, with regard to increasing the total amount of seed oils, some interesting results have been achieved through the overexpression of genes involved in TAG assembly. One of the most successful attempts to increase the amount of seed lipid is the overexpression of a cytosolic yeast, glycerol-3-phosphate dehydrogenase (G3PDH), in the seeds of B. napus, which resulted in a 40% increase in lipid content (191). G3PDH catalyzes the formation of glycerol-3-phosphate, which is needed for TAG formation. This interesting result suggests that genes involved in TAG assembly are of importance for total seed oil production. This is further supported by several other studies in which overexpression of TAG assembly genes resulted in increases in seed oil content. For example, overexpression of glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase, or diacylglycerol acyltransferase (DAGAT) all result in significant increases in plant lipid production (79, 80, 96, 186, 215, 218). Due to the fact that enzymes such as these seem to be good candidates for overexpression strategies with the goal of increasing storage lipid content, an attempt has also been made to use directed evolution to increase the efficiency of one of these enzymes, DAGAT (172).

Another possible approach to increasing the cellular lipid content is blocking metabolic pathways that lead to the accumulation of energy-rich storage compounds, such as starch. For example, two different starch-deficient strains of C. reinhardtii, the sta6 and sta7 mutants, have disruptions in the ADP-glucose pyrophosphorylase or isoamylase genes, respectively (10, 124, 144, 146, 209). Wang et al. (197), as well as unpublished results from our laboratory, have shown that these mutants accumulate increased levels of TAG during nitrogen deprivation. Another starchless mutant of Chlorella pyrenoidosa has also been shown to have elevated polyunsaturated fatty acid content (154).

In addition to what has been accomplished with higher plants, successful modifications have also been achieved with bacteria and yeast to increase and/or modify their lipid content. Due to the ease of genetic engineering with Escherichia coli and Saccharomyces cerevisiae, these modifications include quite comprehensive modulations of entire metabolic pathways, with the simultaneous overexpression or deletion of several key enzymes. Such modifications are, of course, much harder to achieve with microalgae, but they should be attainable with organisms with established protocols for genetic transformation and available selectable markers. One example of a comprehensive modification of E. coli, which resulted in a 20-fold increase in free fatty acid production, entailed overexpression of the lipid biosynthesis genes encoding acetyl-CoA carboxylase, an endogenous thioesterase, and a plant thioesterase, as well as knocking out a gene product involved in β-oxidation of fatty acids, acyl-CoA synthetase (encoded by fadD) (102). Of particular interest in this study is the substantial increase in free fatty acid production that was due to the expression of the two thioesterases. With E. coli it has been shown that long-chain fatty acids can inhibit fatty acid synthesis and that this inhibition can be released by expression of specific thioesterases (84, 193).

Lipid catabolism.A complementary strategy to increase lipid accumulation is to decrease lipid catabolism. In the case of lipid biosynthesis, most of what we know regarding successful strategies to decrease lipid catabolism comes from studies of higher plants and yeast. Genes involved in the activation of both TAG and free fatty acids, as well as genes directly involved in β-oxidation of fatty acids, have been inactivated, sometimes resulting in increased cellular lipid content. To circumvent the current lack of efficient homologous recombination in microalgae, gene inactivation would have to be achieved either through random mutagenesis or through the use of RNA silencing (37, 123, 214). Due to the fact that cells rely on the β-oxidation of fatty acids for cellular energy under certain physiological conditions, knocking out lipid catabolism genes not only may result in increased lipid storage but also could have deleterious effects on cellular growth and proliferation. For example, inactivation of the peroxisomal long-chain acyl-CoA synthetase (LACS) isozymes, LACS6 and LACS7, in A. thaliana inhibits seed lipid breakdown, which increased oil content. However, proper seedling development was also inhibited without the addition of an external carbon source (57). Similar results were achieved through the inactivation of 3-ketoacyl-CoA thiolase (KAT2) in A. thaliana (59). Another potential problem with strategies that involve inhibition of lipid catabolism is that enzymes with overlapping functions exist for many of the steps of β-oxidation, making it difficult to completely abolish these functions. An example is the short-chain acyl-CoA oxidase enzymes ACX3 and ACX4 in A. thaliana. Single mutants of ACX3 or ACX4 have normal lipid breakdown and seedling development, while double mutants are nonviable, putatively due to complete elimination of short-chain acyl-CoA oxidase activity (159).

During diel light-dark cycles, many microalgae initiate TAG storage during the day and deplete those stores at night to support cellular ATP demands and/or cell division. Consequently, inhibition of β-oxidation would prevent the loss of TAG during the night, but most likely at the cost of reduced growth. This strategy, therefore, may not be beneficial for microalgae grown in outdoor open ponds, but it may be a valid strategy to increase lipid production in microalgae grown in photobioreactors with exogenous carbon sources and/or continuous light.

In some studies, inhibition of lipid oxidation has caused unexpected phenotypes. Several publications have shown that knocking out genes involved in β-oxidation in S. cerevisiae not only can lead to increased amounts of intracellular free fatty acids but also results in extracellular fatty acid secretion in some instances (120, 132, 162). The lipid catabolism genes that have been implicated in fatty acid secretion in S. cerevisiae include acyl-CoA oxidase and several acyl-CoA synthetases (see below).

Modification of lipid characteristics.In addition to engineering microalgae for the increased production of lipids, it is also reasonable to attempt to increase the quality of the lipids, with regard to suitability as a diesel fuel feedstock. The carbon chain length and degree of unsaturation of the fatty acids in each microalgal species can affect the cold flow and oxidative stability properties of a biodiesel fuel which is derived from this feedstock. Typically, most microalgal fatty acids have a chain length between 14 and 20; major species are often 16:1, 16:0, and 18:1. Ideal fatty acids for diesel production should be 12:0 and 14:0. The chain lengths of fatty acids are determined by acyl-ACP thioesterases, which release the fatty acid chain from the fatty acid synthase. There are several acyl-ACP thioesterases from a variety of organisms that are specific for certain fatty acid chain lengths, and transgenic overexpression of thioesterases can be used to change fatty acid chain length. Expression of a 12:0-biased thioesterase from Umbellularia californica in both A. thaliana and E. coli drastically changed the lipid profiles in these organisms, with a great increase in the production of lauric acid (193, 194). Similarly, a 14:0-biased thioesterase from Cinnamomum camphorum was expressed in A. thaliana and E. coli, greatly increasing the production of myristic acid (208). Both of these thioesterases are obviously interesting candidates for transgenic overexpression in microalgae since their activities could improve the suitability of microalga-derived diesel feedstock.

Fatty acids of even shorter chain lengths can also be used for production of gasoline and jet fuel. It is possible to use hydrocracking to break down longer hydrocarbons into shorter chain lengths that are more suitable as feedstocks for gasoline or jet fuel, but it may also be possible to reduce production costs through genetically engineering microalgae to directly produce these shorter chain lengths. Transgenic overexpression of an 8:0- and 10:0-biased thioesterase from C. hookeriana in canola has had some success in increasing the production of short chain fatty acids (32). Interestingly, combined overexpression of the 8:0/10:0-biased thioesterase and a ketoacyl ACP synthase (KAS), both from C. hookeriana, increases 8 to 10 fatty acids by an additional 30 to 40% (31).

The potential for modification of lipid content in microalgae.It is reasonable to believe that some of the strategies that result in increased oil seed content in terrestrial plants may be able to increase the lipid content of microalgal cells as well. Many microalgae do not produce large amounts of storage lipids during logarithmic growth. Instead, when they encounter environmental stress, such as a lack of nitrogen, they slow down their proliferation and start producing energy storage products, such as lipids and/or starch (75). It will be interesting to see how overexpression of lipid synthesis pathway genes will affect microalgal proliferation. It may be that increased lipid synthesis will result in a reduction of cell division. In such a case, overexpression of lipid synthesis genes may still be beneficial if they can be controlled by an inducible promoter that can be activated once the microalgal cells have grown to a high density and have entered stationary phase. Examples of inducible promoters in algae include copper-responsive elements in C. reinhardtii (152) and a nitrate-responsive promoter in diatoms (148). Inhibiting lipid catabolism may also cause problems with proliferation and biomass productivity since microalgae often rely on catabolic pathways to provide energy and precursors for cell division.

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Industrial methods for the production of biofuels using energy-rich carbon storage products, such as sugars and lipids, are well established and are currently being used on a large scale in the production of bioethanol from corn grain and biodiesel from oil seed crops. However, it might be possible to introduce biological pathways in microalgal cells that allow for the direct production of fuel products that require very little processing before distribution and use. Several biological pathways have been described for the production of fatty acid esters, alkanes, and alcohols. However, the introduction of metabolic pathways for the direct production of fuels faces many challenges. The product yields for pathways that lead to the accumulation of compounds that are not necessarily useful for the cell are unlikely to be economically viable without comprehensive engineering of many aspects of microalgal metabolism. In addition, many types of fuel products have the potential to be toxic, and tolerant species of microalgae may have to be generated.

Fatty acid ester production.Triacylglycerols can be used for the production of biodiesel through the creation of fatty acid esters. Microalgal lipids can also be used to produce a “green” or renewable diesel through the process of hydrotreating. However, these transformations require additional energy carriers (e.g., methanol or hydrogen) and chemical processing, which increases the cost of biofuel production. Every production step that can be transferred to biological pathways will likely improve the overall economics. An interesting example is the in vivo conversion of fatty acids to fuel by the simultaneous overexpression of the ethanol production genes from Zymomonas mobilis and the wax ester synthase/acyl-CoA-diacylglycerol acyltransferase (WS/DGAT) gene from the Acinetobacter baylyi strain ADP1 in E. coli, which resulted in the synthesis of fatty acid ethyl esters that could be used directly as biodiesel (86). Although the ethyl ester yield from this manipulation was not overly impressive for E. coli, it will be interesting to see if higher productivities can be achieved and what effect fatty acid ester accumulation has on microalgal growth.

Straight-chain alkanes.Short- and medium-chain alkanes have the potential to be used directly as transportation fuel. Since alkanes can be derived from fatty acids, microalgae that are good lipid producers could perhaps be genetically transformed to produce alkanes. This conversion relies on the serial transformation of fatty acids to aldehydes and then to alkanes. The last step is thought to be catalyzed by a decarbonylase enzyme; however, no functional decarbonylase enzyme has been cloned to date, and the actual mechanism for the conversion of aldehyde to alkane remains to be found. Interestingly, a suggested decarbonylase enzyme involved in alkane production has been studied with the green microalga B. braunii, which has the ability to produce very-long-chain alkanes (35). Strains of B. braunii differ in which long-chain hydrocarbons are synthesized, with strain A producing very-long-chain dienes and trienes, while strain B produces very-long-chain triterpenoid hydrocarbons (117). Decarbonylase activity has also been found in the leaves of the pea Pisum sativum (20, 164, 192), and several possible decarbonylases that are thought to be involved in wax formation, including Cer1 and Cer22, have been found in A. thaliana (1, 155). The alkanes that are generated by these putative decarbonylases all have very-long-chain lengths (>22 carbons) and will require further processing for fuel production. A possible example of long-chain-alkane (14- to 22-carbon) production has been reported for the bacterium Vibrio furnissii (137); however, a more recent study disputed these claims (195). Production of shorter-chain-length alkanes that are suitable for direct use as fuel remains an existing goal, and further research is needed to clarify how alkanes are generated and to reveal the precise enzymes involved.

Ethanol, butanol, isopropanol, and other longer-chain alcohols.Ethanol for biofuels is currently produced from the fermentations of food starches or cellulose-derived sugars. Algal starches have been shown to be fermentable by yeast (129), but an approach to directly couple ethanol production to photosynthetic carbon fixation in situ may be preferred. Many microalgae have fermentative metabolic pathways to ethanol, but to couple ethanol production to photoautotrophic metabolism will require changes in regulatory pathways or the insertion of new metabolic pathways. With cyanobacteria, the creation of a pathway for ethanol biosynthesis has been demonstrated, with the insertion of pyruvate decarboxylase and alcohol dehydrogenase from the ethanologenic bacterium Z. mobilis (34, 40). This pathway produces ethanol during photoautotrophic growth and could be incorporated into algae; however, these enzymes are not optimized for performance in oxic conditions and may need to be configured for eukaryotic systems.

As a fuel, ethanol has a lower energy density than gasoline and poor storage properties. Longer-chain alcohols C3 to C5 have higher energy densities that are similar to those of gasoline and are easier to store and transport than ethanol. Recently, many exciting advances toward the biological production of C3 up to C8 alcohols have been achieved. Isopropanol and butanol are naturally produced by bacteria of the genus Clostridium, and production has been industrialized using Clostridium acetobutylicum. Because C. acetobutylicum has a low growth rate and is somewhat difficult to genetically engineer, attempts have been made to express the production pathways for isopropanol and butanol in the more user-friendly host E. coli. For isopropanol production, several combinations of up to five genes from various species of Clostridium were overexpressed in E. coli, resulting in the production of 4.9 g/liter of isopropanol (67). In a similar fashion, six genes encoding the entire pathway for butanol production were transferred from C. acetobutylicum into E. coli by Atsumi et al. (7). With optimization, the overexpression of the butanol production pathway resulted in 1-butanol production of approximately 140 mg/liter. Interestingly, yields were greatly improved by the deletion of 5 host genes that compete with the 1-butanol pathway for acetyl-CoA and NADH, resulting in the production of 550 mg/liter (7). A further increase in production was achieved through the expression of the entire pathway from a single plasmid, resulting in production of 1.2 g/liter 1-butanol (78). Production of biofuels through transgenic overexpression of entire production pathways can cause problems for the host organism when the nonnative enzymes interfere with the host's normal metabolism. An alternative synthetic pathway for the production of butanol utilized the endogenous keto acid pathways for amino acid synthesis. These ubiquitous pathways normally produce amino acids through 2-keto acid precursors. Atsumi et al. proved that it is possible to divert some of the 2-keto acid intermediates from amino acid production into alcohol production, especially that of isobutanol, which was produced at titers up to 22 g/liter (8). This was achieved through the simultaneous transgenic overexpression of a 2-keto-acid decarboxylase and an alcohol dehydrogenase. Using similar approaches, it is possible to obtain longer-chain alcohols as well, and up to C8 alcohols have been synthesized (for a review by Connor and Liao, see reference 28). Some of the 2-keto acid intermediates, such as 2-ketobutyrate, are conserved in microalgae, and it is therefore reasonable to believe that a similar approach to production of alcohols in algae is possible.

Isoprenoids.Isoprenoids, also known as terpenoids, represent an incredibly diverse group of natural compounds, with more than 40,000 different molecules. In microalgae, isoprenoids are synthesized via the methylerythritol (MEP) pathway using glyceraldehydes-3-phosphate and pyruvate to generate the basic building blocks of isoprenoid biosynthesis, isopentyl diphosphate (IPP), and dimethylallyl diphosphate (DMAPP). Molecules that could potentially work as gasoline substitutes, including isopentenol, have been produced by E. coli using isoprenoid biosynthesis pathways. Two enzymes from Bacillus subtilis that utilize IPP and DMAPP for the biosynthesis of isopentenol were overexpressed in E. coli, resulting in production of 112 mg/liter isopentenol (107, 200). Other compounds that could replace diesel and jet fuel can also be produced through isoprenoid pathways (for recent reviews by Fortman et al. and Lee et al., see references 53 and 98, respectively).

Feasibility of direct biological synthesis of fuels.There are many examples of successful pathway manipulation to generate compounds that can be used as fuels. Most of these come from the manipulation of E. coli, in which the overexpression and deletion of entire metabolic pathways are feasible. With advances in genetic transformation methods and increased knowledge regarding expression systems in microalgae, comprehensive modifications, such as those performed with E. coli, may be attempted. It is reasonable to believe that some of the biochemical pathways in microalgae could be leveraged for the direct production of fuels. Considering the reported yields from the various pathways that have been utilized so far, the production of isobutanol through the keto acid pathways should be carefully considered for microalgal systems.

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It is believed that among the most costly downstream processing steps in fuel production using microalgal feedstocks are the harvesting/dewatering steps and the extraction of fuel precursors from the biomass. Based on currently achievable productivities, most microalgae will not grow to a density higher than a few grams of biomass per liter of water. While there are several possible low-cost solutions to concentrating the biomass, including settling and flocculation, these methods are slow and the resulting biomass may still require further dewatering. Alternative methods to concentrate algal biomass include centrifugation and filtration, which are faster, but they are also typically much more expensive and energy intensive. In addition, many microalgal species have a very tough outer cell wall that makes extraction of fuel feedstocks difficult, thereby requiring the use of harsh lysis conditions. One possible solution is to manipulate the biology of microalgal cells to allow for the secretion of fuels or feedstocks directly into the growth medium. There are in fact several pathways in nature that lead to secretion of hydrophobic compounds, including TAGs, free fatty acids, alkanes, and wax esters.

Secretion of free fatty acids in yeast.As mentioned in the section on lipid catabolism, inactivation of genes involved in β-oxidation has been shown to result in fatty acid secretion in some instances. These genes were identified to have a function in fatty acid secretion through the use of a screening method wherein mutated yeast colonies were overlaid with an agar containing a fatty acid auxotrophic yeast strain that requires free fatty acids in order to proliferate. Mutant colonies that secrete fatty acids were thereby identified by the formation of a halo in the overlaid agar (120, 132). Similar screening methods could be utilized to identify microalgae that have the ability to secrete fatty acids. In one of the yeast studies, random mutagenesis resulted in the secretion of TAGs. Unfortunately, neither the genes involved nor the mechanism has been described (132). S. cerevisiae has five genes with fatty acyl-CoA synthetase activity, including those encoding FAA1 and FAA4. Combined inactivation of FAA1 and FAA4, or FAA1 together with acyl-CoA oxidase activity, results in a buildup of intracellular free fatty acids and secretion of free fatty acids. Importantly, the highest levels of fatty acid secretion also seemed to be associated with reduced proliferation (120). This kind of secretion was found to take place mainly during logarithmic growth, whereas the cells started importing free fatty acids in stationary phase (162). The actual mechanisms for TAG and/or free fatty acid secretion in S. cerevisiae in these cases are not known. It is possible that any manipulation that allows yeast cells to accumulate high levels of intracellular free fatty acids will result in secretion of free fatty acids, and it may be possible to reproduce this kind of secretion in microalgae. A similar form of fatty acid secretion has been achieved by Synthetic Genomics with cyanobacteria (158). To improve the rates of secretion and to allow for the secretion of other types of bioenergy carriers, it could be beneficial to investigate some of the efficient mechanisms that exist for the secretion of fatty acids and related compounds in other organisms, which could be transferred to microalgae.

Mechanisms for secretion of lipids and related compounds.There are several examples of established pathways for the secretion of lipophilic compounds. These include the secretion of TAG-containing very-low-density lipid (VLDL) vesicles from hepatocytes, TAG-containing vesicles from mammary glands, and the ATP-binding cassette (ABC) transporter-mediated export of plant waxes, which consist of many types of hydrocarbons. In addition to cellular export pathways, there are also known pathways for intracellular transport of fatty acids between organelles, including import of fatty acids into mitochondria and peroxisomes for β-oxidation, and it may be possible to utilize such pathways for the export of lipids. Several key genes are known for these pathways, and transgenic expression of ABC transporters has been used to enable drug transport, resulting in resistance. However, the successful transgenic expression and utilization of lipid secretion pathways to secrete molecules suitable for biofuel production remain largely to be demonstrated.

Even though many of the genes that are involved in secretion have been identified, the exact mechanisms are generally not known. For example, the secretion of VLDL from hepatocytes has been shown to be affected both by deletion and overexpression of a wide range of genes, such as those encoding apolipoprotein E (ApoE), microsomal triglyceride transfer protein (MTP), triacylglycerol hydrolase (TGH), and arylacetamide deacetylase (AADA) (62, 99, 189, 190). Overexpression of these genes in hepatocytes that already have the capacity to secrete VLDL results in increased secretion; however, it is not known which genes would be needed to enable VLDL secretion from cell types that do not normally secrete VLDL. In a similar fashion, several genes have been identified that are involved in milk TAG vesicle secretion. These genes include those that encode adipophilin (ADPH), xanthine oxidoreductase (XOR), and butyrophilin (BTN) (for a review by McManaman et al., see reference 111). But again, the exact genes that would be needed to enable a functional secretion pathway in another cell type are not known. With further research, it should become clear whether the transfer of these very efficient TAG-secreting pathways is feasible.

What is perhaps a more straightforward approach to enabling secretion of lipids from microalgae is the use of ABC transporters. ABC transporters mediate the export of plant waxes consisting of a multitude of compounds that are derived from very-long-chain fatty acids, including alkanes, ketones, alcohols, aldehydes, alkyl esters, and fatty acids. In A. thaliana there are over 120 different ABC transporters, and in addition to transporting compounds that are related to very-long-chain fatty acids, they are also responsible for the transport of molecules that are produced through the isoprenoid synthesis pathway, including terpenoids and other compounds that are of interest for biofuel production. As with VLDL and milk TAG secretion, the exact mechanisms are not known, but the ABC transporter systems may rely on fewer components, and transgenic expression of ABC transporters has resulted in transport of a variety of compounds, including kanamycin, cholesterol, and sterols (82, 115, 205). Of particular interest are ABC transporters that have been shown to have the ability to transport plant wax components that are derived from very-long-chain fatty acids. These transporters include the A. thaliana Desperado/AtWBC11 transporter and the Cer5/AtWBC12 transporter, both of which have been shown to be important for exporting wax to the epidermis (136, 140). Long-chain fatty acids are imported into the peroxisome for β-oxidation by ABC transporters, and inactivation of the ABC transporter Ped3p or Pxa1 in A. thaliana inhibits peroxisomal uptake of long-chain fatty acids (70, 216). One interesting characteristic of ABC transporters is their promiscuous gating properties. For example, Cer5/AtWBC12 facilitates the export of very-long-chain aldehydes, ketones, alcohols, alkanes, and perhaps fatty acids (140). While wax transporters have not been shown to export products that are derived from medium-chain fatty acids, it would perhaps be possible to use random mutagenesis or directed evolution to generate mutant ABC transporters that have the ability to secrete short- and medium-chain fatty acids.

In summary, lipid secretion is an attractive alternative to harvesting algal biomass that could potentially lower the cost of producing microalga-derived biofuels. However, the current knowledge of secretion pathways is still rather limited and therefore may not necessarily be easily transferred to microalgal cells. In addition, a secretion strategy may not be the best solution when a significant number of contaminating microorganisms are present in the cultivation system. The secretion of the fuel intermediates into the culture medium would provide these microorganisms with a rich source of nutrient, thereby reducing product yields. It is currently possible to induce secretion of free fatty acids from yeast and cyanobacteria, but it remains to be demonstrated at a scale significant for biofuel production from microalgal feedstocks. Since there are several highly efficient pathways for lipid secretion in nature that are being explored with various model organisms, research efforts should be aimed at transferring these pathways into organisms that are good lipid producers.

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[1] Carbohydrates can be metabolized into a variety of biofuels, including ethanol, butanol, H2, lipids, and/or methane. Glucans are stored in microalgae in a variety of ways. The phyla Chlorophyta, Dinophyta, Glaucophyta, and Rhodophyta store glucans in linear α-1,4 and branched α-1,6 glycosidic linkages (10). In Heterokontophyta, Phaeophyceae, and Bacillariophyceae, water-soluble granules of laminarin and chrysolaminarin are synthesized, which are made up of β-1,3 linkages with branching at the C-2 and C-6 positions of glucose (74). In green algae, starch is synthesized and stored within the chloroplast, while it is stored in the cytoplasm in Dinophyta, Glaucophyta, and Rhodophyta and in the periplastidial space in Cryptophyceae (38, 39).

Genetic strategies for increasing glucan storage.The rate-limiting step of starch synthesis (see Fig. 3 for an overview of starch metabolism) is the ADP-glucose pyrophosphorylase (AGPase)-catalyzed reaction of glucose 1-phosphate with ATP, resulting in ADP-glucose and pyrophosphate (176). AGPase is typically a heterotetramer composed of large regulatory and small catalytic subunits and is allosterically activated by 3-phosphoglyceric acid (3-PGA), linking starch synthesis to photosynthesis (209). Polysaccharides are often found surrounding the pyrenoid in microalgae, likely because it is a source of 3-PGA (10).

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FIG. 3.
Starch metabolism in green microalgae. The metabolites and simplified representative pathways in microalgal starch metabolism are shown in black, and enzymes are shown in red. Glucans are added to the water soluble polysaccharide (WSP) by α-1,4 glycosidic linkages (WSP1) until a branching enzyme highly branches the ends (WSP2). Some of these branches are trimmed (WSP3), and this process is repeated until a starch granule is formed. Phosphorolytic [Starch-(P)n] and hydrolytic degradation pathways are shown. αAMY, α-amylase; AGPase, ADP-glucose pyrophosphorylase; βAMY, β-amylases; BE, branching enzymes; DBE, debranching enzymes; DPE, disproportionating enzyme (1 and 2) α-1,4 glucanotransferase; Glc, glucose; GWD, glucan-water dikinases; ISA, isoamylases; MEX1, maltose transporter; MOS, malto-oligosaccharides; PGM, plastidial phosphoglucomutase; P, phosphate; Pi, inorganic phosphate; PPi, pyrophosphate; SP, starch phosphorylases; SS, starch synthases.

Much work has been done on the catalytic and allosteric properties of AGPases in crop plants to increase starch production, with mixed results (174). Designer AGPases, such as those encoded by glgC16 from E. coli (176) or the recombinant rev6 (63), that have successfully increased starch content in other plants should be expressed in microalgae, preferably in a background with no native AGPase activity, such as the C. reinhardtii sta1 or sta6 mutant. An alternative approach for increasing microalgal starch would be to introduce starch-synthesizing enzymes into the cytosol. The cytosol would give more physical space for the starch granules to accumulate (174). A problem for cytosolic starch synthesis could be that because the AGPase is far from the pyrenoid and thus 3-PGA, it may not be activated. This problem could be circumvented by the introduction of an AGPase that does not require 3-PGA, such as the Mos(1-198)/SH2 AGPase, which still has activity even without the presence of an activator (14).

Decreasing starch degradation in microalgae.The precise mechanisms of starch catabolism in green microalgae are largely unknown (10) but are more widely understood for A. thaliana (175). Starch can be degraded by hydrolytic and/or phosphorolytic mechanisms. Hydrolytic starch degradation requires an enzyme capable of hydrolyzing semicrystalline glucans at the surface of the insoluble starch granule. In A. thaliana, α-amylase (AMY3) is thought to participate in starch degradation, and a homologous protein is found in C. reinhardtii (175). Interestingly, starch can be degraded even when all three α-amylases in A. thaliana have been knocked out, indicating alternative mechanisms for starch degradation (207). Plastidial starch degradation is stimulated by phosphorylation of glucose residues at the root of amylopectin by glucan-water dikinases (GWD). GWD catalyzes the transfer of β-phosphate in ATP to the C-6 position of the glucans in amylopectin (156). The C-3 position in the glucan can also be phosphorylated by the phosphoglucan water dikinases (PWD), and both of these phosphorylations are thought to help disrupt the crystalline structure of the starch granules to allow glucan-metabolizing enzymes access (212). In A. thaliana, the disruption of the GWD (sex1 phenotype) results in starch levels that are four to six times higher than those in wild-type leaves (206), while disruptions in PWD result in a less severe starch excess phenotype (156). These phosphorylation steps are critical for starch degradation and are excellent gene knockout targets for a starch accumulation phenotype in microalgae.

Secretion strategies and soluble sugars.Many microalgae have the native ability to secrete fixed carbon products. Mannitol, arabinose, glutamic acid, proline, glycerol, lysine, aspartic acid, and various polysaccharides have been reported to be secreted (71). Ankistrodesmus densus secretes polysaccharides when exposed to light, even during stationary phase (138). Although little is currently known about these secretion events, a further understanding of their regulation and mechanism could potentially be leveraged for continuous biofuel production from secreted saccharides.

The production of soluble sugars may be preferred over polysaccharides because soluble sugars are smaller and easier to process, in addition to likely being more amenable for engineered secretion because many transporters have been described. Maltose, a product of starch degradation in the chloroplast, is transported to the cytosol in green microalgae and land plants by the maltose export protein (MEX1) (38). This protein facilitates bidirectional diffusion and could be leveraged to export maltose out of the cell. Although sucrose has largely been unexplored with microalgae, evidence exists that some of the enzymes involved in sucrose metabolism, such as the sucrose synthetase and sucrose phosphate synthetase, are present (46). The synthesis of sucrose could be exploited by sucrose transporters, such as SUC1 and SUC2 found in A. thaliana, for extracellular excretion. S. cerevisiae cells transformed with SUC1 and SUC2 have been shown to transport sucrose and some maltose across their plasma membranes (160).

In addition to exporting soluble sugars, intracellular sugar accumulation is also a desirable microalgal biofuel trait. Maltose is metabolized in the cytosol by a glucosyltransferase, DPE2, but when it is knocked out in Arabidopsis, it results in a 30-fold increase of maltose in the leaves, enough to cause maltose exportation to the roots, where the concentration is doubled. In addition, when the MEX1 transporter is knocked out there is at least 40 times more maltose in the leaves of the Arabidopsis mutant than in the wild type (103). In sugar cane, an increase in total sugar production has been accomplished by the transgenic expression of a sucrose isomerase from a bacterium. This isomerase converts sucrose to isomaltulose, a nonplant metabolite, and as a result, the total sugar levels of isomaltulose and sucrose are twice as high as those of control plants. This may imply that there is a signaling system that gives negative feedback when sucrose levels reach a certain level, but when sucrose is converted to isomaltulose, the level of sucrose is not detected, allowing for higher levels of total sugar accumulation (202). In microalgae, maltose could be converted to other isoforms that may be silent to the native metabolic regulatory system, which could result in an increase in total sugar content. For example, a glucosyltransferase from a bacterium converting maltose to trehalose (131) could be expressed as a potential strategy to increase total sugar content. A potential side effect of increased trehalose is its ability to induce starch synthesis and AGPase expression, which has been shown with Arabidopsis (199).

Mutant considerations.Mutants that synthesize less starch or have a reduced capacity to degrade starch often have reduced growth rates (175). A. thaliana mutants grown in a diurnal cycle that cannot synthesize or degrade starch grow more slowly than the wild type (17, 18, 213). An A. thaliana plastidial phosphoglucomutase mutant (corresponding to STA5 in C. reinhardtii) had more of a reduced growth rate during short daylight periods compared to long daylight periods, but at continuous light its growth rates were equal (17). These considerations may become issues when microalgae are grown for biofuels and are subject to diurnal light cycles.

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Many eukaryotic microalgae and cyanobacteria have evolved in ecosystems that become depleted of O2, especially during the night, and diverse fermentation metabolisms that can be leveraged in renewable bioenergy strategies are present in these organisms (64, 118, 125). Of particular interest is the ability of many green microalgae to produce H2; however, it should be noted that additional fermentation metabolites, including organic acids and alcohols, are also secreted by many species during anoxia (118, 143). Hydrogen metabolism has been studied extensively with C. reinhardtii (61, 68, 72), resulting in significant advances in both our fundamental understanding of H2 metabolism in this organism (43, 110, 143) and in improvements in overall H2 yields (90, 91, 114). Hydrogenases are classified according to the metal ions at their active sites, and the [NiFe] and [FeFe] hydrogenases are capable of the reversible reduction of protons to H2. These two enzyme classes are phylogenetically distinct, and interestingly, only the [FeFe] hydrogenases have been described in eukaryotic microalgae; whereas, only the [NiFe] hydrogenases have been reported for cyanobacteria. The [FeFe] hydrogenases in many green microalgae are able to effectively couple to the photosynthetic electron transport chain at the level of ferredoxin, providing the means to generate H2 directly from water oxidation. However, all microalgal [FeFe] hydrogenases characterized to date are particularly O2 sensitive, and H2 photoproduction is only transiently observed prior to the accumulation of O2 to inhibitory levels under nutrient-replete conditions (27, 178). In 2000, Melis and coworkers described the use of sulfur deprivation (203), which decreases photosynthetic activity, as an effective means of sustaining H2 photoproduction when respiration is able to consume all of the photosynthetic O2 produced by the cells (114). Recently, it was demonstrated that alginate-immobilized cultures of nutrient-deprived C. reinhardtii could sustain H2 photoproduction even in the presence of an oxygenated atmosphere (90). Genetic techniques have been applied with the aim of increasing H2 photoproduction activity by decreasing light-harvesting antenna size, inhibiting state transitions, and hydrogenase engineering (11, 60, 68, 112). In combination with physiological and biochemical approaches, these studies have rapidly advanced our understanding of H2 metabolism and enzyme maturation (13, 145) in green microalgae, and numerous strategies are emerging to further advance our ability to optimize H2 production in eukaryotic phototrophs.

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The production of any biofuel is dependent on the efficiency of the metabolic pathways that lead to accumulation of storage compounds, such as lipids and starch, as well as on the ability of microalgae to rapidly produce large amounts of biomass. Experiments with small- and large-scale microalgal photobioreactors and molecular research in photosynthetic efficiency have revealed several factors that can limit biomass accumulation. These include stress factors, such as salt concentration, temperature, pH, and light intensity. Depending on the design of the cultivation facilities, it is possible to control these factors to a certain degree through engineering and manipulation of the growth environment, but these manipulations add to the cost of growing microalgae. Therefore, it would be of great benefit to develop genetic strategies to increase the cellular tolerance to a variety of stress factors.

High-light stress.One important consideration is the intensity of light at which a certain strain of microalga reaches its maximum growth rate; this intensity, which corresponds to the maximum photosynthetic efficiency, is usually around 200 to 400 μmol photons m−2 s−1 for most species. Light intensities above the maximum photosynthetic efficiency actually reduce the growth rate, a phenomenon known as photoinhibition. Photosynthetically active radiation intensities from sunlight can exceed 2,000 μmol photons m−2 s−1 during midday (113). Consequently, most microalgae will not grow at maximum efficiency during most of the day. Microalgae are considered great model organisms to study photosynthetic efficiency, and several attempts have been made to improve the photosynthetic efficiency and/or reduce the effects of photoinhibition on microalgal growth. Much of this work has been focused on reducing the size of the chlorophyll antenna or lowering the number of light-harvesting complexes to minimize the absorption of sunlight by individual chloroplasts (97, 126,–,128, 141, 142, 188). This approach may seem counterintuitive, but this strategy may have two positive effects; first, it permits higher light penetration in high-density cultures, and second, it can allow a higher maximum rate of photosynthesis due to the fact that the cells are less likely to be subjected to photoinhibition since their light-harvesting complexes absorb less light (for an excellent review of the subject by Melis, see reference 113). Earlier research relied on random mutagenesis strategies to generate mutants with fewer or smaller chlorophyll antennae, but a recent publication efficiently used an RNAi-based strategy to knock down both LHCI and LHCII in C. reinhardtii (126). This strategy can most likely be applied to many different microalgae more easily than a random mutagenesis approach. It seems clear that manipulation of light-harvesting complexes can lead to increased biomass productivity under high light in controlled laboratory conditions. However, it remains to be seen how well these mutants will perform in larger-scale cultures with more varied conditions and perhaps with competition from wild invasive microalgal species. In one study of algal antenna mutants, no improvement in productivity was observed with outdoor ponds (77). However, they also did not observe any improved productivity in laboratory cultures. With more research, it should become clear whether the current approach can be successfully applied to increase biomass production.

Other stress factors.High light is not the only environmental variable that can cause stress and inhibit microalgal growth. Salt, pH, temperature, and other stimuli can also cause stress to microalgal cultures. Many genes have been identified that are important for withstanding stress conditions. These include genes that are directly involved in scavenging reactive oxygen, such as those encoding glutathione peroxidase and ascorbate peroxidase (168, 182, 204) as well as enzymes that catalyze the production of osmolytes, such as mannitol and ononitol (166, 167, 185), and interestingly, an ATP synthase subunit that is involved in the regulation of intracellular ATP levels and stress tolerance (179). The antistress properties of these genes isolated from bacteria as well as a marine stress-tolerant microalga (Chlamydomonas sp. W80) were demonstrated through transgenic overexpression in several different systems, including tobacco and E. coli, which resulted in increased resistance to several different stressful stimuli, including high salt and low temperature. It is likely that similar improvements can be achieved with microalgae. An additional benefit of increasing stress tolerance is the possibility of growing select microalgae under extreme conditions that limit the proliferation of invasive species.

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Microalgae are an extremely diverse group of organisms, many of which possess novel metabolic features that can be exploited for the production of renewable biofuels. These include (i) high photosynthetic conversion efficiencies, (ii) rapid biomass production rates, (iii) the capacity to produce a wide variety of biofuel feedstocks, and (iv) the ability to thrive in diverse ecosystems. Although microalgae have long been considered a promising platform for the production of biofuels, earlier studies concluded that the economics of microalgal biofuel production needed to be significantly improved. In contrast to these previous efforts, we are now equipped with a wide variety of new genetic tools, genome sequences, and high-throughput analytical techniques that will allow scientists to analyze and manipulate metabolic pathways with unprecedented precision. Promising advances in metabolic engineering allow for not only the increased production of endogenous carbon storage compounds, such as TAGs and starch, but also the direct production, and perhaps secretion, of designer hydrocarbons that may be used directly as fuels. The application of these modern metabolic engineering tools in photosynthetic microalgae has the potential to create important sources of renewable fuel that will not compete with food production or require fresh water and arable land.

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We acknowledge support from the Air Force Office of Scientific Research grant FA9550-05-1-0365 and the Office of Biological and Environmental Research, GTL program, Office of Science, U.S. Department of Energy.

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↵*Corresponding author. Mailing address: Department of Chemistry and Geochemistry, Colorado School of Mines, Golden, CO 80401. Phone: (303) 384-2425. Fax: (303) 273-3629. E-mail: mposewit@mines.edu.
↵▿ Published ahead of print on 5 February 2010.
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Created By: Daylen Gargalis

Spider Silk

Applications of Spider Silk

Humans have been making use of spider silk for thousands of years. The ancient Greeks used cobwebs to stop wounds from bleeding and the Aborigines used silk as fishing lines for small fish. More recently, silk was used as the crosshairs in optical targeting devices such as guns and telescopes until World War II and people of the Solomon Islands still use silk as fish nets.

Current research in spider silk involves its potential use as an incredibly strong and versatile material. The interest in spider silk is mainly due to a combination of its mechanical properties and the non-polluting way in which it is made. The production of modern man-made super-fibres such as Kevlar involves petrochemical processing which contributes to pollution. Kevlar is also drawn from concentrated sulphuric acid. In contrast, the production of spider silk is completely environmentally friendly. It is made by spiders at ambient temperature and pressure and is drawn from water. In addition, silk is completely biodegradable. If the production of spider silk ever becomes industrially viable, it could replace Kevlar and be used to make a diverse range of items such as:Bulletproof vest

[1] Bullet-proof clothing

Wear-resistant lightweight clothing

Ropes, nets, seat belts, parachutesKevlar sail

Rust-free panels on motor vehicles or boats

Biodegradable bottles

Bandages, surgical thread

Artificial tendons or ligaments, supports for weak blood vessels.

However the production of spider silk is not simple and there are inherent problems. Firstly spiders cannot be farmed like silkworms since they are cannibals and will simply eat each other if in close proximity. The silk produced is very fine so 400 spiders would be needed to produce only one square yard of cloth. The silk also hardens when exposed to air which makes it difficult to work with.

E. coliThe alternative approach is to learn how spiders spin silk and then copy them to make synthetic spider silk. The silk itself would also have to be artificially made. Chemical synthesis of spider silk is not viable at present due to the lack of knowledge about silk structure so the replication of silk is currently being achieved using genetic engineering. Randolph V. Lewis, Professor of Molecular Biology at the University of Wyoming in Laramie, has inserted silk genes into Escherichia coli bacteria to successfully produce the repeated segments of spidroin 1 and spidroin 2.

More recently, Nexia Biotechnologies Inc in Montreal, Canada have inserted silk genes into goats to produce silk proteins in their milk. This is hoped to be a better method because protein from bacteria is not as strong due to faulty crosslinking of the proteins and hard white lumps can form. Milk production in mammary glands is similar to silk protein production in spiders so it is thought that proper protein crosslinking could occur in goats.

Soya plantsIt has been suggested that the whole gene sequence might not be needed to produce useful spider silk. Prospects include possible gene insertion into fungi and soya plants. It may also be possible to alter the silk genes for specific purposes. For example altering the genes responsible for camouflaging spider silk in nature could lead to a range of silk colours.

Photograph by Carolyn Merchant

There are still problems with developing synthetic spider silk production. An artificial method of spinning silk remains a mystery. Spider spinning dope is approximately 50% protein but this is too high a concentration to use industrially since the fluid would be too viscous to allow efficient spinning. The silk is also insoluble in water but this can be overcome by attaching soluble amino acids such as histidine or arginine to the ends of the protein molecules. In addition, the silk coagulates if the fluid is stirred so it would have to be redissolved. Current research focuses around these problems and a possible solution would be to adapt the composition of silk proteins to alter its properties. Research is still in its early stages but unravelling the secrets of spider silk is underway.
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