**4. Genetic engineering of microalgae**

#### **4.1 Microalgal lipid biosynthesis**

Although lipid metabolism, in particular the biosynthesis of fatty acids and TAG, is poorly understood in algae, it is generally recognized that the basic pathways for fatty acid and TAG biosynthesis are similar to those demonstrated in higher plants.

Algae synthesize the *de novo* fatty acids in the chloroplast using a single set of enzymes. A simplified schedule for saturated fatty acid biosynthesis is shown in Figure 7. Acetyl-CoA is the basic building block of the acyl chain and serves as a substrate for acetyl CoA

Fig. 7. Simplified overview of saturated fatty acid biosynthesis in algal chloroplast. ACCase, acetyl-CoA carboxylase; ACP, acyl carrier protein; CoA, coenzyme A; ENR, enoyl-ACP reductase; HD, 3-hydroxyacyl-ACP dehydratase; KAR, 3-ketoacyl-ACP reductase; KAS, 3-ketoacyl-ACP synthase; MAT, malonyl-CoA:ACP transacylase.

Genetic engineering is a feasible and complimentary approach to increase algal productivity and improve the economics of algal biodiesel production. This has long been recognized but it seems that so far little progress has been made. The lack of full or near-full genome sequences and robust transformation systems makes genetic engineering of algae lag much behind that of bacteria, fungi and higher eukaryotes. Although certain algal species have been reported for efficient transformation, it proves to be difficult to produce stable transformants of algae. Currently, sophisticated genetic engineering whereby several genes are concurrently down-regulated or overexpressed is only really applicable to the green alga *Chlamydomonas reinhardtii*. This situation, however, is likely to change because of the growing scientific and commercial interest in other algal species that are of great potential

Understanding the algal lipid biosynthesis is of great help to engineer algal lipid production. Although lipid metabolism in algae is not as fully understood as that in higher plants, they have similar lipid biosynthetic pathway as mentioned above. Theoretically, overexpression of the genes involved in fatty acid synthesis is able to increase lipid accumulation, in that fatty acids required as precursors for lipid biosynthesis are produced in excess. However, overexpressoin of the native ACCase, the rate-limiting enzyme catalyzing the first committed step of fatty acid biosynthesis in many organisms, could not increase the lipid production in diatom (Dunahay et al., 1995). It is possible that under high flux conditions through ACCase, the condensing enzymes or other factors may begin to limit fatty acid synthesis rate. Therefore, more complete control may come from certain transcription factors that can increase expression of the entire pathway. Another feasible approach of increasing cellular lipid contents is to inhibit metabolic pathways that lead to other carbon storage compounds, such as starch. Starch synthesis shares common carbon precursors with lipid synthesis in algae. Blocking starch synthesis is able to redirect carbon flux to lipid biosynthetic pathway, resulting in overproduction of fatty acids and thus total lipids (Li et al., 2010a). Neutral lipids in particular TAG surpass other lipids for biodiesel production, attracting the interest of enhancing cellular TAG contents through genetic engineering. Overexpression of genes involved in TAG assembly, e.g., glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase, or diacylglycerol acyltransferase, all significantly increase TAG production in plants. Such strategies may also be applicable to algae for enhancing TAG levels. Commonly, algae produce larger amounts of lipids under unfavorable conditions than logarithmic growing condition. Enhancing lipid biosynthesis through genetic engineering, therefore, is likely to reduce algal proliferation and biomass production. In this context, the use of inducible promoters could overcome the problem because the transgenic expression can only be activated when a high cell density is achieved. The important properties of biodiesel such as cetane number, viscosity, cold flow, oxidative stability, are largely determined by the composition and structure of fatty acid esters which in turn are determined by the characteristics of fatty acids of biodiesel feedstocks, for example carbon chain length and unsaturation degree (Knothe, 2005b). Thus, the genetic modification of algal fatty acid composition is of also great interest. Generally, saturated fatty esters possess high cetane number and superior oxidative stability; whereas unsaturated, especially polyunsaturated fatty esters have improved low-temperature properties (Knothe, 2008). In this regard, it is suggested that the modification of fatty esters, for example the enhanced proportion of oleic acid (C18:1) ester, can provide a compromise solution between oxidative stability and low-temperature properties and therefore promote

**4.2 Genetic engineering of microalgal lipids** 

for industrial applications.

carboxylation and as well as a substrate for the initial condensation reaction. The formation of malonyl CoA from acetyl CoA is generally regarded as the first reaction of fatty acid biosynthesis, which is catalyzed by acetyl CoA carboxylase (ACCase). The malonyl group of malonyl CoA is transferred to a protein co-factor, acyl carrier protein (ACP), resulting in the formation of malonyl ACP that enters into a series of condensation reactions with acyl ACP (or acetyl CoA) acceptors. The first condensation reaction is catalyzed by 3-ketoayl ACP synthase III (KAS III), forming a four-carbon product. KAS I and KAS II catalyze the subsequent condensations. After each condensation, the 3-ketoacyl-ACP product is reduced, dehydrated, and reduced again, by 3-ketoacyl-ACP reductase, 3-hydroxyacyl-ACP dehydratase, and enoyl-ACP reductase, respectively, to form a saturated fatty acid. To produce an unsaturated fatty acid, a double bond is introduced onto the acyl chain by the soluble enzyme stearoyl ACP desaturase (SAD). Unlike plants, some algae produce longchain acyl ACPs (C20-C22) that derive from the further elongation and/or desaturation of C18. The fatty acid elongation is terminated when the acyl group is released from ACP by an acyl-ACP thioesterase that hydrolyzes the acyl ACP and produces free fatty acids or by acyltransferases that transfer the fatty acid from ACP to glycerol-3-phosphate or monoacylglycerol-3-phosphate. These released fatty acids serve as precursors for the synthesis of cellular membranes and neutral storage lipids like TAG.

It has been proposed that the biosynthesis of TAG occurs in cytosol via the direct glycerol pathway (Figure 8). Generally, acyl-CoAs sequentially react with the hydroxyl groups in glycerol-3-phosphate to form phosphatidic acid via lysophosphatidic acid. These two reactions are catalyzed by glycerol-3-phospate acyl transferase and lysophosphatidic acid acyl transferase respectively. Dephosphorylation of phosphatidic acid results in the release of DAG which accepts a third acyl from CoA to form TAG. This final step is catalyzed by diacylglycerol acyltransferase, an enzymatic reaction that is unique to TAG synthesis. In addition, an alternative pathway that is independent of acyl-CoA may also be present in algae for TAG biosynthesis (Dahlqvist et al., 2000). This pathway employs phospholipids as acyl donors and diacylglycerols as the acceptors and might be activated when algal cells are exposed to stress conditions, under which algae usually undergo rapid degradation of the photosynthetic membranes and concurrent accumulation of cytosolic TAG-enriched lipid bodies (Hu et al., 2008b).

Fig. 8. Simplified illustration of the TAG biosynthesis in algae. DAG, diacylglycerol; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; G-3-P, glycerol-3-phosphate; PA, phosphatidic acid; PC, phosphatidylcholine; TAG, triacylglycerol. (1) glycerol-3-phosphate acyl transferase, (2) lysophosphatidic acid acyl transferase, (3) phosphatidic acid phosphatase, (4) diacylglycerol acyl transferase, and (5) phospholipid:diacylglycerol acyltransferase.

carboxylation and as well as a substrate for the initial condensation reaction. The formation of malonyl CoA from acetyl CoA is generally regarded as the first reaction of fatty acid biosynthesis, which is catalyzed by acetyl CoA carboxylase (ACCase). The malonyl group of malonyl CoA is transferred to a protein co-factor, acyl carrier protein (ACP), resulting in the formation of malonyl ACP that enters into a series of condensation reactions with acyl ACP (or acetyl CoA) acceptors. The first condensation reaction is catalyzed by 3-ketoayl ACP synthase III (KAS III), forming a four-carbon product. KAS I and KAS II catalyze the subsequent condensations. After each condensation, the 3-ketoacyl-ACP product is reduced, dehydrated, and reduced again, by 3-ketoacyl-ACP reductase, 3-hydroxyacyl-ACP dehydratase, and enoyl-ACP reductase, respectively, to form a saturated fatty acid. To produce an unsaturated fatty acid, a double bond is introduced onto the acyl chain by the soluble enzyme stearoyl ACP desaturase (SAD). Unlike plants, some algae produce longchain acyl ACPs (C20-C22) that derive from the further elongation and/or desaturation of C18. The fatty acid elongation is terminated when the acyl group is released from ACP by an acyl-ACP thioesterase that hydrolyzes the acyl ACP and produces free fatty acids or by acyltransferases that transfer the fatty acid from ACP to glycerol-3-phosphate or monoacylglycerol-3-phosphate. These released fatty acids serve as precursors for the

It has been proposed that the biosynthesis of TAG occurs in cytosol via the direct glycerol pathway (Figure 8). Generally, acyl-CoAs sequentially react with the hydroxyl groups in glycerol-3-phosphate to form phosphatidic acid via lysophosphatidic acid. These two reactions are catalyzed by glycerol-3-phospate acyl transferase and lysophosphatidic acid acyl transferase respectively. Dephosphorylation of phosphatidic acid results in the release of DAG which accepts a third acyl from CoA to form TAG. This final step is catalyzed by diacylglycerol acyltransferase, an enzymatic reaction that is unique to TAG synthesis. In addition, an alternative pathway that is independent of acyl-CoA may also be present in algae for TAG biosynthesis (Dahlqvist et al., 2000). This pathway employs phospholipids as acyl donors and diacylglycerols as the acceptors and might be activated when algal cells are exposed to stress conditions, under which algae usually undergo rapid degradation of the photosynthetic membranes and concurrent accumulation of cytosolic TAG-enriched lipid

Fig. 8. Simplified illustration of the TAG biosynthesis in algae. DAG, diacylglycerol; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; G-3-P, glycerol-3-phosphate; PA, phosphatidic acid; PC, phosphatidylcholine; TAG, triacylglycerol. (1) glycerol-3-phosphate

acyl transferase, (2) lysophosphatidic acid acyl transferase, (3) phosphatidic acid phosphatase, (4) diacylglycerol acyl transferase, and (5) phospholipid:diacylglycerol

synthesis of cellular membranes and neutral storage lipids like TAG.

bodies (Hu et al., 2008b).

acyltransferase.

#### **4.2 Genetic engineering of microalgal lipids**

Genetic engineering is a feasible and complimentary approach to increase algal productivity and improve the economics of algal biodiesel production. This has long been recognized but it seems that so far little progress has been made. The lack of full or near-full genome sequences and robust transformation systems makes genetic engineering of algae lag much behind that of bacteria, fungi and higher eukaryotes. Although certain algal species have been reported for efficient transformation, it proves to be difficult to produce stable transformants of algae. Currently, sophisticated genetic engineering whereby several genes are concurrently down-regulated or overexpressed is only really applicable to the green alga *Chlamydomonas reinhardtii*. This situation, however, is likely to change because of the growing scientific and commercial interest in other algal species that are of great potential for industrial applications.

Understanding the algal lipid biosynthesis is of great help to engineer algal lipid production. Although lipid metabolism in algae is not as fully understood as that in higher plants, they have similar lipid biosynthetic pathway as mentioned above. Theoretically, overexpression of the genes involved in fatty acid synthesis is able to increase lipid accumulation, in that fatty acids required as precursors for lipid biosynthesis are produced in excess. However, overexpressoin of the native ACCase, the rate-limiting enzyme catalyzing the first committed step of fatty acid biosynthesis in many organisms, could not increase the lipid production in diatom (Dunahay et al., 1995). It is possible that under high flux conditions through ACCase, the condensing enzymes or other factors may begin to limit fatty acid synthesis rate. Therefore, more complete control may come from certain transcription factors that can increase expression of the entire pathway. Another feasible approach of increasing cellular lipid contents is to inhibit metabolic pathways that lead to other carbon storage compounds, such as starch. Starch synthesis shares common carbon precursors with lipid synthesis in algae. Blocking starch synthesis is able to redirect carbon flux to lipid biosynthetic pathway, resulting in overproduction of fatty acids and thus total lipids (Li et al., 2010a). Neutral lipids in particular TAG surpass other lipids for biodiesel production, attracting the interest of enhancing cellular TAG contents through genetic engineering. Overexpression of genes involved in TAG assembly, e.g., glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase, or diacylglycerol acyltransferase, all significantly increase TAG production in plants. Such strategies may also be applicable to algae for enhancing TAG levels. Commonly, algae produce larger amounts of lipids under unfavorable conditions than logarithmic growing condition. Enhancing lipid biosynthesis through genetic engineering, therefore, is likely to reduce algal proliferation and biomass production. In this context, the use of inducible promoters could overcome the problem because the transgenic expression can only be activated when a high cell density is achieved. The important properties of biodiesel such as cetane number, viscosity, cold flow, oxidative stability, are largely determined by the composition and structure of fatty acid esters which in turn are determined by the characteristics of fatty acids of biodiesel feedstocks, for example carbon chain length and unsaturation degree (Knothe, 2005b). Thus, the genetic modification of algal fatty acid composition is of also great interest. Generally, saturated fatty esters possess high cetane number and superior oxidative stability; whereas unsaturated, especially polyunsaturated fatty esters have improved low-temperature properties (Knothe, 2008). In this regard, it is suggested that the modification of fatty esters, for example the enhanced proportion of oleic acid (C18:1) ester, can provide a compromise solution between oxidative stability and low-temperature properties and therefore promote

Abou-Shanab, R.A.I.; Hwang, J.-H.; Cho, Y.; Min, B.& Jeon, B.-H. (2011). Characterization of

Afify, A.; Shalaby, E.A.& Shanab, S.M.M. (2010). Enhancement of biodiesel production from

Ahmad, A.L.; Yasin, N.H.M.; Derek, C.J.C.& Lim, J.K. (2011). Microalgae as a sustainable

Araujo, G.S.; Matos, L.J.B.L.; Gonçalves, L.R.B.; Fernandes, F.A.N.& Farias, W.R.L. (2011).

Banerjee, A.& Chakraborty, R. (2009). Parametric sensitivity in transesterification of waste

Bigogno, C.; Khozin-Goldberg, I.; Boussiba, S.; Vonshak, A.& Cohen, Z. (2002). Lipid and

Boussiba, S.; Vonshak, A.; Cohen, Z.; Avissar, Y.& Richmond, A. (1987). Lipid and biomass production by the halotolerant microalga *Nannochloropsis salina*. *Biomass*, 12, 37-47. Brennan, L.& Owende, P. (2010). Biofuels from microalgae--A review of technologies for

Brown, M.R.; Dunstan, G.A.; Norwood, S.J.& Miller, K.A. (1996). Effects of harvest stage and

Canakci, M. (2007). The potential of restaurant waste lipids as biodiesel feedstocks.

Chaiklahan, R.; Chirasuwan, N.; Loha, V.& Bunnag, B. (2008). Lipid and fatty acids extraction from the cyanobacterium *Spirulina*. *Scienceasia*, 34, 299-305. Checkbiotech (2009) Massive increase in global biofuel production. Available from: http://bioenergy.checkbiotech.org/news/massive\_increase\_global\_biofuel\_produ

Chen, C.-Y.; Yeh, K.-L.; Aisyah, R.; Lee, D.-J.& Chang, J.-S. (2011). Cultivation,

Chen, F.& Johns, M. (1991). Effect of C/N ratio and aeration on the fatty acid composition of heterotrophic *Chlorella sorokiniana*. *Journal of Applied Phycology*, 3, 203-209. Chen, G.-Q.; Jiang, Y.& Chen, F. (2008). Salt-induced alterations in lipid composition of diatom *Nitzshia laevis* (Bacillariophyceae). *Journal of Phycology*, 44, 1309-1314. Cheng, Y.; Zhou, W.; Gao, C.; Lan, K.; Gao, Y.& Wu, Q. (2009). Biodiesel production from

photobioreactor design and harvesting of microalgae for biodiesel production: A

Jerusalem artichoke (*Helianthus Tuberosus L*.) tuber by heterotrophic microalgae *Chlorella protothecoides*. *Journal of Chemical Technology & Biotechnology*, 84, 777-781. Chinnasamy, S.; Bhatnagar, A.; Hunt, R.W.& Das, K.C. (2010). Microalgae cultivation in a

wastewater dominated by carpet mill effluents for biofuel applications. *Bioresource* 

plant source of arachidonic acid. *Phytochemistry*, 60, 497-503.

different species of algae. *Grasas Y Aceites*, 61, 416-422.

*Reviews*, 15, 584-593.

*Recycling*, 53, 490-497.

*of Phycology*, 32, 64-73.

*Technology*, 101, 3097-3105.

ction.

10.1016/j.biortech.2011.01.089.

*Sustainable Energy Reviews*, 14, 557-577.

*Bioresource Technology*, 98, 183-190.

critical review. *Bioresource Technology*, 102, 71-81.

microalgal species isolated from fresh water bodies as a potential source for biodiesel production. *Applied Energy*, In Press, DOI: 10.1016/j.apenergy.2011.01.060.

energy source for biodiesel production: A review. *Renewable and Sustainable Energy* 

Bioprospecting for oil producing microalgal strains: Evaluation of oil and biomass production for ten microalgal strains. *Bioresource Technology*, In Press, DOI:

cooking oil for biodiesel production--A review. *Resources, Conservation and* 

fatty acid composition of the green oleaginous alga *Parietochloris incisa*, the richest

production, processing, and extractions of biofuels and co-products. *Renewable and* 

light on the biochemical composition of the diatom *Thalassiosira pseudonana*. *Journal* 

the quality of biodiesel (Knothe, 2008, 2009). Oleic acid is converted to linoleic acid (C18:2) in a single desaturation step, catalyzed by a Δ12 desaturase enzyme encoded by the *FAD2* gene. Inactivation of this desaturation step can greatly increase the proportion of oleic acid in soybean and may represent a possible strategy for elevated accumulation of oleic acid in algae.

Genetic engineering can also be used potentially to improve tolerance of algae to stress factors such as temperature, salinity and pH. These improved attributes will allow for the cost reduction in algal biomass production and be beneficial for growing selected algae under extreme conditions that limit the proliferation of invasive species. Photoinhibition is another technical challenge to be addressed by genetic engineering. When the light intensities exceed the value for maximum photosynthetic efficiency, algae show photoinhibition, a common phenomenon for phototrophy under which the growth rate slows down. Engineered algae with a higher threshold of light inhibition will significantly improve the economics of biodiesel production.

Engineering algae for biodiesel production is currently still in its infancy. Significant advances have only been achieved in the genetic manipulation of some model algae. It is likely that many of these advances can be extended to industrially important algal species in the future, making it possible to use modified algae as cell factories for commercial biodiesel production. Nevertheless, many challenges yet remain open and should be addressed before profitable algal biodiesel become possible.
