Eukaryotic Cell 2010 Radakovits 486 501

Genetic Engineering of Algae for Enhanced Biofuel ProductionRandor 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. Downloaded from http://ec.asm.org/ on October 29, 2012 by guest Updated information and services can be found at: http://ec.asm.org/content/9/4/486 These include: REFERENCES This article cites 215 articles, 78 of which can be accessed free at: http://ec.asm.org/content/9/4/486#ref-list-1 Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» CONTENT ALERTS Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ It is postulated that a light-harvesting footprint of at least 20. Consequently.000 square miles will be required to satisfy most of the current U. including biodiesel and ethanol. which is similar to that found in higher plants. E-mail: mposewit@mines . 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. polysaccharides. even modest improvements in photon conversion efficiencies will dramatically reduce the land area and cost required to produce biofuels. and the dangers of increasing atmospheric CO2 levels. it may be possible to leverage the metabolic pathways of microalgae to produce a wide variety of biofuels (Fig. 1617 Cole Blvd. Golden.2 and Matthew C. and bacteria through genetic engineering.00364-09 Copyright © 2010.EUKARYOTIC CELL. and many eukaryotic microalgae have the ability to store significant amounts of energy-rich compounds. superior growth rates. Department of Energy’s Aquatic Species Program analyzed approximately 3..asm. All Rights Reserved.. both microalgae and macroalgae (i. the presence of invasive species in large-scale ponds. the reality of peak oil in the near future. low light penetrance in dense microalgal cultures. Interest in a variety of renewable biofuels has been rejuvenated due to the instability of petroleum fuel costs. No. including the accumulation of significant quantities of triacylglycerol. Fax: (303) 273-3629. and ability to store or secrete energy-rich hydrocarbons. and the diversion of central metabolic intermediates into fungible biofuels. Jinkerson. the synthesis of storage starch (amylopectin and amylose). 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. 9. most have drawbacks that have prevented the emergence of an economically viable algal biofuel industry. yeast. several technical barriers need to be overcome before microalgae can be used as an economically viable biofuel feedstock (139). It is likely that many of these advances can be extended to industrially relevant organisms. Vol. In contrast to corn-based ethanol or soy/ palm-based biodiesel. diesel fuel surrogates. To advance the utilization of microalgae in biofuel production. Mailing address: Department of Chemistry and Geochemistry. Colorado 804012 There are currently intensive global research efforts aimed at increasing and modifying the accumulation of lipids. Apr.1128/EC. Colorado School of Mines. and this is likely only a small fraction of the total number of available species (75). However. Colorado School of Mines. Phone: (303) 384-2425. Photosynthetic microorganisms are attracting considerable interest within these efforts due to their relatively high photosynthetic conversion efficiencies.. with diatoms being especially likely candidates. transportation fuel demand (41).1 Al Darzins. Published ahead of print on 5 February 2010. using waste or salt water (41). Although the application of genetic engineering to improve energy production phenotypes in eukaryotic microalgae is in its infancy. a reliance on unstable foreign petroleum resources. Posewitz1* Department of Chemistry and Geochemistry. 2010. Although these efforts demonstrated that many species of microalgae have properties that are desirable for biofuel production. Over 40. 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. that can be utilized for the production of several distinct biofuels. . eukaryotic microalgae possess several unique metabolic attributes of relevance to biofuel production.00 doi:10. American Society for Microbiology.org/ on October 29. considering their lipid profiles and productivity (153). including increased lipid and carbohydrate 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. 1500 Illinois St. improved H2 yields.edu. p. Many improvements have been realized. Golden. In addition. These include developing low-energy methods to harvest microalgal cells. Golden.e. continued bioprospecting efforts and the development and engineering of select microalgal strains are required to im486 Downloaded from http://ec. Therefore. hydrocarbons. 1). 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. 4 Genetic Engineering of Algae for Enhanced Biofuel Production Randor Radakovits. diverse metabolic capabilities.000 different microalgae for their potential to produce biofuels. and the ability to efficiently couple photosynthetic electron transport to H2 production. seaweeds). and other energy storage compounds in photosynthetic organisms. Colorado 80401.1 Robert E.1 and National Renewable Energy Laboratory. Relative to cyanobacteria. and/or alkanes. and the potentially poor cold flow properties of most microalga-derived biodiesel. Several species have biomass production rates that can surpass those of terrestrial plants (41). Photosynthetic algae. have been of considerable interest as a possible biofuel resource for decades (165). starch-derived alcohols. 2012 by guest * Corresponding author. the lack of cost-effective bioenergy carrier extraction techniques. difficulties in consistently producing biomass at a large scale in highly variable outdoor conditions.S. it is clear that investigation of the potential of living microalgae to produce biofuels should be a priority. such as triacylglycerol (TAG) and starch. CO 80401. The U. If ancient algae are responsible for creating substantial crude oil deposits.000 species of algae have been described. alcohols.S. and numerous additional species have subsequently been investigated (165). It is believed that a large portion of crude oil is of microalgal origin. 486–501 1535-9778/10/$12. reinhardtii (116. ongoing microalgal genome sequencing projects include those for Fragilariopsis cylindrus. Historically. including those for C. However. Micromonas pusilla. Cyanidioschyzon merolae (109). and Aureococcus anophageferrens (100).org/ on October 29. Botryococcus braunii. ER. particularly diploid diatoms. 24.asm. In addition. the green alga Chlamydomonas reinhardtii has been the focus of most molecular and genetic phycological research. most of the tools for the expression of transgenes and gene knockdown have been developed for and are specific for this species. Current commercial agriculture crops have been cultivated for thousands of years. Porphyra purpurea. Currently. prove the yields of bioenergy carriers. 1. Ostreococcus tauri (36). Microalgal metabolic pathways that can be leveraged for biofuel production. nuclear. 9. Thalassiosira pseudonana (6). the full potential of genetic engineering in some microalgal species. Thalassiosira rotula. 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. there are several completed and ongoing efforts to se- . Galdieria sulphuraria. Volvox carteri. and several more are being sequenced. Phaeodactylum tricornutum (15). Expressed sequence tag (EST) databases have been established. despite the recent advances in biotechnological approaches. endoplasmic reticulum. Ostreococcus lucimarinus (135). with desired traits selected over time. 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. Therefore. It stands to reason that with microalgae. 2010 MINIREVIEWS 487 Downloaded from http://ec. and Micromonas pusilla (201).VOL. Dunaliella salina. 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. and chloroplast genomes from several microalgae have been sequenced. tools are now also being rapidly developed for diatoms and other algae that are of greater interest for industrial applications. Chlorella vulgaris. 25). mitochondrial. thereby allowing useful traits or mutations to be easily combined (5. 2012 by guest FIG. Pseudo-nitzschia. Microalgal genomes. However. can be fully realized only if conventional breeding methods become firmly established. Since the topic of microalgal sexual breeding is beyond the scope of this review. 171). GENETIC ENGINEERING OF MICROALGAE Significant advances in microalgal genomics have been achieved during the last decade. Porphyra miniata (92). 119). the actual number of antibiotics that work with a specific strain may be much more limited.488 MINIREVIEWS EUKARYOT. 45. 45. reinhardtii that are reportedly more specific and stable than traditional RNA interference (RNAi) approaches (123. 83. 52. More than 30 different strains of microalgae have been transformed successfully to date. and Porphyridium sp. but in some cases only transient expression was observed. Rhodophytes. G418 (45. 173). 29.org/ on October 29. and dinoflagellates (2. and the method of transformation has to be carefully selected and optimized for each microalga. 3. 2 for a simplified overview of lipid biosynthesis pathways. 87. tricornutum (2. biolistic microparticle bombardment (2. 65. 187. However. 49. Heterokontophytes that have reportedly been transformed include Nannochloropsis oculata (21). and Agrobacterium tumefaciens-mediated gene transfer (23. In addition. and D. Efficient isolation of genetic transformants is greatly facilitated by the use of selection markers. 210). 54–56. 52. 54. and Symbiodinium microadriaticum (119). diatoms such as T. chloramphenicol (184). 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. and phaeophytes. 210. 85. 83. as well as dynamic transcriptomes from many different microalgae (4. 214. such as C. 51. it is possible to achieve transformation of the chloroplast through homologous recombination (for a review by Marín-Navarro et al. 181. it makes it difficult to delete specific target genes. diatoms. 26. from either the nucleus or the plastid. Gracilaria changii (58). Both the quantity and the quality of diesel precursors from a specific strain are closely linked to how lipid metabolism is controlled. A variety of transformation methods have been used to transfer DNA into microalgal cells. such as Laminaria japonica (150) and Undaria pinnatifada (151). including antibiotic resistance and/or fluorescent/biochemical markers. 66. reinhardtii. 101. streptomycin (42). and inclusion of speciesspecific 5 .asm. Chlorella kessleri (49). 130. 149. 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. reinhardtii and P. 30. 211. 104. antibiotics like nourseothricin and G418 are much less effective in salt-containing media and are not ideal for use with marine algae (210). carteri (81. The efficiency of transformation seems to be strongly species dependent. Due to the fact that many microalgae are resistant to a wide range of antibiotics. Therefore. Ulva lactuca (76). 58. 187. 21–23. see reference 48) demonstrate that the stability of expression can be improved through proper codon usage. GENETIC ENGINEERING OF THE LIPID METABOLISM Understanding microalgal lipid metabolism is of great interest for the ultimate production of diesel fuel surrogates. and rapeseed (Brassica napus). and brown (Phaeophyta) algae. 76. 88. 42). 49. 37. Ohlrogge and Jaworski have proposed that the fatty acid supply helps determine the regulation of oil synthesis (134). Haematococcus pluvialis (177. 88. reinhardtii (for a recent review by Eichler-Stahlberg et al. 44. 3 . 170. 69). P. as well as pathways that modify the length and saturation of fatty acids. Some progress in homologous recombination has been made with the nuclear genome of C. In recent years. hygromycin (12). merolae (121). including bleomycin (2. Chlorella saccharophila (108). Lipid biosynthesis and catabolism. 196. have not been as thoroughly investigated for algae as they have for terrestrial plants. paromomycin (81. the mechanisms for RNA silencing have been studied with microalgae. Lipid biosynthesis. Homologous recombination has also been reported for the red microalga C. Navicula saprophila (45). Several different antibiotic resistance genes have been used successfully for microalgal transformant selection. Dinoflagellates that have been transformed include Amphidinium sp. 147. 81. -glucuronidase (22. Transgene expression and protein localization in the chloroplast is needed for the proper function of many metabolic genes of interest for biofuel production. 87. 55. 214). (95). 42. 171. which has been transformed using a variety of methods (44. Methods developed primarily with C. Chlorella ellipsoidea (22. Kappaphycus alvarezii (94). and intron sequences. 69. 22. 180. 211). 148. transformation resulted in stable expression of transgenes. 85. 93). 92). 181. vulgaris (30. 163. and others. 122. 148. 151). 123. 210. reinhardtii. 211). electroporation (21. 148. C. 93. 88. In many cases. 170). 210. 83). While this may be suitable for transgene expression or for random mutagenesis screens. Nuclear transformation of microalgae generally results in the random integration of transgenes. pseudonana (147). 148). 170. 56. Cyclotella cryptica (45). Cylindrotheca fusiformis (52. 121. Members of the chlorophyte group that have been transformed include C. 56. 23. Another option for gene inactivation is the use of RNA silencing to knock down gene expression. several publications have used plastid targeting sequences to translocate proteins to the chloroplast (3. 49. the use of strong endogenous promoters. therefore. including agitation in the presence of glass beads or silicon carbide whiskers (44. 9. and green fluorescent protein (GFP) (23. 133. 123. salina (181. tricornutum (16. 108. -galactosidase (58. 161. Porphyra yezoensis (23). spectinomycin (19. reinhardtii. but the efficiency remains low (217). merolae (121). Successful genetic transformation has been reported for the green (Chlorophyta). some efforts have been made to increase the expression of enzymes Downloaded from http://ec. 50. 169. red (Rhodophyta). 163). 73. 183). and Thalassiosira weissflogii (51). many of the genes involved in lipid metabolism in terrestrial plants have homologs in the sequenced microalgal genomes. 150. 198). 51. Methods for transformation and expression. 214). 187). Chlorella sorokiniana (30). such as Arabidopsis thaliana. Other markers that have been used include luciferase (51. Several of these transgenic overexpression strategies have resulted in the increased production of triacylglycerols in seeds and in other plant tissues. Recent improvements in gene knockdown strategies include the development of highthroughput artificial-micro-RNA (armiRNA) techniques for C. The only euglenid that has been transformed to date is Euglena gracilis (42). 26. 65). have also been transformed. 2012 by guest . V. 210). 105. 108. While chloroplast transformation has not been demonstrated with diatoms. soy bean (Glycine max). 151. 210). In C. 87. 217). nourseothricin (210). See Fig. 119. euglenids. 92–95. 184).. Dunaliella viridis (180). 83). 37. 183. CELL quence plastid and mitochondrial genomes. 183. see reference 106). 26. and RNA silencing has been used successfully with both C.. in the seeds of B. overexpressed native ACCase in the diatom C. glycerol-3-phosphate acyltransferase. G3PDH catalyzes the formation of glycerol-3-phosphate. One of the most successful attempts to increase the amount of seed lipid is the overexpression of a cytosolic yeast. GPAT. fatty acyl-ACP thioesterase. with regard to increasing the total amount of seed oils. Dunahay et al. HD. triacylglycerols. One early committing step in fatty acid synthesis is the conversion of acetyl-coenzyme A (CoA) to malonyl-CoA. 9. which resulted in a 40% increase in lipid content (191). acyl carrier protein. lyso-phosphatidic acid acyltransferase.org/ on October 29.0116 to 0. several attempts to utilize ACCase overexpression to increase lipid content in various systems have been somewhat disappointing. However. 2010 MINIREVIEWS 489 Downloaded from http://ec. the effect of ACCase overexpression in potato tubers. 2012 by guest FIG. was not successful in increasing lipid production. napus. LPAAT. 3-ketoacyl-acylcarrier protein synthase III (KASIII).0580 mg g 1 fresh weight). that are involved in the pathways of fatty acid synthesis. glycerol-3-phosphate dehydrogenase (G3PDH). thaliana has been overexpressed in B. Free fatty acids are synthesized in the chloroplast. napus. Interestingly. which is needed for TAG formation. TAG. ACCase from A. Despite a 2. PDH. Overexpression of ACCase in the oleaginous seeds of B. KAR. LPAT. enoyl-ACP reductase. This interesting result suggests that genes involved in TAG assembly are of importance for total seed oil production. 157). 3-hydroxyacylACP dehydratase. no increased lipid production could be observed (165). lyso-phosphatidylcholine acyltransferase.asm. CoA. A. coenzyme A. KAS. 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. and B. cryptica (45). which is considered the first committed step in fatty acid biosynthesis in many organisms. Another attempt to increase expression of a protein involved in fatty acid synthesis. G3PDH. DHAP.VOL. pyruvate dehydrogenase complex. While increasing the expression of genes involved in fatty acid synthesis has had small successes. malonyl-CoA:ACP transacylase. thaliana.or 3-fold increase in ACCase activity. ENR. KASIII from spinach (Spinacia oleracea) or Cuphea hookeriana was expressed in tobacco (Nicotiana tabacum). MAT. 2. some interesting results have been achieved through the overexpression of genes involved in TAG assembly. catalyzed by acetyl-CoA carboxylase (ACCase). FAT. napus and Solanum tuberosum (potato) (89. resulted in a 5-fold increase in TAG content (from 0. 3-ketoacyl-ACP synthase. 3-ketoacyl-ACP reductase. 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. dihydroxyacetone phosphate. ACCase. diacylglycerol acyltransferase. DAGAT. gycerol-3-phosphate dehydrogenase. respectively). acetyl-CoA carboxylase. ACP. This is . Simplified overview of the metabolites and representative pathways in microalgal lipid biosynthesis shown in black and enzymes shown in red. while TAGs may be assembled at the ER. resulting in either no change or reduced seed oil content (33). a tissue that normally is very starch rich and lipid poor. 144. thaliana and E. 193). 209). CELL further supported by several other studies in which overexpression of TAG assembly genes resulted in increases in seed oil content. thaliana inhibits seed lipid breakdown. However. overexpression of glycerol-3-phosphate acyltransferase. major species are often 16:1. 218). sometimes resulting in increased cellular lipid content. 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. For example. of course. 2012 by guest . The chain lengths of fatty acids are determined by acyl-ACP thioesterases. 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. Similarly. For example. but it may be a valid strategy to increase lipid production in microalgae grown in photobioreactors with exogenous carbon sources and/or continuous light. such as starch. and transgenic overexpression of thioesterases can be used to change fatty acid chain length. Another possible approach to increasing the cellular lipid content is blocking metabolic pathways that lead to the accumulation of energy-rich storage compounds. Consequently. therefore. 80. thaliana and E. Lipid catabolism. 132. making it difficult to completely abolish these functions. two different starch-deficient strains of C. may not be beneficial for microalgae grown in outdoor open ponds. 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. 194). Expression of a 12:0-biased thioesterase from Umbellularia californica in both A. putatively due to complete elimination of shortchain acyl-CoA oxidase activity (159). thaliana (59). Both of these thioesterases are obviously Downloaded from http://ec. Several publications have shown that knocking out genes involved in -oxidation in S. it is also reasonable to attempt to increase the quality of the lipids.490 MINIREVIEWS EUKARYOT. have disruptions in the ADPglucose pyrophosphorylase or isoamylase genes. In addition to what has been accomplished with higher plants. as well as unpublished results from our laboratory. In some studies. DAGAT (172). There are several acyl-ACP thioesterases from a variety of organisms that are specific for certain fatty acid chain lengths. proper seedling development was also inhibited without the addition of an external carbon source (57). 96. in A. Wang et al. In addition to engineering microalgae for the increased production of lipids. 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. the sta6 and sta7 mutants. 215. Ideal fatty acids for diesel production should be 12:0 and 14:0. (197). gene inactivation would have to be achieved either through random mutagenesis or through the use of RNA silencing (37. Due to the ease of genetic engineering with Escherichia coli and Saccharomyces cerevisiae. knocking out lipid catabolism genes not only may result in increased lipid storage but also could have deleterious effects on cellular growth and proliferation. Such modifications are. Single mutants of ACX3 or ACX4 have normal lipid breakdown and seedling development. This strategy. and a plant thioesterase. Due to the fact that cells rely on the -oxidation of fatty acids for cellular energy under certain physiological conditions. 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. inhibition of -oxidation would prevent the loss of TAG during the night.asm. as well as genes directly involved in -oxidation of fatty acids. most of what we know regarding successful strategies to decrease lipid catabolism comes from studies of higher plants and yeast. reinhardtii. thaliana. have shown that these mutants accumulate increased levels of TAG during nitrogen deprivation. coli. A complementary strategy to increase lipid accumulation is to decrease lipid catabolism. 162). while double mutants are nonviable. 123. Another starchless mutant of Chlorella pyrenoidosa has also been shown to have elevated polyunsaturated fatty acid content (154). a 14:0-biased thioesterase from Cinnamomum camphorum was expressed in A.org/ on October 29. have been inactivated. Modification of lipid characteristics. respectively (10. coli drastically changed the lipid profiles in these organisms. these modifications include quite comprehensive modulations of entire metabolic pathways. An example is the short-chain acyl-CoA oxidase enzymes ACX3 and ACX4 in A. In the case of lipid biosynthesis. coli. or diacylglycerol acyltransferase (DAGAT) all result in significant increases in plant lipid production (79. and 18:1. as well as knocking out a gene product involved in -oxidation of fatty acids. One example of a comprehensive modification of E. with regard to suitability as a diesel fuel feedstock. but they should be attainable with organisms with established protocols for genetic transformation and available selectable markers. acyl-CoA synthetase (encoded by fadD) (102). successful modifications have also been achieved with bacteria and yeast to increase and/or modify their lipid content. LACS6 and LACS7. For example. 124. During diel light-dark cycles. 186. 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. inactivation of the peroxisomal long-chain acylCoA synthetase (LACS) isozymes. 214). 146. Typically. much harder to achieve with microalgae. Genes involved in the activation of both TAG and free fatty acids. greatly increasing the production of myristic acid (208). many microalgae initiate TAG storage during the day and deplete those stores at night to support cellular ATP demands and/or cell division. inhibition of lipid oxidation has caused unexpected phenotypes. with a great increase in the production of lauric acid (193. lysophosphatidic acid acyltransferase. cerevisiae include acyl-CoA oxidase and several acyl-CoA synthetases (see below). The lipid catabolism genes that have been implicated in fatty acid secretion in S. which resulted in a 20-fold increase in free fatty acid production. an endogenous thioesterase. Similar results were achieved through the inactivation of 3-ketoacyl-CoA thiolase (KAT2) in A. but most likely at the cost of reduced growth. To circumvent the current lack of efficient homologous recombination in microalgae. which increased oil content. most microalgal fatty acids have a chain length between 14 and 20. with the simultaneous overexpression or deletion of several key enzymes. which release the fatty acid chain from the fatty acid synthase. entailed overexpression of the lipid biosynthesis genes encoding acetyl-CoA carboxylase. an attempt has also been made to use directed evolution to increase the efficiency of one of these enzymes. 16:0. however. reinhardtii (152) and a nitrate-responsive promoter in diatoms (148). but to couple ethanol production to photoautotrophic metabolism will require changes in regulatory pathways or the insertion of new metabolic pathways. 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. the introduction of metabolic pathways for the direct production of fuels faces many challenges. Transgenic overexpression of an 8:0. In such a case. DIRECT BIOLOGICAL SYNTHESIS OF BIOFUELS Industrial methods for the production of biofuels using energy-rich carbon storage products. microalgae that are good lipid producers could perhaps be genetically transformed to produce alkanes. Fatty acids of even shorter chain lengths can also be used for production of gasoline and jet fuel. and alcohols.VOL. However. 155). methanol or hydrogen) and chemical processing. which increases the cost of biofuel production. hookeriana in canola has had some success in increasing the production of short chain fatty acids (32). but an approach to directly couple ethanol production to photosynthetic carbon fixation in situ may be preferred. such as sugars and lipids. With cyanobacteria. both from C. no functional decarbonylase enzyme has been cloned to date. however. and tolerant species of microalgae may have to be generated. a more recent study disputed these claims (195). the creation of a Downloaded from http://ec. Every production step that can be transferred to biological pathways will likely improve the overall economics. Instead. but it may also be possible to reduce production costs through genetically engineering microalgae to directly produce these shorter chain lengths. Short.and 10:0-biased thioesterase from C. Microalgal lipids can also be used to produce a “green” or renewable diesel through the process of hydrotreating. which resulted in the synthesis of fatty acid ethyl esters that could be used directly as biodiesel (86). Interestingly. which has the ability to produce very-long-chain alkanes (35). Many microalgae have fermentative metabolic pathways to ethanol. alkanes. However. a suggested decarbonylase enzyme involved in alkane production has been studied with the green microalga B. while strain B produces very-long-chain triterpenoid hydrocarbons (117). Algal starches have been shown to be fermentable by yeast (129). braunii differ in which long-chain hydrocarbons are synthesized. Strains of B. Triacylglycerols can be used for the production of biodiesel through the creation of fatty acid esters. Since alkanes can be derived from fatty acids. and further research is needed to clarify how alkanes are generated and to reveal the precise enzymes involved. and other longer-chain alcohols. these transformations require additional energy carriers (e. Fatty acid ester production. The potential for modification of lipid content in microalgae. butanol. 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. combined overexpression of the 8:0/10:0-biased thioesterase and a ketoacyl ACP synthase (KAS). This conversion relies on the serial transformation of fatty acids to aldehydes and then to alkanes. Ethanol. hookeriana. isopropanol.g. 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. such as lipids and/or starch (75). many types of fuel products have the potential to be toxic. The last step is thought to be catalyzed by a decarbonylase enzyme. 192). including Cer1 and Cer22. In addition. 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. 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. they slow down their proliferation and start producing energy storage products. 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. Production of shorter-chainlength alkanes that are suitable for direct use as fuel remains an existing goal. Several biological pathways have been described for the production of fatty acid esters. Decarbonylase activity has also been found in the leaves of the pea Pisum sativum (20. However.asm. braunii. such as a lack of nitrogen. 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. A possible example of long-chain-alkane (14. 164. 2010 MINIREVIEWS 491 interesting candidates for transgenic overexpression in microalgae since their activities could improve the suitability of microalga-derived diesel feedstock. and the actual mechanism for the conversion of aldehyde to alkane remains to be found. It will be interesting to see how overexpression of lipid synthesis pathway genes will affect microalgal proliferation. Although the ethyl ester yield from this manipulation was not overly impressive for E. and several possible decarbonylases that are thought to be involved in wax formation. 9. Interestingly.. thaliana (1. 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. coli. 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.org/ on October 29. with strain A producing very-long-chain dienes and trienes. increases 8 to 10 fatty acids by an additional 30 to 40% (31). The alkanes that are generated by these putative decarbonylases all have very-longchain lengths ( 22 carbons) and will require further processing for fuel production. have been found in A. Examples of inducible promoters in algae include copper-responsive elements in C. when they encounter environmental stress. Many microalgae do not produce large amounts of storage lipids during logarithmic growth. 2012 by guest .and medium-chain alkanes have the potential to be used directly as transportation fuel. It may be that increased lipid synthesis will result in a reduction of cell division. Ethanol for biofuels is currently produced from the fermentations of food starches or cellulose-derived sugars.to 22-carbon) production has been reported for the bacterium Vibrio furnissii (137). acetobutylicum has a low growth rate and is somewhat difficult to genetically engineer. coli. Alternative methods to concentrate algal biomass include centrifugation and filtration. thereby requiring the use of harsh lysis conditions. which are faster. such as those performed with E. are conserved in microalgae. 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. Feasibility of direct biological synthesis of fuels. however. Interestingly. acetobutylicum into E. the production of isobutanol through the keto acid pathways should be carefully considered for microalgal systems. In a similar fashion. 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.9 g/liter of isopropanol (67). with more than 40. in which the overexpression and deletion of entire metabolic pathways are feasible. In microalgae. Two enzymes from Bacillus subtilis that utilize IPP and DMAPP for the biosynthesis of isopentenol were overexpressed in E. such as 2-ketobutyrate. Using similar approaches. including isopentenol. 40). attempts have been made to express the production pathways for isopropanol and butanol in the more user-friendly host E. SECRETION OF TRIACYLGLYCEROL. Isoprenoids. 2012 by guest . free fatty acids. and it is therefore reasonable to believe that a similar approach to production of alcohols in algae is possible. may be attempted. Atsumi et al. several combinations of up to five genes from various species of Clostridium were overexpressed in E. 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. these methods are slow and the resulting biomass may still require further dewatering. CELL pathway for ethanol biosynthesis has been demonstrated. There are in fact several pathways in nature that lead to secretion of hydrophobic compounds. including settling and flocculation. and up to C8 alcohols have been synthesized (for a review by Connor and Liao. mobilis (34. 132). isoprenoids are synthesized via the methylerythritol (MEP) pathway using glyceraldehydes-3-phosphate and pyruvate to generate the basic building blocks of isoprenoid biosynthesis. Similar screening methods could be utilized to identify mi- Downloaded from http://ec. A further increase in production was achieved through the expression of the entire pathway from a single plasmid. With optimization. With advances in genetic transformation methods and increased knowledge regarding expression systems in microalgae. represent an incredibly diverse group of natural compounds. see references 53 and 98. Other compounds that could replace diesel and jet fuel can also be produced through isoprenoid pathways (for recent reviews by Fortman et al. see reference 28). An alternative synthetic pathway for the production of butanol utilized the endogenous keto acid pathways for amino acid synthesis. This was achieved through the simultaneous transgenic overexpression of a 2-keto-acid decarboxylase and an alcohol dehydrogenase. resulting in the production of 4. This pathway produces ethanol during photoautotrophic growth and could be incorporated into algae. the overexpression of the butanol production pathway resulted in 1-butanol production of approximately 140 mg/liter. coli. it is possible to obtain longer-chain alcohols as well. It is reasonable to believe that some of the biochemical pathways in microalgae could be leveraged for the direct production of fuels. While there are several possible low-cost solutions to concentrating the biomass. These ubiquitous pathways normally produce amino acids through 2-keto acid precursors. proved that it is possible to divert some of the 2-keto acid intermediates from amino acid production into alcohol production. Because C. Recently. 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. also known as terpenoids. alkanes.asm. many exciting advances toward the biological production of C3 up to C8 alcohols have been achieved. Mutant colonies that secrete fatty acids were thereby identified by the formation of a halo in the overlaid agar (120. most microalgae will not grow to a density higher than a few grams of biomass per liter of water. (7).492 MINIREVIEWS EUKARYOT. with the insertion of pyruvate decarboxylase and alcohol dehydrogenase from the ethanologenic bacterium Z. There are many examples of successful pathway manipulation to generate compounds that can be used as fuels. have been produced by E. coli using isoprenoid biosynthesis pathways. especially that of isobutanol. ethanol has a lower energy density than gasoline and poor storage properties. Molecules that could potentially work as gasoline substitutes. Isoprenoids. coli. Most of these come from the manipulation of E. including TAGs. resulting in production of 112 mg/liter isopentenol (107. which was produced at titers up to 22 g/liter (8). 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.2 g/liter 1-butanol (78). In addition. Isopropanol and butanol are naturally produced by bacteria of the genus Clostridium. and wax esters.. ALKANES. comprehensive modifications. resulting in production of 1. isopentyl diphosphate (IPP). 200). For isopropanol production. Considering the reported yields from the various pathways that have been utilized so far. Based on currently achievable productivities. and production has been industrialized using Clostridium acetobutylicum. yields were greatly improved by the deletion of 5 host genes that compete with the 1-butanol pathway for acetyl-CoA and NADH. but they are also typically much more expensive and energy intensive. Some of the 2-keto acid intermediates. As a fuel. Secretion of free fatty acids in yeast.org/ on October 29. six genes encoding the entire pathway for butanol production were transferred from C. coli. respectively). these enzymes are not optimized for performance in oxic conditions and may need to be configured for eukaryotic systems. coli. and dimethylallyl diphosphate (DMAPP). resulting in the production of 550 mg/liter (7). inactivation of genes involved in -oxidation has been shown to result in fatty acid secretion in some instances. FREE FATTY ACIDS.000 different molecules. and Lee et al. many microalgal species have a very tough outer cell wall that makes extraction of fuel feedstocks difficult. As mentioned in the section on lipid catabolism. coli by Atsumi et al. and transgenic expression of ABC transporters has been used to enable drug transport. While wax transporters have not been shown to export products that are derived from medium-chain fatty acids. However. and transgenic expression of ABC transporters has resulted in transport of a variety of compounds. 190). the secretion of VLDL from hepatocytes has been shown to be affected both by deletion and overexpression of a wide range of genes. The secretion of the fuel intermediates into the culture medium would provide these microorganisms with a rich source of nutrient. cerevisiae has five genes with fatty acyl-CoA synthetase activity. To improve the rates of secretion and to allow for the secretion of other types of bioenergy carriers. In addition to cellular export pathways. including import of fatty acids into mitochondria and peroxisomes for -oxidation. and fatty acids. including terpenoids and other compounds that are of interest for biofuel production. the exact genes that would be needed to enable a functional secretion pathway in another cell type are not known. There are several examples of established pathways for the secretion of lipophilic compounds. random mutagenesis resulted in the secretion of TAGs. which consist of many types of hydrocarbons. a secretion strategy may not be the best solution when a significant number of contaminating microorganisms are present in the cultivation system. results in a buildup of intracellular free fatty acids and secretion of free fatty acids. however. and sterols (82. and inactivation of the ABC transporter Ped3p or Pxa1 in A. alcohols. Downloaded from http://ec. These genes include those that encode adipophilin (ADPH). and it may be possible to reproduce this kind of secretion in microalgae. However. ABC transporters mediate the export of plant waxes consisting of a multitude of compounds that are derived from very-long-chain fatty acids. Mechanisms for secretion of lipids and related compounds. alkyl esters. which could be transferred to microalgae. the exact mechanisms are not known. several genes have been identified that are involved in milk TAG vesicle secretion. What is perhaps a more straightforward approach to enabling secretion of lipids from microalgae is the use of ABC transporters. and arylacetamide deacetylase (AADA) (62. ketones. cerevisiae in these cases are not known. it should become clear whether the transfer of these very efficient TAG-secreting pathways is feasible. or FAA1 together with acyl-CoA oxidase activity. whereas the cells started importing free fatty acids in stationary phase (162). In a similar fashion. and butyrophilin (BTN) (for a review by McManaman et al. These transporters include the A. As with VLDL and milk TAG secretion. 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 perhaps fatty acids (140). thereby reducing product yields. cholesterol. thaliana Desperado/ AtWBC11 transporter and the Cer5/AtWBC12 transporter. such as those encoding apolipoprotein E (ApoE). 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. In summary. ketones. they are also responsible for the transport of molecules that are produced through the isoprenoid synthesis pathway. but the ABC transporter systems may rely on fewer components. it would perhaps be possible to use random mutagenesis or directed evolution to generate mutant ABC transporters that have the ability to secrete short. the current knowledge of secretion pathways is still rather limited and therefore may not necessarily be easily transferred to microalgal cells.org/ on October 29. including those encoding FAA1 and FAA4. both of which have been shown to be important for exporting wax to the epidermis (136. 205). alkanes. In one of the yeast studies. Importantly. Unfortunately. Long-chain fatty acids are imported into the peroxisome for -oxidation by ABC transporters. and the ATP-binding cassette (ABC) transporter-mediated export of plant waxes. The actual mechanisms for TAG and/or free fatty acid secretion in S. These include the secretion of TAG-containing very-low-density lipid (VLDL) vesicles from hepatocytes. there are also known pathways for intracellular transport of fatty acids between organelles. research efforts should be aimed at transferring these pathways into organisms that are good lipid producers. Since there are several highly efficient pathways for lipid secretion in nature that are being explored with various model organisms. neither the genes involved nor the mechanism has been described (132). 115. It is currently possible to induce secretion of free fatty acids from yeast and cyanobacteria. 140). Even though many of the genes that are involved in secretion have been identified. the highest levels of fatty acid secretion also seemed to be associated with reduced proliferation (120). But again. 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. With further research. the exact mechanisms are generally not known. In addition. but it remains to be demonstrated at a scale significant for biofuel production from microalgal feedstocks. 99. 9. it is not known which genes would be needed to enable VLDL secretion from cell types that do not normally secrete VLDL. alcohols. and in addition to transporting compounds that are related to very-long-chain fatty acids. 2012 by guest . Combined inactivation of FAA1 and FAA4. and it may be possible to utilize such pathways for the export of lipids. This kind of secretion was found to take place mainly during logarithmic growth. thaliana there are over 120 different ABC transporters.. resulting in resistance. In A. Cer5/AtWBC12 facilitates the export of very-long-chain aldehydes. 216). 2010 MINIREVIEWS 493 croalgae that have the ability to secrete fatty acids.and medium-chain fatty acids.asm. xanthine oxidoreductase (XOR). microsomal triglyceride transfer protein (MTP). One interesting characteristic of ABC transporters is their promiscuous gating properties. including alkanes. Overexpression of these genes in hepatocytes that already have the capacity to secrete VLDL results in increased secretion.VOL. For example. triacylglycerol hydrolase (TGH). the successful transgenic expression and utilization of lipid secretion pathways to secrete molecules suitable for biofuel production remain largely to be demonstrated. including kanamycin. thaliana inhibits peroxisomal uptake of long-chain fatty acids (70. For example. S. TAG-containing vesicles from mammary glands. see reference 111). aldehydes. lipid secretion is an attractive alternative to harvesting algal biomass that could potentially lower the cost of producing microalga-derived biofuels. Several key genes are known for these pathways. A similar form of fatty acid secretion has been achieved by Synthetic Genomics with cyanobacteria (158). 189. phosphate. glucose. In green algae. lipids. Some of these branches are trimmed (WSP3).6 glycosidic linkages (10). Genetic strategies for increasing glucan storage. disproportionating enzyme (1 and 2) -1. -amylases. Much work has been done on the catalytic and allosteric properties of AGPases in crop plants to increase starch production. malto-oligosaccharides. and enzymes are shown in red. GWD. isoamylases. inorganic phosphate. A problem for cytosolic starch synthesis could be that because the AGPase is far from the pyrenoid and thus 3-PGA. 2012 by guest FIG. AGPase is typically a heterotetramer composed of large regulatory and small catalytic subunits and is allosterically activated by 3-phosphoglyceric acid (3-PGA). DBE. MEX1. Polysaccharides are often found surrounding the pyrenoid in microalgae. such as those encoded by glgC16 from E. that have successfully increased starch content in other plants should be expressed in microalgae.org/ on October 29. likely because it is a source of 3-PGA (10). ISA. PPi. The phyla Chlorophyta. Glucans are stored in microalgae in a variety of ways. SS. while it is stored in the cytoplasm in Dinophyta. coli (176) or the recombinant rev6 (63). Glaucophyta. glucan-water dikinases. resulting in ADP-glucose and pyrophosphate (176). starch phosphorylases. BE. AMY. AMY. linking starch synthesis to photosynthesis (209). ADP-glucose pyrophosphorylase. such as the C. pyrophosphate. Glc. debranching enzymes. with mixed results (174). Dinophyta. and/or methane. water-soluble granules of laminarin and chrysolaminarin are synthesized. and Rhodophyta and in the periplastidial space in Cryptophyceae (38. including ethanol. SP. Glaucophyta. MOS.494 MINIREVIEWS EUKARYOT. 3 for an overview of starch metabolism) is the ADP-glucose pyrophosphorylase (AGPase)-catalyzed reaction of glucose 1-phosphate with ATP. and Rhodophyta store glucans in linear -1.3 linkages with branching at the C-2 and C-6 positions of glucose (74). -amylase. This problem could be circumvented by .4 and branched -1. An alternative approach for increasing microalgal starch would be to introduce starch-synthesizing enzymes into the cytosol. CELL Downloaded from http://ec. AGPase. plastidial phosphoglucomutase. Glucans are added to the water soluble polysaccharide (WSP) by -1. 3. P. it may not be activated. reinhardtii sta1 or sta6 mutant. The metabolites and simplified representative pathways in microalgal starch metabolism are shown in black. GENETIC MODIFICATION OF CARBOHYDRATE METABOLISM Carbohydrates can be metabolized into a variety of biofuels. The cytosol would give more physical space for the starch granules to accumulate (174). and this process is repeated until a starch granule is formed. butanol. 39).asm.4 glycosidic linkages (WSP1) until a branching enzyme highly branches the ends (WSP2). Phosphorolytic [Starch-(P)n] and hydrolytic degradation pathways are shown. branching enzymes. Starch metabolism in green microalgae. Designer AGPases. maltose transporter. In Heterokontophyta. starch is synthesized and stored within the chloroplast. Pi. Phaeophyceae. PGM. preferably in a background with no native AGPase activity. which are made up of -1. DPE. and Bacillariophyceae. starch synthases. The ratelimiting step of starch synthesis (see Fig. H2.4 glucanotransferase. a further understanding of their regulation and mechanism could potentially be leveraged for continuous biofuel production from secreted saccharides. and the [NiFe] and [FeFe] hydrogenases are capable of the reversible reduction of protons to H2. Secretion strategies and soluble sugars. In addition to exporting soluble sugars. while disruptions in PWD result in a less severe starch excess phenotype (156). 213). A. only the [NiFe] hydrogenases have been reported for cyanobacteria. Hydrogen metabolism has been studied extensively with C. intracellular sugar accumulation is also a desirable microalgal biofuel trait. 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). thaliana. thaliana have been knocked out. Mutants that synthesize less starch or have a reduced capacity to degrade starch often have reduced growth rates (175). but when it is knocked out in Arabidopsis. This protein facilitates bidirectional diffusion and could be leveraged to export maltose out of the cell. aspartic acid. In A. Starch can be degraded by hydrolytic and/or phosphorolytic mechanisms. arabinose. but when sucrose is converted to isomaltulose. an increase in total sugar production has been accomplished by the transgenic expression of a sucrose isomerase from a bacterium. even during stationary phase (138). thaliana plastidial phosphoglucomutase mutant (corresponding to STA5 in C. In sugar cane. for extracellular excretion. 2012 by guest . The synthesis of sucrose could be exploited by sucrose transporters. These two enzyme classes are phylogenetically distinct. thaliana (175). a product of starch degradation in the chloroplast. which could result in an increase in total sugar content. In microalgae. 68. only the [FeFe] hydrogenases have been described in eukaryotic microalgae. glycerol. especially during the night. and as a result. thaliana mutants grown in a diurnal cycle that cannot synthesize or degrade starch grow more slowly than the wild type (17. and diverse fermentation metabolisms that can be leveraged in renewable bioenergy strategies are present in these organisms (64. These considerations may become issues when microalgae are grown for biofuels and are subject to diurnal light cycles. are present (46). This isomerase converts sucrose to isomaltulose. Of particular interest is the ability of many green microalgae to produce H2. A potential side effect of increased trehalose is its ability to induce starch synthesis and AGPase expression. 9. thaliana. including organic acids and alcohols. 110. These phosphorylation steps are critical for starch degradation and are excellent gene knockout targets for a starch accumulation phenotype in microalgae. where the concentration is doubled. Hydrolytic starch degradation requires an enzyme capable of hydrolyzing semicrystalline glucans at the surface of the insoluble starch granule. GWD catalyzes the transfer of -phosphate in ATP to the C-6 position of the glucans in amylopectin (156). MICROALGAL HYDROGEN PRODUCTION Many eukaryotic microalgae and cyanobacteria have evolved in ecosystems that become depleted of O2. This may imply that there is a signaling system that gives negative feedback when sucrose levels reach a certain level. 114). glutamic acid. 143) and in improvements in overall H2 yields (90. the level of sucrose is not detected. Mannitol. Hydrogenases are classified according to the metal ions at their active sites. maltose could be converted to other isoforms that may be silent to the native metabolic regulatory system. 18. and a homologous protein is found in C. 91. The C-3 position in the glucan can also be phosphorylated by the phosphoglucan water dikinases (PWD). 143). however. An A. Mutant considerations. In A. The [FeFe] hydrogenases in many green microalgae are able to effectively couple to the photo- Downloaded from http://ec. 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. and interestingly. -amylase (AMY3) is thought to participate in starch degradation. proline. 125). starch can be degraded even when all three -amylases in A. The production of soluble sugars may be preferred over polysaccharides because soluble sugars are smaller and easier to process. cerevisiae cells transformed with SUC1 and SUC2 have been shown to transport sucrose and some maltose across their plasma membranes (160). reinhardtii (61. Decreasing starch degradation in microalgae. Although little is currently known about these secretion events. a nonplant metabolite. but at continuous light its growth rates were equal (17). are also secreted by many species during anoxia (118. 72). whereas. Interestingly. Maltose. it results in a 30-fold increase of maltose in the leaves. reinhardtii (175). in addition to likely being more amenable for engineered secretion because many transporters have been described. resulting in significant advances in both our fundamental understanding of H2 metabolism in this organism (43. In addition. it should be noted that additional fermentation metabolites. and both of these phosphorylations are thought to help disrupt the crystalline structure of the starch granules to allow glucan-metabolizing enzymes access (212).VOL. The precise mechanisms of starch catabolism in green microalgae are largely unknown (10) but are more widely understood for A. Malt- ose is metabolized in the cytosol by a glucosyltransferase. which has been shown with Arabidopsis (199). allowing for higher levels of total sugar accumulation (202). S. enough to cause maltose exportation to the roots. Ankistrodesmus densus secretes polysaccharides when exposed to light. which still has activity even without the presence of an activator (14). lysine. the total sugar levels of isomaltulose and sucrose are twice as high as those of control plants. such as the Mos(1-198)/SH2 AGPase. 118. such as the sucrose synthetase and sucrose phosphate synthetase. evidence exists that some of the enzymes involved in sucrose metabolism. thaliana. such as SUC1 and SUC2 found in A. indicating alternative mechanisms for starch degradation (207).org/ on October 29. Although sucrose has largely been unexplored with microalgae. 2010 MINIREVIEWS 495 the introduction of an AGPase that does not require 3-PGA. a glucosyltransferase from a bacterium converting maltose to trehalose (131) could be expressed as a potential strategy to increase total sugar content.asm. DPE2. and various polysaccharides have been reported to be secreted (71). For example. 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). is transported to the cytosol in green microalgae and land plants by the maltose export protein (MEX1) (38). Plastidial starch degradation is stimulated by phosphorylation of glucose residues at the root of amylopectin by glucan-water dikinases (GWD). these studies have rapidly advanced our understanding of H2 metabolism and enzyme maturation (13. 141. Many genes have been identified that are important for withstanding stress conditions. 185). such as salt concentration. High light is not the only environmental variable that can cause stress and inhibit microalgal growth.org/ on October 29. However. an ATP synthase subunit that is involved in the regulation of intracellular ATP levels and stress tolerance (179). such as those encoding glutathione peroxidase and ascorbate peroxidase (168. most microalgae will not grow at maximum efficiency during most of the day. including high salt and low temperature. this intensity. 68. and light intensity. which decreases photosynthetic activity. and (iv) the ability to thrive in diverse ecosystems. Earlier research relied on random mutagenesis strategies to generate mutants with fewer or smaller chlorophyll antennae. These include (i) high photosynthetic conversion efficiencies.000 mol photons m 2 s 1 during midday (113). High-light stress. first. as an effective means of sustaining H2 photoproduction when respiration is able to consume all of the photosynthetic O2 produced by the cells (114). and second. it should become clear whether the current approach can be successfully applied to increase biomass production. However. 2012 by guest . W80) were demonstrated through transgenic overexpression in several different systems. Depending on the design of the cultivation facilities. they also did not observe any improved productivity in laboratory cultures. but a recent publication efficiently used an RNAi-based strategy to knock down both LHCI and LHCII in C. Light intensities above the maximum photosynthetic efficiency actually reduce the growth rate. 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. Therefore.asm. 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. it was demonstrated that alginate-immobilized cultures of nutrient-deprived C. Consequently. pH. 60. 145) in green microalgae. it is possible to control these factors to a certain degree through engineering and manipulation of the growth environment. as well as on the ability of microalgae to rapidly produce large amounts of biomass. One important consideration is the intensity of light at which a certain strain of microalga reaches its maximum growth rate. (ii) rapid biomass production rates. 182. (iii) the capacity to produce a wide variety of biofuel feedstocks. 188). It seems clear that manipulation of light-harvesting complexes can lead to increased biomass productivity under high light in controlled laboratory conditions. earlier studies concluded that the economics of microalgal Downloaded from http://ec. Other stress factors. such as mannitol and ononitol (166. and H2 photoproduction is only transiently observed prior to the accumulation of O2 to inhibitory levels under nutrient-replete conditions (27. 142. including tobacco and E. no improvement in productivity was observed with outdoor ponds (77). it would be of great benefit to develop genetic strategies to increase the cellular tolerance to a variety of stress factors. CONCLUSION Microalgae are an extremely diverse group of organisms. and interestingly. 204) as well as enzymes that catalyze the production of osmolytes. 112). These include genes that are directly involved in scavenging reactive oxygen. it permits higher light penetration in high-density cultures. Genetic techniques have been applied with the aim of increasing H2 photoproduction activity by decreasing light-harvesting antenna size. but these manipulations add to the cost of growing microalgae. 178). inhibiting state transitions. reinhardtii (126). and numerous strategies are emerging to further advance our ability to optimize H2 production in eukaryotic phototrophs. Photosynthetically active radiation intensities from sunlight can exceed 2. temperature. many of which possess novel metabolic features that can be exploited for the production of renewable biofuels. CELL synthetic electron transport chain at the level of ferredoxin. providing the means to generate H2 directly from water oxidation. An additional benefit of increasing stress tolerance is the possibility of growing select microalgae under extreme conditions that limit the proliferation of invasive species. Experiments with small. In combination with physiological and biochemical approaches. In 2000. and hydrogenase engineering (11. 126–128. such as lipids and starch. coli. Microalgae are considered great model organisms to study photosynthetic efficiency. However. all microalgal [FeFe] hydrogenases characterized to date are particularly O2 sensitive. is usually around 200 to 400 mol photons m 2 s 1 for most species. 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. and other stimuli can also cause stress to microalgal cultures. which resulted in increased resistance to several different stressful stimuli. reinhardtii could sustain H2 photoproduction even in the presence of an oxygenated atmosphere (90). Melis and coworkers described the use of sulfur deprivation (203). These include stress factors. 167. In one study of algal antenna mutants. but this strategy may have two positive effects. which corresponds to the maximum photosynthetic efficiency. see reference 113). a phenomenon known as photoinhibition. pH. Salt. and several attempts have been made to improve the photosynthetic efficiency and/or reduce the effects of photoinhibition on microalgal growth.496 MINIREVIEWS EUKARYOT. Recently. The antistress properties of these genes isolated from bacteria as well as a marine stresstolerant microalga (Chlamydomonas sp. temperature. It is likely that similar improvements can be achieved with microalgae. 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. This strategy can most likely be applied to many different microalgae more easily than a random mutagenesis approach. 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