利用基因工程技术提高微藻油脂含量的研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2015.04.010

摘要/Abstract

摘要： 利用微藻油脂制备生物柴油因具有重要的战略意义而受到世界各国的重视，成为近年来的研究热点。利用微藻制备生物柴油具有生长周期短、易于大规模培养、能大量吸收CO₂及不占用耕地等优点。但是，由于对藻类油脂合成代谢中的调节机制了解不多，导致微藻基因组研究相对滞后，极大地限制了微藻生物能源的大规模开发和利用。随着现代生物技术的发展，通过基因工程、代谢工程等方法调控微藻脂类的合成代谢，提高藻类含油量和生物量已成为可能。概述了微藻中油脂的合成代谢，归纳总结利用基因工程技术提高微藻油脂含量的研究进展，为获得含油量高的工程微藻及微藻制备生物柴油提供技术储备。

关键词: 微藻, 基因工程, 生物柴油, 油脂

Abstract: In recent years, microalgae oil biodiesel has become a hot spot because of its strategic importance. Microalgae are a promising feedstock for biodiesel due to their short growth cycle, easy to mass culture, ability to absorb CO₂ and no taking farmland, etc. However, large-scale development and utilization of microalgae biomass energy is limited by less knowledge about metabolic mechanisms of lipid synthesis and lagging research of genome in microalgae. With the development of modern biotechnology, improving lipid content and biomass of microalgae may achieve through genetic engineering and metabolic engineering methods. This review describes recent efforts to metabolic mechanisms on lipid biosynthesis and genetic engineering techniques on increasing lipid content, which provide technical reserves for attaining high lipid transgenic microalgae and producing microalgae biodiesel.

Key words: microalgae, genetic engineering, biodiesel, lipid

李逸, 王潮岗胡章立. 利用基因工程技术提高微藻油脂含量的研究进展[J]. 生物技术通报, 2015, 31(3): 70-81.

Li Yi, Wang Chaogang, Hu Zhangli. Research Advances of Genetic Engineering of Microalgae for Improving Lipid Production[J]. Biotechnology Bulletin, 2015, 31(3): 70-81.

参考文献

[1] Radakovits R, Jinkerson RE, Darzins A, et al. Genetic engineering of algae for enhanced biofuel production[J]. Eukaryotic Cell, 2010, 9(4)：486-501.
[2] Zhang FY, Yang MF, Xu YN. Silencing of DGAT1 in tobacco causes a reduction in seed oil content[J]. Plant Science, 2005, 169(4)：689-694.
[3] 陈锦清, 郎春秀, 胡张华, 等. 反义PEP基因调控油菜籽粒蛋白质/油脂含量比率的研究[J]. 农业生物技术学报, 1999, 7(4)：316-320.
[4] 姚茹, 程丽华, 徐新华, 等. 微藻的高油脂化技术研究进展[J]. 化学进展, 2010, 22(6)：1221-1232.
[5] 夏金兰, 万民熙, 王润民, 等. 微藻生物柴油的现状与进展[J]. 中国生物工程杂志, 2009, 29(7)：118-126.
[6] Merchant SS, Prochnik SE, Vallon O, et al. The Chlamydomonas genome reveals the evolution of key animal and plant functions[J]. Science, 2007, 318(5848)：245-250.
[7] 王金娜, 严小军, 周成旭, 等. 产油微藻的筛选及中性脂动态积累过程的检测[J]. 生物物理学报, 2010, 26(6)：472-480.
[8] 朱顺妮, 王忠铭, 尚常花, 等. 微藻脂肪合成与代谢调控[J]. 化学进展, 2011, 23(10)：2169-2176.
[9] 李兴军, 林文亚. 利用遗传工程提高油料作物含油量的研究进展[J]. 粮食科技与经济, 2010, 35(6)：33-36.
[10] Hu Q, Sommerfeld M, Jarvis E, et al. Microalgal triacylglycerols as feedstocks for biofuel production：perspectives and advances[J]. The Plant Journal, 2008, 54：621-639.
[11] 赫冬梅, 段舜山. 代谢调控在微藻油脂累积中的作用[J]. 生态科学, 2009, 28(1)：85-89.
[12] Georgianna DR, Mayfield SP. Exploiting diversity and synthetic biology for the production of algal biofuels[J]. Nature, 2012, 488：329-330.
[13] Sheehan J, Dunahay T, Benemann J, et al. A look back at the US Department of Energy’s aquatic species program：biodiesel from algae[M]. Colorado：National Renewable Energy Laboratory, 1998：328.
[14] Michinaka Y, Shimauchi T, Aki T, et al. Extracellular secretion of free fatty acids by disruption of a fatty acyl-CoA synthetase gene in Saccharomyces cerevisiae[J]. Journal of Bioscience and Bioengineering, 2003, 95(5)：435-440.
[15] 于水燕, 赵权宇, 史吉平. 固碳产油微藻的基因工程改造[J]. 中国生物工程杂志, 2012, 32(12)：117-124.
[16] Sinetova MA, Kupriyanova EV, Markelova AG, et al. Identification and functional role of the carbonic anhydrase Cah3 in thylakoid membranes of pyrenoid of Chlamydomonas reinhardtii[J]. Biochimica et Biophysica Acta, 2012, 1817：1248-1255.
[17] Fulda M, Schnurr J, Abbadi A, et al. Peroxisomal acyl-CoA synthetase activity is essential for seedling development in Arabidopsis thaliana[J]. The Plant Cell, 2004, 16(2)：394-405.
[18] Germain V, Rylott EL, Larson TR, et al. Requirement for 3-ketoacyl-CoA thiolase-2 in peroxisome development, fatty acid β-oxidation and breakdown of triacylglycerol in lipid bodies of Arabidopsis seedlings[J]. The Plant Journal, 2001, 28(1)：1-12.
[19] Rylott EL, Rogers CA, Gilday AD, et al. Arabidopsis mutants in short- and medium-chain acyl-CoA oxidase activities accumulate acyl-CoAs and reveal that fatty acid β-oxidation is essential for embryo development[J]. The Journal of Biological Chemistry, 2003, 278：21370-21377.
[20] Shi S, Chen Y, Siewers V, et al. Improving production of malonyl coenzyme A-derived metabolites by abolishing Snf1-dependent regulation of Acc1[J]. MBio, 2014, 5：1-8.
[21] Davis MS, Solbiati J, Cronan JE. Overproduction of acetyl-CoA carboxylase activity increases the rate of fatty acid biosynthesis in Escherichia coli[J]. Journal of Biological Chemistry, 2000, 275：28593-28598.
[22] Dunahay TG, Jarvis EE, Roessler PG. Genetic transformation of the diatoms Cyclotella cryptica and Navicula saprophila[J]. Journal of Phycology, 1995, 31(6)：1004-1012.
[23] Dehesh K, Tai H, Edwards P, et al. Overexpression of 3-ketoacyl-acyl-carrier protein synthase IIIs in plants reduces the rate of lipid synthesis[J]. Plant Physiology, 2001, 125：1103-1114.
[24] Griffiths MJ, Harrison STL. Lipid productivity as a key character-istic for choosing algal species for biodiesel production[J]. Journal of Applied Phycology, 2009, 21：493-507.
[25] Mutanda T, Ramesh D, Karthikeyan S, et al. Bioprospecting for hyper-lipid producing microalgal strains for sustainable biofuel production[J]. Bioresource Technology, 2011, 102：57-70.
[26] Larkum AWD, Ross IL, Kruse O, et al. Selection, breeding and engineering of microalgae for bioenergy and biofuel production[J]. Trends in Biotechnology, 2011, 30：198-205.
[27] 冯国栋, 程丽华, 徐新华, 等. 微藻高油脂化基因工程研究策略[J]. 化学进展, 2012, 24：1413-1426.
[28] Kim JW. Topical prostaglandin analogue drugs inhibit adipocyte differentiation[J]. Korean Journal of Ophthalmology, 2014, 28：257-264.
[29] Yi B, Wang J, Wang S, et al. Overexpression of Banna mini-pig inbred line fatty acid binding protein 3 promotes adipogenesis in 3T3-L1 preadipocytes[J]. Cell Biology International, 2014, 38：918-923.
[30] Vigeolas H, Waldeck P, Zank T, et al. Increasing seed oil content in oil-seed rape(Brassica napus L. )by over-expression of a yeast glycerol-3-phosphate dehydrogenase under the control of a seed-specific promoter[J]. Plant Biotechnology Journal, 2007, 5：431-441.
[31] Misra N, Panda PK. In search of actionable targets for agrigenomics and microalgal biofuel production：sequence-structural diversity studies on algal and higher plants with a focus on GPAT protein[J]. OMICS：A Journal of Integrative Biology, 2013, 17：173-186.
[32] Jain RK, Coffey M, Lai K, et al. Enhancement of seed oil content by expression of glycerol-3-phosphate acyltransferase genes[J]. Biochemical Society Transactions, 2000, 28：958-961.
[33] Zou J, Katavic V, Giblin EM, et al. Modification of seed oil content and acyl composition in the brassicaceae by expression of a yeast sn-2 acyltransferase gene[J]. The Plant Cell, 1997, 9：909-923.
[34] Knutzon DS, Hayes TR, Wyrick A, et al. Lysophosphatidic acid acyltransferase from coconut endosperm mediates the insertion of laurate at the sn-2 position of triacylglycerols in lauric rapeseed oil and can increase total laurate levels[J]. Plant Physiology, 1999, 120：739-746.
[35] Lv H, Qu G, Qi X, et al. Transcriptome analysis of Chlamydomonas reinhardtii during the process of lipid accumulation[J]. Genomics, 2013, 101：229-237.
[36] Pingitore P, Pirazzi C, Mancina RM, et al. Recombinant PNPLA3 protein shows triglyceride hydrolase activity and its I148M mutation results in loss of function[J]. Biochimica et Biophysica Acta, 2014, 1841：574-580.
[37] Eto M, Shindou H, Shimizu T. A novel lysophosphatidic acid acyl-transferase enzyme(LPAAT4)with a possible role for incorpora-ting docosahexaenoic acid into brain glycerophospholipids[J]. Biochemical and Biophysical Research Communications, 2014, 443：718-724.
[38] Lardizabal K, Effertz R, Levering C, et al. Expression of Umbelopsis ramanniana DGAT2A in seed increases oil in soybean[J]. Plant Physiology, 2008, 148：89-96.
[39] Zheng P, Allen WB, Roesler K, et al. A phenylalanine in DGAT is a key determinant of oil content and composition in maize[J]. Nature Genetics, 2008, 40：367-372.
[40] Bouvier-Navé P, Benveniste P, Oelkers P, et al. Expression in yeast and tobacco of plant cDNAs encoding acyl CoA：diacylglycerol acyltransferase[J]. European Journal of Biochemistry, 2000, 267：85-96.
[41] Jako C, Kumar A, Wei Y, et al. Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight[J]. Plant Physiology, 2001, 126：861-874.
[42] Deng X, Li Y, Fei X. The mRNA abundance of pepc2 gene is negatively correlated with oil content in Chlamydomonas reinhardtii[J]. Biomass and Bioenergy, 2011, 35(5)：1811-1817.
[43] Deng X, Cai J, Li Y, et al. Expression and knockdown of the PEPC1 gene affect carbon flux in the biosynthesis of triacylglycerols by the green alga Chlamydomonas reinhardtii[J]. Biotechnology Letters, 2014, 108：56-67.
[44] De Riso V, Raniello R, Maumus F, et al. Gene silencing in the marine diatom Phaeodactylum tricornutum[J]. Nucleic Acids Research, 2009, 57：66-78.
[45] Molnar A, Bassett A, Thuenemann E, et al. Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii[J]. The Plant Journal, 2009, 58(1)：165-174.
[46] Tong J, Zhang GM, Wang XF, et al. Cloning of citrate synthase gene in rapeseed(Brassica napus L. )and its expression under stresses[J]. Acta Agronomica Sinica, 2009, 35：33-40.
[47] Hu LH, Wu HM, Zhou ZM, et al. Introduction of citrate synthase gene(CS)into an elite indica rice restorer line Minghui 86 by agrobacterium -mediated method[J]. Molecular Plant Breeding, 2006, 4：160-166.
[48] Barone P, Rosellini D, LaFayette P, et al. Bacterial citrate synthase expression and soil aluminum tolerance in transgenic alfalfa[J]. Plant Cell Reports, 2008, 27(5)：893-901.
[49] Chi GH, Zhou XL, Li MY, et al. Cloning and bioinformatics analysis of MaGCS encoding a homolog citrate synthase from banana[J]. Chinese Journal of Tropical Agriculture, 2009, 29：12-18.
[50] Zhang XM, Du LQ, Sun GM, et al. Changes in organic acid concentrations and the relative enzyme activities during the development of Cayenne pineapple fruit[J]. Journal of Fruit Science, 2007, 24：381-384.
[51] Taylor BF. Fine control of citrate synthase activity in blue-green algae[J]. Arch Mikrobiol, 1973, 92(3)：245-249.
[52] Deng X, Cai J, Fei X. Effect of the expression and knockdown of citrate synthase gene on carbon flux during triacylglycerol biosynthesis by green algae Chlamydomonas reinhardtii[J]. BMC Biochemistry, 2013, 14：38-49.
[53] Zhao T, Wang W, Bai X, et al. Gene silencing by artificial microRNAs in Chlamydomonas[J]. The Plant Journal, 2009, 58(1)：157-164.
[54] Nojima Y, Kibayashi A, Matsuzaki H, et al. Isolation and characterization of triacylglycerol-secreting mutant strain from yeast, Saccharomyces cerevisiae[J]. The Journal of General and Applied Microbiology, 1999, 45：1-6.
[55] Scharnewski M, Pongdontri P, Mora G, et al. Mutants of Saccharomyces cerevisiae deficient in acyl-CoA synthetases secrete fatty acids due to interrupted fatty acid recycling[J]. FEBS Journal, 2008, 275(11)：2765-2778.
[56] Ramazanov A, Ramazanov Z. Isolation and characterization of a starchless mutant of Chlorella pyrenoidosa STL-PI with a high growth rate, and high protein and polyunsaturated fatty acid content[J]. Phycological Research, 2006, 54(4)：255-259.
[57] 刘飞飞, 李秀波, 方仙桃, 等. 三角褐指藻产油突变株的筛选[J]. 水生生物学报, 2013, 37(4)：799-802.
[58] Roessler PG, Chen Y, Liu B, et al. Secretion of fatty acids by photosynthetic microorganisms：US, US20080333280[P]. 2009-12-3.
[59] Gibbons GF, Islam K, Pease RJ. Mobilisation of triacylglycerol stores[J]. Biochimica et Biophysica Acta, 2000, 1483(1)：37-57.
[60] Lehner R, Vance DE. Cloning and expression of a cDNA encoding a hepatic microsomal lipase that mobilizes stored triacylglycerol[J]. Biochemical Journal, 1999, 343：1-10.
[61] Tietge UJF, Bakillah A, Maugeais C, et al. Hepatic overexpression of microsomal triglyceride transfer protein(MTP)results in increased in vivo secretion of VLDL triglycerides and apolipoprotein B[J]. The Journal of Lipid Research, 1999, 40：2134-2139.
[62] McManaman JL, Russell TD, Schaack J, et al. Molecular determinants of milk lipid secretion[J]. Journal of Mammary Gland Biology and Neoplasia, 2007, 12(4)：259-268.
[63] Panikashvili D, Savaldi-Goldstein S, Mandel T, et al. The Arabidopsis DESPERADO/AtWBC11 transporter is required for cutin and wax secretion[J]. Plant Physiology, 2007, 145：1345-1360.
[64] Pighin JA, Zheng H, Balakshin LJ, et al. Plant cuticular lipid export requires an ABC transporter[J]. Science, 2004, 306：702-704.
[65] Janvilisri T, Venter H, Shahi S, et al. Sterol transport by the human breast cancer resistance protein(ABCG2)expressed in Lactococcus lactis[J]. The Journal of Biological Chemistry, 2003, 278：20645-20651.
[66] Mentewab A, Stewart CN. Overexpression of an Arabidopsis thaliana ABC transporter confers kanamycin resistance to transgenic plants[J]. Nature Biotechnology, 2005, 23：1177-1180.
[67] Yu L, Gupta S, Xu F, et al. Expression of ABCG5 and ABCG8 is required for regulation of biliary cholesterol secretion[J]. The Journal of Biological Chemistry, 2005, 280：8742-8747.
[68] Hayashi M, Nito K, Takei-Hoshi R, et al. Ped3p is a peroxisomal ATP-binding cassette transporter that might supply substrates for fatty acid beta-oxidation[J]. Plant and Cell Physiology, 2002, 43(1)：1-11.
[69] Zolman BK, Silva ID, Bartel B. The Arabidopsis pxa1 mutant is defective in an ATP-binding cassette transporter-like protein required for peroxisomal fatty acid beta-oxidation[J]. Plant Physiology, 2001, 127(3)：1266-1278.