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Genetic Engineering of Algae for Enhanced Biofuel Production

Randor Radakovits, Robert E. Jinkerson, Al Darzins and Matthew C. Posewitz Eukaryotic Cell 2010, 9(4):486. DOI: 10.1128/EC.00364-09. Published Ahead of Print 5 February 2010.

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1500 Illinois St. continued bioprospecting efforts and the development and engineering of select microalgal strains are required to im486 Downloaded from http://ec. yeast. Golden. including the accumulation of significant quantities of triacylglycerol. Golden. several technical barriers need to be overcome before microalgae can be used as an economically viable biofuel feedstock (139). and many eukaryotic microalgae have the ability to store significant amounts of energy-rich compounds. and numerous additional species have subsequently been investigated (165).S. Photosynthetic microorganisms are attracting considerable interest within these efforts due to their relatively high photosynthetic conversion efficiencies. It is believed that a large portion of crude oil is of microalgal origin.. 2012 by guest * Corresponding author. Photosynthetic algae. and the dangers of increasing atmospheric CO2 levels. Apr. Golden. polysaccharides. These include developing low-energy methods to harvest microalgal cells. 1617 Cole Blvd. 4 Genetic Engineering of Algae for Enhanced Biofuel Production Randor Radakovits. Published ahead of print on 5 February 2010. considering their lipid profiles and productivity (153). Interest in a variety of renewable biofuels has been rejuvenated due to the instability of petroleum fuel costs.1 Robert E. and other energy storage compounds in photosynthetic organisms. It is likely that many of these advances can be extended to industrially relevant organisms. superior growth rates. Relative to cyanobacteria. Although these efforts demonstrated that many species of microalgae have properties that are desirable for biofuel production. The U. 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.00 doi:10.. difficulties in consistently producing biomass at a large scale in highly variable outdoor conditions. 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. it is clear that investigation of the potential of living microalgae to produce biofuels should be a priority. transportation fuel demand (41).edu. Jinkerson. and bacteria through genetic engineering. Department of Energy’s Aquatic Species Program analyzed approximately 3.asm.1128/EC. eukaryotic microalgae possess several unique metabolic attributes of relevance to biofuel production. including biodiesel and ethanol.1 Al Darzins. such as triacylglycerol (TAG) and starch. 486–501 1535-9778/10/$12. the lack of cost-effective bioenergy carrier extraction techniques. However. Colorado School of Mines. the presence of invasive species in large-scale ponds. improved H2 yields. diverse metabolic capabilities. It is postulated that a light-harvesting footprint of at least 20. using waste or salt water (41). and the ability to efficiently couple photosynthetic electron transport to H2 production. Several species have biomass production rates that can surpass those of terrestrial plants (41). have been of considerable interest as a possible biofuel resource for decades (165). and ability to store or secrete energy-rich hydrocarbons. the synthesis of storage starch (amylopectin and amylose). it may be possible to leverage the metabolic pathways of microalgae to produce a wide variety of biofuels (Fig. In addition. alcohols. CO 80401. Colorado School of Mines. including increased lipid and carbohydrate production. Therefore.S. Mailing address: Department of Chemistry and Geochemistry. even modest improvements in photon conversion efficiencies will dramatically reduce the land area and cost required to produce biofuels.000 different microalgae for their potential to produce biofuels. p. which is similar to that found in higher plants. . that can be utilized for the production of several distinct biofuels. To advance the utilization of microalgae in biofuel production.org/ on October 29. Colorado 804012 There are currently intensive global research efforts aimed at increasing and modifying the accumulation of lipids. E-mail: mposewit@mines .e. 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. In contrast to corn-based ethanol or soy/ palm-based biodiesel. American Society for Microbiology. Vol. diesel fuel surrogates. with diatoms being especially likely candidates.EUKARYOTIC CELL.000 species of algae have been described. Over 40. Although the application of genetic engineering to improve energy production phenotypes in eukaryotic microalgae is in its infancy. and this is likely only a small fraction of the total number of available species (75). and/or alkanes. Colorado 80401. most have drawbacks that have prevented the emergence of an economically viable algal biofuel industry. 9. seaweeds)..000 square miles will be required to satisfy most of the current U. a reliance on unstable foreign petroleum resources. No. starch-derived alcohols. Fax: (303) 273-3629. Phone: (303) 384-2425. 1).1 and National Renewable Energy Laboratory. and the diversion of central metabolic intermediates into fungible biofuels. 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. both microalgae and macroalgae (i.2 and Matthew C. hydrocarbons.00364-09 Copyright © 2010. If ancient algae are responsible for creating substantial crude oil deposits. Many improvements have been realized. the reality of peak oil in the near future. 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. Posewitz1* Department of Chemistry and Geochemistry. All Rights Reserved. 2010. low light penetrance in dense microalgal cultures. Consequently. and the potentially poor cold flow properties of most microalga-derived biodiesel.

org/ on October 29. ER. Thalassiosira pseudonana (6). 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. most of the tools for the expression of transgenes and gene knockdown have been developed for and are specific for this species. can be fully realized only if conventional breeding methods become firmly established. Currently. Pseudo-nitzschia. Chlorella vulgaris. In addition. Volvox carteri. and chloroplast genomes from several microalgae have been sequenced. and several more are being sequenced. Ostreococcus tauri (36). Galdieria sulphuraria. Dunaliella salina. Porphyra purpurea. 24. despite the recent advances in biotechnological approaches. reinhardtii (116. there are several completed and ongoing efforts to se- . 2012 by guest FIG. Ostreococcus lucimarinus (135). particularly diploid diatoms. and the availability of rapid large-scale sequencing technology represents a revolution in microalga research. 2010 MINIREVIEWS 487 Downloaded from http://ec.VOL. the green alga Chlamydomonas reinhardtii has been the focus of most molecular and genetic phycological research. including those for C.asm. It stands to reason that with microalgae. Therefore. nuclear. endoplasmic reticulum. 171). Access to microalgal genome sequences that are of interest for academic or industrial applications greatly facilitates genetic manipulation. Microalgal metabolic pathways that can be leveraged for biofuel production. tools are now also being rapidly developed for diatoms and other algae that are of greater interest for industrial applications. Current commercial agriculture crops have been cultivated for thousands of years. Botryococcus braunii. prove the yields of bioenergy carriers. Thalassiosira rotula. 1. However. 25). and Micromonas pusilla (201). thereby allowing useful traits or mutations to be easily combined (5. Phaeodactylum tricornutum (15). and Aureococcus anophageferrens (100). the full potential of genetic engineering in some microalgal species. Since the topic of microalgal sexual breeding is beyond the scope of this review. ongoing microalgal genome sequencing projects include those for Fragilariopsis cylindrus. GENETIC ENGINEERING OF MICROALGAE Significant advances in microalgal genomics have been achieved during the last decade. Expressed sequence tag (EST) databases have been established. However. mitochondrial. 9. it would be beneficial to use genetic engineering in an attempt to bypass such a lengthy selection process. Several nuclear genome sequencing projects have now been completed. Historically. with desired traits selected over time. Micromonas pusilla. Microalgal genomes. Cyanidioschyzon merolae (109).

such as C. 56. 149. 187. tricornutum (16. Gracilaria changii (58). 88. but the efficiency remains low (217). 3 . Efficient isolation of genetic transformants is greatly facilitated by the use of selection markers. 210). 123. 148). and Porphyridium sp. Cyclotella cryptica (45). 211. transformation resulted in stable expression of transgenes. Chlorella ellipsoidea (22. including antibiotic resistance and/or fluorescent/biochemical markers. 87. 211). 85. C. 21–23. 119). have also been transformed. 119. euglenids.. 169. In many cases. therefore. and Agrobacterium tumefaciens-mediated gene transfer (23. Recent improvements in gene knockdown strategies include the development of highthroughput artificial-micro-RNA (armiRNA) techniques for C. streptomycin (42). Other markers that have been used include luciferase (51. Porphyra miniata (92). tricornutum (2. 123. Dinoflagellates that have been transformed include Amphidinium sp. and inclusion of speciesspecific 5 . 52. 150. 108. 26. 45. 93). -glucuronidase (22. 58. the actual number of antibiotics that work with a specific strain may be much more limited. 130. some efforts have been made to increase the expression of enzymes Downloaded from http://ec. and D. (95). 37. 88. 51. 214). Transgene expression and protein localization in the chloroplast is needed for the proper function of many metabolic genes of interest for biofuel production. 196. 210. reinhardtii (for a recent review by Eichler-Stahlberg et al. While this may be suitable for transgene expression or for random mutagenesis screens. it is possible to achieve transformation of the chloroplast through homologous recombination (for a review by Marín-Navarro et al. 69). and RNA silencing has been used successfully with both C. While chloroplast transformation has not been demonstrated with diatoms. 121. Rhodophytes. electroporation (21. 183). 9. 26. paromomycin (81. Some progress in homologous recombination has been made with the nuclear genome of C. P. In addition. 210). 161. 93. 147. 2012 by guest . V. Chlorella sorokiniana (30). 210). and brown (Phaeophyta) algae. In recent years. 101. Navicula saprophila (45). 22. 183. Heterokontophytes that have reportedly been transformed include Nannochloropsis oculata (21). 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. Chlorella kessleri (49). 214. 170. 148.asm. many of the genes involved in lipid metabolism in terrestrial plants have homologs in the sequenced microalgal genomes. 29. Several different antibiotic resistance genes have been used successfully for microalgal transformant selection. but in some cases only transient expression was observed. the use of strong endogenous promoters. Cylindrotheca fusiformis (52. such as Laminaria japonica (150) and Undaria pinnatifada (151). 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. In C. as well as pathways that modify the length and saturation of fatty acids. 92). 104. 37. 49. 105. 44. reinhardtii. 83. G418 (45. Methods developed primarily with C. 148. diatoms. The only euglenid that has been transformed to date is Euglena gracilis (42). chloramphenicol (184). 65). and green fluorescent protein (GFP) (23. 163. biolistic microparticle bombardment (2. Nuclear transformation of microalgae generally results in the random integration of transgenes. and the method of transformation has to be carefully selected and optimized for each microalga. 210. diatoms such as T. including bleomycin (2. 26. Dunaliella viridis (180). Several of these transgenic overexpression strategies have resulted in the increased production of triacylglycerols in seeds and in other plant tissues. reinhardtii that are reportedly more specific and stable than traditional RNA interference (RNAi) approaches (123. Therefore. 87. 69. Successful genetic transformation has been reported for the green (Chlorophyta). reinhardtii and P. Haematococcus pluvialis (177. 83. Kappaphycus alvarezii (94). several publications have used plastid targeting sequences to translocate proteins to the chloroplast (3. 54. Homologous recombination has also been reported for the red microalga C. salina (181. 52. 217). see reference 106). pseudonana (147). 50. and Thalassiosira weissflogii (51). Members of the chlorophyte group that have been transformed include C. Ulva lactuca (76). as well as dynamic transcriptomes from many different microalgae (4. 184). see reference 48) demonstrate that the stability of expression can be improved through proper codon usage. and intron sequences. 76. 65. hygromycin (12). such as Arabidopsis thaliana.. 198). carteri (81. 171. 92–95.org/ on October 29. 151. reinhardtii. and others. 42. 42). Chlorella saccharophila (108). the mechanisms for RNA silencing have been studied with microalgae. CELL quence plastid and mitochondrial genomes. 45. 183. Lipid biosynthesis and catabolism. 54–56. Lipid biosynthesis. 122. 85. Ohlrogge and Jaworski have proposed that the fatty acid supply helps determine the regulation of oil synthesis (134). 66. it makes it difficult to delete specific target genes. 187. See Fig. 81. -galactosidase (58. and rapeseed (Brassica napus). 181. 214). 211). nourseothricin (210). 173). 73. 87. 180. 30. 49. soy bean (Glycine max). 56. 2 for a simplified overview of lipid biosynthesis pathways. 151). 3. which has been transformed using a variety of methods (44. 51. 55. have not been as thoroughly investigated for algae as they have for terrestrial plants. Porphyra yezoensis (23). 83). However. Another option for gene inactivation is the use of RNA silencing to knock down gene expression. antibiotics like nourseothricin and G418 are much less effective in salt-containing media and are not ideal for use with marine algae (210). 133. spectinomycin (19. 170. 23. 170). 210. GENETIC ENGINEERING OF THE LIPID METABOLISM Understanding microalgal lipid metabolism is of great interest for the ultimate production of diesel fuel surrogates. and dinoflagellates (2. vulgaris (30. and Symbiodinium microadriaticum (119). reinhardtii. 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. merolae (121). 181. More than 30 different strains of microalgae have been transformed successfully to date. Both the quantity and the quality of diesel precursors from a specific strain are closely linked to how lipid metabolism is controlled. 148. 108. red (Rhodophyta). Methods for transformation and expression. and phaeophytes. The efficiency of transformation seems to be strongly species dependent. merolae (121). from either the nucleus or the plastid. 187). Due to the fact that many microalgae are resistant to a wide range of antibiotics. 88.488 MINIREVIEWS EUKARYOT. 163). 83). 49.

catalyzed by acetyl-CoA carboxylase (ACCase). Dunahay et al. acetyl-CoA carboxylase.asm. HD. 3-hydroxyacylACP dehydratase. which is considered the first committed step in fatty acid biosynthesis in many organisms. 2012 by guest FIG. gycerol-3-phosphate dehydrogenase. ACP. Another attempt to increase expression of a protein involved in fatty acid synthesis. resulting in either no change or reduced seed oil content (33). several attempts to utilize ACCase overexpression to increase lipid content in various systems have been somewhat disappointing. 3-ketoacyl-ACP synthase. KAS. LPAT. KASIII from spinach (Spinacia oleracea) or Cuphea hookeriana was expressed in tobacco (Nicotiana tabacum). While increasing the expression of genes involved in fatty acid synthesis has had small successes. lyso-phosphatidylcholine acyltransferase. napus. in the seeds of B. Free fatty acids are synthesized in the chloroplast. resulted in a 5-fold increase in TAG content (from 0. G3PDH catalyzes the formation of glycerol-3-phosphate.or 3-fold increase in ACCase activity. which is needed for TAG formation. TAG. malonyl-CoA:ACP transacylase. fatty acyl-ACP thioesterase. ENR. lyso-phosphatidic acid acyltransferase.VOL. 9. was not successful in increasing lipid production. PDH.0580 mg g 1 fresh weight). enoyl-ACP reductase. CoA. Interestingly. glycerol-3-phosphate dehydrogenase (G3PDH). triacylglycerols. FAT. Overexpression of ACCase in the oleaginous seeds of B. G3PDH. This interesting result suggests that genes involved in TAG assembly are of importance for total seed oil production. DHAP. 157).org/ on October 29. GPAT. napus. 3-ketoacyl-ACP reductase. and B. MAT. One of the most successful attempts to increase the amount of seed lipid is the overexpression of a cytosolic yeast. the effect of ACCase overexpression in potato tubers. Simplified overview of the metabolites and representative pathways in microalgal lipid biosynthesis shown in black and enzymes shown in red. that are involved in the pathways of fatty acid synthesis. respectively). dihydroxyacetone phosphate. napus and Solanum tuberosum (potato) (89. thaliana. Despite a 2. 2. However. DAGAT. acyl carrier protein. diacylglycerol acyltransferase. no increased lipid production could be observed (165). This is . ACCase from A. with regard to increasing the total amount of seed oils. a tissue that normally is very starch rich and lipid poor. KAR.0116 to 0. cryptica (45). ACCase. A. thaliana has been overexpressed in B. One early committing step in fatty acid synthesis is the conversion of acetyl-coenzyme A (CoA) to malonyl-CoA. which resulted in a 40% increase in lipid content (191). 3-ketoacyl-acylcarrier protein synthase III (KASIII). 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. while TAGs may be assembled at the ER. overexpressed native ACCase in the diatom C. 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. pyruvate dehydrogenase complex. glycerol-3-phosphate acyltransferase. 2010 MINIREVIEWS 489 Downloaded from http://ec. coenzyme A. LPAAT. some interesting results have been achieved through the overexpression of genes involved in TAG assembly.

thaliana inhibits seed lipid breakdown.asm. and transgenic overexpression of thioesterases can be used to change fatty acid chain length. LACS6 and LACS7. much harder to achieve with microalgae. cerevisiae include acyl-CoA oxidase and several acyl-CoA synthetases (see below). 123. Similar results were achieved through the inactivation of 3-ketoacyl-CoA thiolase (KAT2) in A. proper seedling development was also inhibited without the addition of an external carbon source (57). 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. Ideal fatty acids for diesel production should be 12:0 and 14:0. coli. such as starch. with a great increase in the production of lauric acid (193. (197). The chain lengths of fatty acids are determined by acyl-ACP thioesterases. as well as genes directly involved in -oxidation of fatty acids. as well as knocking out a gene product involved in -oxidation of fatty acids. 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. 124. With E. 96. knocking out lipid catabolism genes not only may result in increased lipid storage but also could have deleterious effects on cellular growth and proliferation. reinhardtii. with regard to suitability as a diesel fuel feedstock. 132. Another possible approach to increasing the cellular lipid content is blocking metabolic pathways that lead to the accumulation of energy-rich storage compounds. but it may be a valid strategy to increase lipid production in microalgae grown in photobioreactors with exogenous carbon sources and/or continuous light. For example. have disruptions in the ADPglucose pyrophosphorylase or isoamylase genes. gene inactivation would have to be achieved either through random mutagenesis or through the use of RNA silencing (37. have been inactivated. Consequently. a 14:0-biased thioesterase from Cinnamomum camphorum was expressed in A. During diel light-dark cycles. In addition to engineering microalgae for the increased production of lipids. In some studies. 214). which release the fatty acid chain from the fatty acid synthase. 16:0. have shown that these mutants accumulate increased levels of TAG during nitrogen deprivation. An example is the short-chain acyl-CoA oxidase enzymes ACX3 and ACX4 in A. Another starchless mutant of Chlorella pyrenoidosa has also been shown to have elevated polyunsaturated fatty acid content (154). 194). 193). with the simultaneous overexpression or deletion of several key enzymes. thaliana. 215. thaliana and E. therefore. overexpression of glycerol-3-phosphate acyltransferase. and a plant thioesterase. Similarly. This strategy. two different starch-deficient strains of C. In the case of lipid biosynthesis. To circumvent the current lack of efficient homologous recombination in microalgae. 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. Wang et al. successful modifications have also been achieved with bacteria and yeast to increase and/or modify their lipid content. many microalgae initiate TAG storage during the day and deplete those stores at night to support cellular ATP demands and/or cell division. In addition to what has been accomplished with higher plants. acyl-CoA synthetase (encoded by fadD) (102). inhibition of lipid oxidation has caused unexpected phenotypes. The lipid catabolism genes that have been implicated in fatty acid secretion in S. coli drastically changed the lipid profiles in these organisms. 144. Another potential problem with strategies that involve inhibition of lipid catabolism is that enzymes with overlapping functions exist for many of the steps of -oxidation. DAGAT (172). major species are often 16:1. Such modifications are. 80. an attempt has also been made to use directed evolution to increase the efficiency of one of these enzymes. One example of a comprehensive modification of E. most of what we know regarding successful strategies to decrease lipid catabolism comes from studies of higher plants and yeast. 218). as well as unpublished results from our laboratory. the sta6 and sta7 mutants. entailed overexpression of the lipid biosynthesis genes encoding acetyl-CoA carboxylase. lysophosphatidic acid acyltransferase. 209). but most likely at the cost of reduced growth. making it difficult to completely abolish these functions. 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. A complementary strategy to increase lipid accumulation is to decrease lipid catabolism. For example. Typically. 146. may not be beneficial for microalgae grown in outdoor open ponds. Lipid catabolism. thaliana (59). 186. Expression of a 12:0-biased thioesterase from Umbellularia californica in both A. or diacylglycerol acyltransferase (DAGAT) all result in significant increases in plant lipid production (79. of course. 2012 by guest . Both of these thioesterases are obviously Downloaded from http://ec. Genes involved in the activation of both TAG and free fatty acids. while double mutants are nonviable. which increased oil content. CELL further supported by several other studies in which overexpression of TAG assembly genes resulted in increases in seed oil content. Due to the ease of genetic engineering with Escherichia coli and Saccharomyces cerevisiae. these modifications include quite comprehensive modulations of entire metabolic pathways. However. most microalgal fatty acids have a chain length between 14 and 20. which resulted in a 20-fold increase in free fatty acid production. greatly increasing the production of myristic acid (208). thaliana and E. Modification of lipid characteristics. 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. Due to the fact that cells rely on the -oxidation of fatty acids for cellular energy under certain physiological conditions. coli. sometimes resulting in increased cellular lipid content. Single mutants of ACX3 or ACX4 have normal lipid breakdown and seedling development. There are several acyl-ACP thioesterases from a variety of organisms that are specific for certain fatty acid chain lengths. putatively due to complete elimination of shortchain acyl-CoA oxidase activity (159). it is also reasonable to attempt to increase the quality of the lipids.org/ on October 29. but they should be attainable with organisms with established protocols for genetic transformation and available selectable markers. an endogenous thioesterase. and 18:1. inactivation of the peroxisomal long-chain acylCoA synthetase (LACS) isozymes. inhibition of -oxidation would prevent the loss of TAG during the night. 162). in A.490 MINIREVIEWS EUKARYOT. Several publications have shown that knocking out genes involved in -oxidation in S. respectively (10. For example.

However. alkanes. Fatty acid ester production. Ethanol for biofuels is currently produced from the fermentations of food starches or cellulose-derived sugars.asm.to 22-carbon) production has been reported for the bacterium Vibrio furnissii (137). 155). both from C. Triacylglycerols can be used for the production of biodiesel through the creation of fatty acid esters. however. a suggested decarbonylase enzyme involved in alkane production has been studied with the green microalga B..VOL. Although the ethyl ester yield from this manipulation was not overly impressive for E. coli. thaliana (1. Straight-chain alkanes. 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. The last step is thought to be catalyzed by a decarbonylase enzyme. Transgenic overexpression of an 8:0. methanol or hydrogen) and chemical processing. such as lipids and/or starch (75). but it may also be possible to reduce production costs through genetically engineering microalgae to directly produce these shorter chain lengths. but an approach to directly couple ethanol production to photosynthetic carbon fixation in situ may be preferred. but to couple ethanol production to photoautotrophic metabolism will require changes in regulatory pathways or the insertion of new metabolic pathways. and several possible decarbonylases that are thought to be involved in wax formation. and the actual mechanism for the conversion of aldehyde to alkane remains to be found. coli. Many microalgae do not produce large amounts of storage lipids during logarithmic growth. Interestingly. DIRECT BIOLOGICAL SYNTHESIS OF BIOFUELS Industrial methods for the production of biofuels using energy-rich carbon storage products. Strains of B. 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. hookeriana. and further research is needed to clarify how alkanes are generated and to reveal the precise enzymes involved. increases 8 to 10 fatty acids by an additional 30 to 40% (31). no functional decarbonylase enzyme has been cloned to date. reinhardtii (152) and a nitrate-responsive promoter in diatoms (148). 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. while strain B produces very-long-chain triterpenoid hydrocarbons (117). Short. Examples of inducible promoters in algae include copper-responsive elements in C.g. In such a case. Many microalgae have fermentative metabolic pathways to ethanol. 2012 by guest . 9. Ethanol. Several biological pathways have been described for the production of fatty acid esters. Instead. such as sugars and lipids. 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. However. The potential for modification of lipid content in microalgae. the creation of a Downloaded from http://ec. the introduction of metabolic pathways for the direct production of fuels faces many challenges. a more recent study disputed these claims (195). with strain A producing very-long-chain dienes and trienes. 2010 MINIREVIEWS 491 interesting candidates for transgenic overexpression in microalgae since their activities could improve the suitability of microalga-derived diesel feedstock. Microalgal lipids can also be used to produce a “green” or renewable diesel through the process of hydrotreating. which increases the cost of biofuel production. Every production step that can be transferred to biological pathways will likely improve the overall economics. The alkanes that are generated by these putative decarbonylases all have very-longchain lengths ( 22 carbons) and will require further processing for fuel production. these transformations require additional energy carriers (e. Since alkanes can be derived from fatty acids. such as a lack of nitrogen. With cyanobacteria. when they encounter environmental stress. Fatty acids of even shorter chain lengths can also be used for production of gasoline and jet fuel. However. It may be that increased lipid synthesis will result in a reduction of cell division. 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. Production of shorter-chainlength alkanes that are suitable for direct use as fuel remains an existing goal. microalgae that are good lipid producers could perhaps be genetically transformed to produce alkanes. and other longer-chain alcohols. 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. 164. and alcohols. A possible example of long-chain-alkane (14. isopropanol. have been found in A. they slow down their proliferation and start producing energy storage products. 192). Decarbonylase activity has also been found in the leaves of the pea Pisum sativum (20. including Cer1 and Cer22. butanol. This conversion relies on the serial transformation of fatty acids to aldehydes and then to alkanes. 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. it will be interesting to see if higher productivities can be achieved and what effect fatty acid ester accumulation has on microalgal growth. combined overexpression of the 8:0/10:0-biased thioesterase and a ketoacyl ACP synthase (KAS). Interestingly. In addition. braunii.and medium-chain alkanes have the potential to be used directly as transportation fuel. which has the ability to produce very-long-chain alkanes (35). and tolerant species of microalgae may have to be generated. hookeriana in canola has had some success in increasing the production of short chain fatty acids (32). 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. many types of fuel products have the potential to be toxic. braunii differ in which long-chain hydrocarbons are synthesized. It will be interesting to see how overexpression of lipid synthesis pathway genes will affect microalgal proliferation.and 10:0-biased thioesterase from C. Algal starches have been shown to be fermentable by yeast (129). which resulted in the synthesis of fatty acid ethyl esters that could be used directly as biodiesel (86).org/ on October 29.

and Lee et al. Considering the reported yields from the various pathways that have been utilized so far. these enzymes are not optimized for performance in oxic conditions and may need to be configured for eukaryotic systems. 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. This pathway produces ethanol during photoautotrophic growth and could be incorporated into algae. Feasibility of direct biological synthesis of fuels. and up to C8 alcohols have been synthesized (for a review by Connor and Liao. These ubiquitous pathways normally produce amino acids through 2-keto acid precursors. Secretion of free fatty acids in yeast. An alternative synthetic pathway for the production of butanol utilized the endogenous keto acid pathways for amino acid synthesis. In addition. such as 2-ketobutyrate. CELL pathway for ethanol biosynthesis has been demonstrated. coli using isoprenoid biosynthesis pathways.asm. see references 53 and 98. As a fuel. ALKANES. however. yields were greatly improved by the deletion of 5 host genes that compete with the 1-butanol pathway for acetyl-CoA and NADH. Atsumi et al. and dimethylallyl diphosphate (DMAPP). including settling and flocculation. acetobutylicum into E. Other compounds that could replace diesel and jet fuel can also be produced through isoprenoid pathways (for recent reviews by Fortman et al. Isoprenoids..000 different molecules. (7). resulting in the production of 4. alkanes. respectively). the production of isobutanol through the keto acid pathways should be carefully considered for microalgal systems. attempts have been made to express the production pathways for isopropanol and butanol in the more user-friendly host E. Using similar approaches. but they are also typically much more expensive and energy intensive. With optimization. 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. in which the overexpression and deletion of entire metabolic pathways are feasible. coli. see reference 28). coli. There are many examples of successful pathway manipulation to generate compounds that can be used as fuels. comprehensive modifications. As mentioned in the section on lipid catabolism. isopentyl diphosphate (IPP). such as those performed with E. Recently. proved that it is possible to divert some of the 2-keto acid intermediates from amino acid production into alcohol production. Based on currently achievable productivities. resulting in production of 112 mg/liter isopentenol (107. Most of these come from the manipulation of E. It is reasonable to believe that some of the biochemical pathways in microalgae could be leveraged for the direct production of fuels. and it is therefore reasonable to believe that a similar approach to production of alcohols in algae is possible. coli. including TAGs.9 g/liter of isopropanol (67). mobilis (34. AND WAX ESTERS 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. Molecules that could potentially work as gasoline substitutes. While there are several possible low-cost solutions to concentrating the biomass. most microalgae will not grow to a density higher than a few grams of biomass per liter of water. are conserved in microalgae. In microalgae. 200). may be attempted. coli. which was produced at titers up to 22 g/liter (8). inactivation of genes involved in -oxidation has been shown to result in fatty acid secretion in some instances. Mutant colonies that secrete fatty acids were thereby identified by the formation of a halo in the overlaid agar (120. it is possible to obtain longer-chain alcohols as well. 2012 by guest . coli by Atsumi et al. and production has been industrialized using Clostridium acetobutylicum. thereby requiring the use of harsh lysis conditions. Interestingly. coli. resulting in the production of 550 mg/liter (7). which are faster. many microalgal species have a very tough outer cell wall that makes extraction of fuel feedstocks difficult. Isoprenoids. A further increase in production was achieved through the expression of the entire pathway from a single plasmid. have been produced by E. these methods are slow and the resulting biomass may still require further dewatering. several combinations of up to five genes from various species of Clostridium were overexpressed in E. free fatty acids. acetobutylicum has a low growth rate and is somewhat difficult to genetically engineer. 40). 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. six genes encoding the entire pathway for butanol production were transferred from C. Isopropanol and butanol are naturally produced by bacteria of the genus Clostridium. There are in fact several pathways in nature that lead to secretion of hydrophobic compounds. and wax esters. with more than 40. In a similar fashion. isoprenoids are synthesized via the methylerythritol (MEP) pathway using glyceraldehydes-3-phosphate and pyruvate to generate the basic building blocks of isoprenoid biosynthesis. including isopentenol. many exciting advances toward the biological production of C3 up to C8 alcohols have been achieved. For isopropanol production. 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. This was achieved through the simultaneous transgenic overexpression of a 2-keto-acid decarboxylase and an alcohol dehydrogenase. represent an incredibly diverse group of natural compounds. the overexpression of the butanol production pathway resulted in 1-butanol production of approximately 140 mg/liter.2 g/liter 1-butanol (78). Two enzymes from Bacillus subtilis that utilize IPP and DMAPP for the biosynthesis of isopentenol were overexpressed in E. especially that of isobutanol.492 MINIREVIEWS EUKARYOT. 132). Alternative methods to concentrate algal biomass include centrifugation and filtration. with the insertion of pyruvate decarboxylase and alcohol dehydrogenase from the ethanologenic bacterium Z. resulting in production of 1. SECRETION OF TRIACYLGLYCEROL. Some of the 2-keto acid intermediates. Similar screening methods could be utilized to identify mi- Downloaded from http://ec. With advances in genetic transformation methods and increased knowledge regarding expression systems in microalgae. Because C.org/ on October 29. ethanol has a lower energy density than gasoline and poor storage properties. FREE FATTY ACIDS. also known as terpenoids.

org/ on October 29. and the ATP-binding cassette (ABC) transporter-mediated export of plant waxes. 9. and sterols (82. 99. Downloaded from http://ec. but the ABC transporter systems may rely on fewer components. With further research. cerevisiae in these cases are not known. see reference 111). What is perhaps a more straightforward approach to enabling secretion of lipids from microalgae is the use of ABC transporters. results in a buildup of intracellular free fatty acids and secretion of free fatty acids. While wax transporters have not been shown to export products that are derived from medium-chain fatty acids. 216). cholesterol. These transporters include the A. alkanes. Combined inactivation of FAA1 and FAA4. 140). research efforts should be aimed at transferring these pathways into organisms that are good lipid producers.and medium-chain fatty acids. but it remains to be demonstrated at a scale significant for biofuel production from microalgal feedstocks. It is currently possible to induce secretion of free fatty acids from yeast and cyanobacteria. For example. In A. including import of fatty acids into mitochondria and peroxisomes for -oxidation. and arylacetamide deacetylase (AADA) (62. including alkanes. There are several examples of established pathways for the secretion of lipophilic compounds. Several key genes are known for these pathways. Long-chain fatty acids are imported into the peroxisome for -oxidation by ABC transporters. 189. One interesting characteristic of ABC transporters is their promiscuous gating properties. xanthine oxidoreductase (XOR). and it may be possible to reproduce this kind of secretion in microalgae. Cer5/AtWBC12 facilitates the export of very-long-chain aldehydes. ABC transporters mediate the export of plant waxes consisting of a multitude of compounds that are derived from very-long-chain fatty acids. TAG-containing vesicles from mammary glands. the exact genes that would be needed to enable a functional secretion pathway in another cell type are not known. As with VLDL and milk TAG secretion. and it may be possible to utilize such pathways for the export of lipids. thaliana there are over 120 different ABC transporters.. cerevisiae has five genes with fatty acyl-CoA synthetase activity. and perhaps fatty acids (140).VOL. But again. a secretion strategy may not be the best solution when a significant number of contaminating microorganisms are present in the cultivation system. 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. such as those encoding apolipoprotein E (ApoE). it should become clear whether the transfer of these very efficient TAG-secreting pathways is feasible. however. These genes include those that encode adipophilin (ADPH). In summary. 115. A similar form of fatty acid secretion has been achieved by Synthetic Genomics with cyanobacteria (158). neither the genes involved nor the mechanism has been described (132). and fatty acids. However. Mechanisms for secretion of lipids and related compounds. For example. In a similar fashion. which consist of many types of hydrocarbons. 190). and inactivation of the ABC transporter Ped3p or Pxa1 in A. several genes have been identified that are involved in milk TAG vesicle secretion. alcohols. triacylglycerol hydrolase (TGH). they are also responsible for the transport of molecules that are produced through the isoprenoid synthesis pathway. alkyl esters. the successful transgenic expression and utilization of lipid secretion pathways to secrete molecules suitable for biofuel production remain largely to be demonstrated. resulting in resistance. both of which have been shown to be important for exporting wax to the epidermis (136. 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 in addition to transporting compounds that are related to very-long-chain fatty acids. it is not known which genes would be needed to enable VLDL secretion from cell types that do not normally secrete VLDL. Even though many of the genes that are involved in secretion have been identified. microsomal triglyceride transfer protein (MTP). and transgenic expression of ABC transporters has been used to enable drug transport. random mutagenesis resulted in the secretion of TAGs. the exact mechanisms are generally not known. and butyrophilin (BTN) (for a review by McManaman et al. However. 205). including terpenoids and other compounds that are of interest for biofuel production. the current knowledge of secretion pathways is still rather limited and therefore may not necessarily be easily transferred to microalgal cells. 2012 by guest . ketones. Unfortunately. including those encoding FAA1 and FAA4. thaliana Desperado/ AtWBC11 transporter and the Cer5/AtWBC12 transporter. and transgenic expression of ABC transporters has resulted in transport of a variety of compounds.asm. Importantly. This kind of secretion was found to take place mainly during logarithmic growth. including kanamycin. whereas the cells started importing free fatty acids in stationary phase (162). To improve the rates of secretion and to allow for the secretion of other types of bioenergy carriers. The actual mechanisms for TAG and/or free fatty acid secretion in S. aldehydes. the secretion of VLDL from hepatocytes has been shown to be affected both by deletion and overexpression of a wide range of genes. In one of the yeast studies. the highest levels of fatty acid secretion also seemed to be associated with reduced proliferation (120). Overexpression of these genes in hepatocytes that already have the capacity to secrete VLDL results in increased secretion. there are also known pathways for intracellular transport of fatty acids between organelles. Since there are several highly efficient pathways for lipid secretion in nature that are being explored with various model organisms. 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. S. thaliana inhibits peroxisomal uptake of long-chain fatty acids (70. or FAA1 together with acyl-CoA oxidase activity. These include the secretion of TAG-containing very-low-density lipid (VLDL) vesicles from hepatocytes. thereby reducing product yields. the exact mechanisms are not known. ketones. 2010 MINIREVIEWS 493 croalgae that have the ability to secrete fatty acids. In addition to cellular export pathways. alcohols. The secretion of the fuel intermediates into the culture medium would provide these microorganisms with a rich source of nutrient. lipid secretion is an attractive alternative to harvesting algal biomass that could potentially lower the cost of producing microalga-derived biofuels. it would perhaps be possible to use random mutagenesis or directed evolution to generate mutant ABC transporters that have the ability to secrete short. In addition. which could be transferred to microalgae.

coli (176) or the recombinant rev6 (63). DPE. that have successfully increased starch content in other plants should be expressed in microalgae. AMY. This problem could be circumvented by . pyrophosphate. H2. AGPase is typically a heterotetramer composed of large regulatory and small catalytic subunits and is allosterically activated by 3-phosphoglyceric acid (3-PGA). isoamylases. -amylases. Some of these branches are trimmed (WSP3). 39). butanol. PGM. Glucans are stored in microalgae in a variety of ways. Much work has been done on the catalytic and allosteric properties of AGPases in crop plants to increase starch production. and enzymes are shown in red.4 glycosidic linkages (WSP1) until a branching enzyme highly branches the ends (WSP2). branching enzymes. P. SS. DBE. inorganic phosphate. SP. AGPase. such as the C. Glucans are added to the water soluble polysaccharide (WSP) by -1. starch synthases. An alternative approach for increasing microalgal starch would be to introduce starch-synthesizing enzymes into the cytosol. lipids. CELL Downloaded from http://ec.org/ on October 29. Polysaccharides are often found surrounding the pyrenoid in microalgae. Glaucophyta. The phyla Chlorophyta. plastidial phosphoglucomutase. glucan-water dikinases. MEX1. The ratelimiting step of starch synthesis (see Fig. In green algae. malto-oligosaccharides. starch phosphorylases. MOS. starch is synthesized and stored within the chloroplast. PPi. Phaeophyceae. The cytosol would give more physical space for the starch granules to accumulate (174). disproportionating enzyme (1 and 2) -1. AMY. it may not be activated.4 and branched -1. Dinophyta. linking starch synthesis to photosynthesis (209). Glaucophyta. A problem for cytosolic starch synthesis could be that because the AGPase is far from the pyrenoid and thus 3-PGA. which are made up of -1. Phosphorolytic [Starch-(P)n] and hydrolytic degradation pathways are shown. Genetic strategies for increasing glucan storage. and Bacillariophyceae. including ethanol. resulting in ADP-glucose and pyrophosphate (176). ADP-glucose pyrophosphorylase. GENETIC MODIFICATION OF CARBOHYDRATE METABOLISM Carbohydrates can be metabolized into a variety of biofuels. Starch metabolism in green microalgae. while it is stored in the cytoplasm in Dinophyta.4 glucanotransferase. The metabolites and simplified representative pathways in microalgal starch metabolism are shown in black. BE.asm. glucose. and Rhodophyta and in the periplastidial space in Cryptophyceae (38. and this process is repeated until a starch granule is formed. and/or methane. such as those encoded by glgC16 from E. Glc. phosphate. maltose transporter. reinhardtii sta1 or sta6 mutant. water-soluble granules of laminarin and chrysolaminarin are synthesized. ISA. 3.494 MINIREVIEWS EUKARYOT.3 linkages with branching at the C-2 and C-6 positions of glucose (74). In Heterokontophyta. with mixed results (174). GWD. and Rhodophyta store glucans in linear -1. -amylase. likely because it is a source of 3-PGA (10). 3 for an overview of starch metabolism) is the ADP-glucose pyrophosphorylase (AGPase)-catalyzed reaction of glucose 1-phosphate with ATP. 2012 by guest FIG. debranching enzymes. Pi. Designer AGPases. preferably in a background with no native AGPase activity.6 glycosidic linkages (10).

and both of these phosphorylations are thought to help disrupt the crystalline structure of the starch granules to allow glucan-metabolizing enzymes access (212). These considerations may become issues when microalgae are grown for biofuels and are subject to diurnal light cycles. MICROALGAL HYDROGEN PRODUCTION Many eukaryotic microalgae and cyanobacteria have evolved in ecosystems that become depleted of O2. a further understanding of their regulation and mechanism could potentially be leveraged for continuous biofuel production from secreted saccharides. 9. 2010 MINIREVIEWS 495 the introduction of an AGPase that does not require 3-PGA. and various polysaccharides have been reported to be secreted (71). proline. S. 143) and in improvements in overall H2 yields (90. 110. A potential side effect of increased trehalose is its ability to induce starch synthesis and AGPase expression. thaliana mutants grown in a diurnal cycle that cannot synthesize or degrade starch grow more slowly than the wild type (17. such as the sucrose synthetase and sucrose phosphate synthetase. thaliana. In A. are also secreted by many species during anoxia (118. Although little is currently known about these secretion events. which could result in an increase in total sugar content. Starch can be degraded by hydrolytic and/or phosphorolytic mechanisms.org/ on October 29. This isomerase converts sucrose to isomaltulose. Hydrolytic starch degradation requires an enzyme capable of hydrolyzing semicrystalline glucans at the surface of the insoluble starch granule. it should be noted that additional fermentation metabolites. 72). but when it is knocked out in Arabidopsis. is transported to the cytosol in green microalgae and land plants by the maltose export protein (MEX1) (38). while disruptions in PWD result in a less severe starch excess phenotype (156). 125). resulting in significant advances in both our fundamental understanding of H2 metabolism in this organism (43. the total sugar levels of isomaltulose and sucrose are twice as high as those of control plants. Mutant considerations. 91. but at continuous light its growth rates were equal (17). In addition to exporting soluble sugars. 118. and the [NiFe] and [FeFe] hydrogenases are capable of the reversible reduction of protons to H2. The [FeFe] hydrogenases in many green microalgae are able to effectively couple to the photo- Downloaded from http://ec. indicating alternative mechanisms for starch degradation (207). 2012 by guest . A. allowing for higher levels of total sugar accumulation (202). This protein facilitates bidirectional diffusion and could be leveraged to export maltose out of the cell. and diverse fermentation metabolisms that can be leveraged in renewable bioenergy strategies are present in these organisms (64. The precise mechanisms of starch catabolism in green microalgae are largely unknown (10) but are more widely understood for A. a nonplant metabolite. This may imply that there is a signaling system that gives negative feedback when sucrose levels reach a certain level. 213). Mutants that synthesize less starch or have a reduced capacity to degrade starch often have reduced growth rates (175). the level of sucrose is not detected. glutamic acid. only the [FeFe] hydrogenases have been described in eukaryotic microalgae. for extracellular excretion. Although sucrose has largely been unexplored with microalgae. starch can be degraded even when all three -amylases in A. whereas. thaliana (175). a product of starch degradation in the chloroplast. which still has activity even without the presence of an activator (14). where the concentration is doubled. These two enzyme classes are phylogenetically distinct. aspartic acid. glycerol. Hydrogenases are classified according to the metal ions at their active sites. and as a result. The synthesis of sucrose could be exploited by sucrose transporters. thaliana plastidial phosphoglucomutase mutant (corresponding to STA5 in C. and interestingly. in addition to likely being more amenable for engineered secretion because many transporters have been described. lysine. arabinose. DPE2. Of particular interest is the ability of many green microalgae to produce H2. such as the Mos(1-198)/SH2 AGPase. Decreasing starch degradation in microalgae. Interestingly. an increase in total sugar production has been accomplished by the transgenic expression of a sucrose isomerase from a bacterium. however. enough to cause maltose exportation to the roots. In addition. thaliana have been knocked out. 18.VOL. which has been shown with Arabidopsis (199). thaliana. a glucosyltransferase from a bacterium converting maltose to trehalose (131) could be expressed as a potential strategy to increase total sugar content. -amylase (AMY3) is thought to participate in starch degradation. and a homologous protein is found in C. An A. These phosphorylation steps are critical for starch degradation and are excellent gene knockout targets for a starch accumulation phenotype in microalgae. Maltose. The C-3 position in the glucan can also be phosphorylated by the phosphoglucan water dikinases (PWD). Mannitol. are present (46). In microalgae. The production of soluble sugars may be preferred over polysaccharides because soluble sugars are smaller and easier to process. 143). In A. Hydrogen metabolism has been studied extensively with C. reinhardtii (61. Plastidial starch degradation is stimulated by phosphorylation of glucose residues at the root of amylopectin by glucan-water dikinases (GWD). 68. maltose could be converted to other isoforms that may be silent to the native metabolic regulatory system. GWD catalyzes the transfer of -phosphate in ATP to the C-6 position of the glucans in amylopectin (156). especially during the night. Ankistrodesmus densus secretes polysaccharides when exposed to light. Malt- ose is metabolized in the cytosol by a glucosyltransferase. 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). thaliana.asm. cerevisiae cells transformed with SUC1 and SUC2 have been shown to transport sucrose and some maltose across their plasma membranes (160). intracellular sugar accumulation is also a desirable microalgal biofuel trait. In sugar cane. Many microalgae have the native ability to secrete fixed carbon products. reinhardtii) had more of a reduced growth rate during short daylight periods compared to long daylight periods. but when sucrose is converted to isomaltulose. reinhardtii (175). including organic acids and alcohols. evidence exists that some of the enzymes involved in sucrose metabolism. 114). even during stationary phase (138). it results in a 30-fold increase of maltose in the leaves. such as SUC1 and SUC2 found in A. Secretion strategies and soluble sugars. 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). only the [NiFe] hydrogenases have been reported for cyanobacteria. For example.

Experiments with small. 204) as well as enzymes that catalyze the production of osmolytes. Consequently. In one study of algal antenna mutants. This strategy can most likely be applied to many different microalgae more easily than a random mutagenesis approach. Other stress factors.496 MINIREVIEWS EUKARYOT. but a recent publication efficiently used an RNAi-based strategy to knock down both LHCI and LHCII in C. Melis and coworkers described the use of sulfur deprivation (203). but this strategy may have two positive effects. including tobacco and E. The antistress properties of these genes isolated from bacteria as well as a marine stresstolerant microalga (Chlamydomonas sp. (ii) rapid biomass production rates. (iii) the capacity to produce a wide variety of biofuel feedstocks. and light intensity. see reference 113). In 2000. These include (i) high photosynthetic conversion efficiencies. However. it should become clear whether the current approach can be successfully applied to increase biomass production. it was demonstrated that alginate-immobilized cultures of nutrient-deprived C. An additional benefit of increasing stress tolerance is the possibility of growing select microalgae under extreme conditions that limit the proliferation of invasive species.000 mol photons m 2 s 1 during midday (113). 178). as an effective means of sustaining H2 photoproduction when respiration is able to consume all of the photosynthetic O2 produced by the cells (114). an ATP synthase subunit that is involved in the regulation of intracellular ATP levels and stress tolerance (179). 141. reinhardtii (126). 182. such as salt concentration. CONCLUSION Microalgae are an extremely diverse group of organisms. 126–128. this intensity. Microalgae are considered great model organisms to study photosynthetic efficiency. it would be of great benefit to develop genetic strategies to increase the cellular tolerance to a variety of stress factors. reinhardtii could sustain H2 photoproduction even in the presence of an oxygenated atmosphere (90). Earlier research relied on random mutagenesis strategies to generate mutants with fewer or smaller chlorophyll antennae.and large-scale microalgal photobioreactors and molecular research in photosynthetic efficiency have revealed several factors that can limit biomass accumulation. 167. Although microalgae have long been considered a promising platform for the production of biofuels. which resulted in increased resistance to several different stressful stimuli. 185). a phenomenon known as photoinhibition. Light intensities above the maximum photosynthetic efficiency actually reduce the growth rate. and (iv) the ability to thrive in diverse ecosystems. This approach may seem counterintuitive. first. CELL synthetic electron transport chain at the level of ferredoxin. With more research. 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 absorp- tion of sunlight by individual chloroplasts (97. and several attempts have been made to improve the photosynthetic efficiency and/or reduce the effects of photoinhibition on microalgal growth. it remains to be seen how well these mutants will perform in largerscale cultures with more varied conditions and perhaps with competition from wild invasive microalgal species. no improvement in productivity was observed with outdoor ponds (77). High-light stress. providing the means to generate H2 directly from water oxidation. 60. such as mannitol and ononitol (166. such as lipids and starch. inhibiting state transitions. as well as on the ability of microalgae to rapidly produce large amounts of biomass. Therefore. it is possible to control these factors to a certain degree through engineering and manipulation of the growth environment. coli. Salt. 188). 142. IMPROVED GROWTH CAPACITY THROUGH INCREASED STRESS TOLERANCE OR INCREASED PHOTOSYNTHETIC EFFICIENCY The production of any biofuel is dependent on the efficiency of the metabolic pathways that lead to accumulation of storage compounds. However. it permits higher light penetration in high-density cultures. It seems clear that manipulation of light-harvesting complexes can lead to increased biomass productivity under high light in controlled laboratory conditions. pH.asm. all microalgal [FeFe] hydrogenases characterized to date are particularly O2 sensitive. and second. earlier studies concluded that the economics of microalgal Downloaded from http://ec. these studies have rapidly advanced our understanding of H2 metabolism and enzyme maturation (13. One important consideration is the intensity of light at which a certain strain of microalga reaches its maximum growth rate. and interestingly. pH. However. temperature. and other stimuli can also cause stress to microalgal cultures. such as those encoding glutathione peroxidase and ascorbate peroxidase (168. These include genes that are directly involved in scavenging reactive oxygen. and numerous strategies are emerging to further advance our ability to optimize H2 production in eukaryotic phototrophs. and hydrogenase engineering (11. and H2 photoproduction is only transiently observed prior to the accumulation of O2 to inhibitory levels under nutrient-replete conditions (27. temperature. These include stress factors. which decreases photosynthetic activity. 112). 2012 by guest . they also did not observe any improved productivity in laboratory cultures. but these manipulations add to the cost of growing microalgae. Depending on the design of the cultivation facilities. In combination with physiological and biochemical approaches.org/ on October 29. many of which possess novel metabolic features that can be exploited for the production of renewable biofuels. 145) in green microalgae. It is likely that similar improvements can be achieved with microalgae. is usually around 200 to 400 mol photons m 2 s 1 for most species. Photosynthetically active radiation intensities from sunlight can exceed 2. including high salt and low temperature. which corresponds to the maximum photosynthetic efficiency. W80) were demonstrated through transgenic overexpression in several different systems. most microalgae will not grow at maximum efficiency during most of the day. High light is not the only environmental variable that can cause stress and inhibit microalgal growth. Many genes have been identified that are important for withstanding stress conditions. Recently. 68. Genetic techniques have been applied with the aim of increasing H2 photoproduction activity by decreasing light-harvesting antenna size. 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.

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