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• 综述与评论 •

微藻高油脂化基因工程研究策略

冯国栋1, 程丽华*2, 徐新华2, 张林1, 陈欢林1   

  1. 1. 浙江大学化学工程与生物工程学系 杭州 310027;
    2. 浙江大学环境工程系 杭州 310058
  • 收稿日期:2011-09-01 修回日期:2012-01-01 出版日期:2012-07-24 发布日期:2012-06-30
  • 通讯作者: 程丽华 E-mail:chenglihua@zju.edu.cn
  • 基金资助:

    国家自然科学基金项目(No.21076177, 21106130)和浙江省自然科学基金项目(No.Y4100222)资助

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导出 被引次数 ( 17 | 6 )

Strategies in Genetic Engineering of Microalgae for High-Lipid Production

Feng Guodong1, Cheng Lihua2, Xu Xinhua2, Zhang Lin1, Chen Huanlin1   

  1. 1. Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China;
    2. Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, China
  • Received:2011-09-01 Revised:2012-01-01 Online:2012-07-24 Published:2012-06-30
石化能源危机与全球气候变化已成为人类的两大重要难题。生物柴油作为可替代普通柴油的环境友好且可再生的能源受到普遍关注。相比植物油和动物脂肪生产,藻类油脂产率高且容易培养,被认为是未来生物柴油发展的重要原料之一。通过转基因技术强化油脂代谢途径,提高富油微藻含油量,已经成为新的研究热点。已有植物研究表明,增强甘油酯酰基转移酶表达,可以提高Kennedy途径代谢中间体通量,从而增加甘油三酯(TAG)的积累。本文综述了微藻油脂代谢途径的国内外研究现状和提高油脂积累的代谢调控策略;详细阐述了基于植物油脂合成强化的成功经验,通过增强微藻Kennedy途径对提高TAG生物合成的重要作用;讨论了当前转基因微藻的遗传转化方法及其需要解决的关键性科学技术问题;分析了基因工程技术调控微藻脂类代谢途径生产高油脂的可能性,并对该研究的发展进行了展望。
关键词： 富油微藻, 三羧酸甘油酯, 生物柴油, 油脂合成代谢, 基因工程
Fossil energy crisis and global climate change are two major challenges in the 21st century. Biodiesel, as an environmentally friendly and renewable alternative energy source to petroleum diesel, has drawn worldwide attention. Compared with vegetable oil and animal fats, algal lipid is considered to be one of future biodiesel feedstocks because of the microalgae characterized in its easy cultivation and high lipid content. The genetic engineering has now become a new research hotpoint to enhance the lipid metabolism pathway, thus to improve the lipid content of oil-rich microalgae. In this paper, the advances of lipid biosynthesis pathway in microalgae, and the metabolic regulation strategies for increasing lipid accumulation are reviewed. The importance of Kennedy pathway enhancement in the microalgal TAG biosynthesis is elaborated based on the fact that the enhanced glycerol acyltransferase expression in plants can increase the metabolism of Kennedy pathway intermediates flux, thereby increasing the triacylglycerols (TAG) accumulation. The current genetic transformation methods of microalgae and their key scientific and technical issues, as well as the possibility and prospects of genetic engineering metabolic pathways to regulate the microalgae lipid production are further discussed in detail. Contents
1 Introduction
2 TAG biosynthesis pathways
3 Kennedy pathway strengthened TAG biosynthesis
3.1 Glycerol-3-phosphate dehydrogenase (G3PDH)
3.2 Glycerol-3-phosphate acyltransferase (GPAT)
3.3 Lysophosphatidic acid acyltransferase (LPAAT)
3.4 Diacylglycerol acyltransferase (DGAT)
4 Genetic transformation of oil-rich microalgae
4.1 Genetic transformation methods
4.2 Selection markers and promoters
5 Factors affecting protein expression
5.1 Codon usage
5.2 Gene position and copy number
5.3 Protein stability
6 Conclusions and outlook
Key words: high-lipid microalgae, triacylglycerols, biodiesel, lipid storage metabolism, genetic engineering

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[1] Demirbas A, Demirbas M F. Energ. Convers. Manage., 52(1): 163-170
[2] Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M. Plant J., 2008, 54: 621-639
[3] Williams P J B, Laurens L M L. Energy Environ. Sci., 2010, 3: 554-590
[4] Radakovits R, Jinkerson R E, Darzins A, Posewitz M C. Eukaryot. Cell, 2010, 9(4): 486-501
[5] Radakovits R, Eduafo P M, Posewitz M C. Metab. Eng., 2011, 13(1): 89-95
[6] Roessler P G. Arch. Biochem. Biophys., 1988, 267(2): 521-528
[7] Dunahay T, Jarvis E, Dais S, Roessler P. Appl. Biochem. Biotech., 1996, 57/58(1): 223-231
[8] Sheehan J, Dunahay T, Benemann J, Roessler P. A Look Back at the US Department of Energy's Aquatic Species Program: Biodiesel from Algae. NREL/TP-580-24190, 1998, 67-139
[9] 王镜岩(Wang J Y), 朱圣庚(Zhu S G), 徐长法(Xu C F). 生物化学(Biochemistry). 第3版(3rd ed). 北京: 高等教育出版社(Beijing: Higher Education Press), 2002
[10] Remize F, Barnavon L, Dequin S. Metab. Eng., 2001, 3(4): 301-312
[11] Vigeolas H, Waldeck P, Zank T, Geigenberger P. Plant Biotechnol. J., 2007, 5(3): 431-441
[12] Murata N, Sato N, Takahashi N, Hamazaki Y. Plant Cell Physiol., 1982, 23(6): 1071-1079
[13] Murata N. Plant Cell Physiol., 1983, 24(1): 81-86
[14] Murata N, Yamaya J. Plant Physiol., 1984, 74(4): 1016-1024
[15] Jain R K, Coffey M, Lai K, Kumar A, MacKenzie S L. Biochem. Soc. T., 2000, 28(6): 958-961
[16] Mputu M N, Rhazi L, Vasseur G, Vu T D, Gontier E, Thomasset B. Biochimie, 2009, 91(6): 703-710
[17] Zou J, Wei Y, Jako C, Kumar A, Selvaraj G, Taylor D C. Plant J., 1999, 19(6): 645-653
[18] Taylor D C, Katavic V, Zou J, Mackenzie S L, Keller W A, An J, Friesen W, Barton D L, Pedersen K K, Giblin E M, Ge Y, Dauk M, Sonntag C, Luciw T, Males D. Mol. Breeding, 2002, 8(4): 317-322
[19] Knutzon D S, Hayes T R, Wyrick A, Xiong H, Davies H M, Voelker T A. Plant Physiol., 1999, 120(3): 739-746
[20] Maisonneuve S, Bessoule J J, Lessire R, Delseny M, Roscoe T. J. Plant Physiol., 2010, 152(2): 670-684
[21] Merchant S S, Prochnik S E, Rokhsar D S, et al. Science, 2007, 318(5848): 245-251
[22] Yen C L E, Monetti M, Burri B J, Farese R V. J. Lipid Res., 2005, 46(7): 1502-1511
[23] Lardizabal K D, Mai J T, Wagner N W, Wyrick A, Voelker T, Hawkins D J. J. Biol. Chem., 2001, 276(42): 38862-38869
[24] Cases S, Stone S J, Zhou P, Yen E, Tow B, Lardizabal K D, Voelker T, Farese R V. J. Biol. Chem., 2001, 276(42): 38870-38876
[25] Shockey J M, Gidda S K, Chapital D C, Kuan J C, Dhanoa P K, Bland J M, Rothstein S J, Mullen R T, Dyer J M. Plant Cell, 2006, 18(9): 2294-2313
[26] Saha S, Enugutti B, Rajakumari S, Rajasekharan R. Plant Physiol., 2006, 141(4): 1533-1543
[27] Kalscheuer R, Steinbüchel A. J. Biol. Chem., 2003, 278(10): 8075-8082
[28] Courchesne N M D, Parisien A, Wang B, Lan C Q. J. Biotechnol., 2009, 141(1/2): 31-41
[29] 姚茹(Yao R), 程丽华(Cheng L H), 徐新华(Xu X H), 张林(Zhang L), 陈欢林(Chen H L). 化学进展(Progress in Chemistry), 2010, 22(6): 1221-1232
[30] Jako C, Kumar A, Wei Y, Zou J, Barton D L, Giblin E M, Covello P S, Taylor D C. Plant Physiol., 2001, 126(2): 861-874
[31] Zheng P, Allen W B, Roesler K, Williams M E, Zhang S, Li J, Glassman K, Ranch J, Nubel D, Solawetz W, Bhattramakki D, Llaca V, Deschamps S, Zhong G Y, Tarczynski M C, Shen B. Nat. Genet., 2008, 40(3): 367-372
[32] Weselake R J, Taylor D C, Rahman M H, Shah S, Laroche A, McVetty P B, Harwood J L. Biotechnol. Adv., 2009, 27(6): 866-878
[33] Baud S, Lepiniec L. Prog. Lipid Res., 2010, 49(3): 235-249
[34] Wagner M, Hoppe K, Czabany T, Heilmann M, Daum G, Feussner I, Fulda M. Plant Physiol. Bioch., 2010, 48(6): 407-416
[35] Khozin-Goldberg I, Cohen Z. Biochimie, 2011, 93(1): 91-100
[36] Xu J, Zheng Z, Zou J. Botany, 2009, 87(6): 544-551
[37] Zou J, Xu J, Zheng Z. WO/2009/085169, 2009
[38] Li Y T, Han D X, Hu G R, Sommerfeld M, Hu Q. Biotechnol. Bioeng., 2010, 107(2): 258-268
[39] Xiong W, Gao C F, Yan D, Wu C, Wu Q Y. Bioresource Technol., 2010, 101(7): 2287-2293
[40] Walker T L, Purton S, Becker D K, Collet C. Plant Cell Rep., 2005, 24: 629-641
[41] Potvin G, Zhang Z S. Biotechnol. Adv., 2010, 28(6): 910-918
[42] Mayfield S P, Kindle K L. Proc. Natl. Acad. Sci. USA, 1990, 87: 2087-2091
[43] Boynton J E, Gillham N W, Harris E H, Hosler J P, Johnson A M, Jones A R, Randolph-Anderson B L, Robertson D, Klein T M, Shark K B, Sanford J C. Science, 1988, 240: 1534-1538
[44] El-Sheekh M M. Folia Microbiol., 2000, 45: 496-504
[45] Schiedlmeier B, Schmitt R, Muller W, Kirk M M, Gruber H, Mages W, Kirk D L. Proc. Natl. Acad. Sci. USA, 1994, 91: 5080-5084
[46] Dawson H N, Burlingame R, Cannons A C. Curr. Microbiol., 1997, 35: 356-362
[47] Chen Y, Li W B, Bai Q H, Sun Y R. Chin. J. Oceanol. Limnol., 1998, 16(Suppl.1): 47-49
[48] 陈颖(Chen Y). 中国科学院博士论文(Doctoral Dissertation of Chinese Academy of Sciences), 1999
[49] Chen Y, Wang Y Q, Sun Y R, Zhang L M, Li W B. Curr. Genet., 2001, 39: 365-370
[50] 张小宇(Zhang X Y). 北京林业大学硕士论文(Master Dissertation of Beijing Forestry University), 2006
[51] 安洋(An Y). 华东理工大学硕士论文(Master Dissertation of East China University of Science and Technology), 2008
[52] 任雪艳(Ren X Y), 王恒强(Wang H Q), 祝建波(Zhu J B), 孔庆军(Kong Q J). 中国兽医科学(Chinese Veterinary Science), 2010, 40(1): 41-44
[53] El-Sheekh M M. Biol. Plantarum, 1999, 42: 209-216
[54] Teng C, Qin S, Liu J, Yu D, Liang C, Tseng C. J. Appl. Phycol., 2002, 14: 497-500
[55] Apt K E, Kroth-Pancic P G, Grossman A R. Mol. Gen. Genet., 1996, 252: 572-579
[56] Boynton J E, Gillham N W. Methods Enzymol., 1993, 217: 510-536
[57] Lerche K, Hallmann A. BMC Biotechnol., 2009, 9: 64
[58] Kindle K L. Proc. Natl. Acad. Sci. USA, 1990, 87: 1228-1232
[59] Kindle K L, Richards K L, Stern D B. Proc. Natl. Acad. Sci. USA, 1990, 88: 1721-1725
[60] Feng S, Xue L, Liu H, Lu P. Mol. Bio. Rep., 2009, 36: 1433-1439
[61] Dunahay T G. Biotechniques, 1993, 15: 452-460
[62] Wang K, Drayton P, Frame B, Dunwell J, Thompson J. In Vitro Cell. Dev. Biol., 1995, 31: 101-104
[63] Geng D G, Han Y, Wang Y Q, Wang P, Zhang L M, Li W B, Sun Y R. Acta Bot. Sin., 2004, 46: 342-346
[64] Sun Y, Yang Z, Gao X, Li Q, Zhang Q, Xu Z. Mol. Biotechnol., 2005, 30: 185-192
[65] Wang T Y, Xue L X, Hou W H, Yang B S, Chai Y R, Ji X, Wang Y F. Appl. Microbiol. Biotechnol., 2007, 76: 651-657
[66] Sun G H, Zhang X C, Sui Z H, Mao Y X. Marine Biotechnol., 2008, 10: 219-226
[67] Sun Y, Gao X S, Li Q Y, Zhang Q Q, Xu Z K. Gene, 2006, 377: 140-149
[68] Walker T L, Becker D K, Dale J L, Collet C. J. Appl. Phycol., 2005, 17: 363-368
[69] Tang D K H, Qiao S Y, Wu M. Biochem. Mol. Biol. Int., 1995, 36: 1025-1035
[70] Shimogawara K, Fujiwara S, Grossman A, Usuda H. Genetics, 1998, 148: 1821-1828
[71] Kovar J L, Zhang J, Funke R P, Weeks D P. Plant J., 2002, 29: 109-117
[72] Ladygin V G. Microbiol., 2003, 72: 585-591
[73] Ladygin V G. Process Biochem., 2004, 39: 1685-1691
[74] Chow K C, Tung W L. Plant Cell Rep., 1999, 18: 778-780
[75] 王逸云(Wang Y Y). 大连理工大学博士论文(Doctoral Dissertation of Dalian University of Technology), 2005
[76] Wang C, Wang Y, Su Q, Gao X. Biotechnol. Bioproc. Eng., 2007, 12: 180-183
[77] Chen H L, Li S S, Huang R, Tsai H J. J. Phycol., 2008, 44: 768-776
[78] Li S S, Tsai H J. Fish Shellfish Immunol., 2009, 26: 316-325
[79] Kumar S V, Misquitta R W, Reddy V S, Rao B J, Rajam M V. Plant Sci., 2004, 166: 731-738
[80] Kathiresan S, Chandrashekar A, Ravishankar A, Sarada R. J. Phycol., 2009, 45: 642-649
[81] Kathiresan S, Sarada R. J. Appl. Phycol., 2009, 21: 553-558
[82] Lutz K A, Maliga P. Curr. Opin. Biotechnol., 2007, 18: 107-114
[83] Leon-Banares R, Gonzalez-Ballester D, Galvan A, Fernandez E. Trends Biotechnol., 2004, 22(1): 45-52
[84] Griesbeck C, Kobl I, Heitzer M. Mol. Biotechnol., 2006, 34: 213-223
[85] Debuchy R, Purton S, Rochaix J D. EMBO J., 1989, 8: 2803-2809
[86] Remacle C, Cline S, Boutaffala L, Gabilly S, Larosa V, Barbieri M R, Coosemans N, Hamel P P. Eukaryot. Cell, 2009, 8: 1460-1463
[87] Kindle K L, Schnell R A, Fernandez E, Lefebvre P A. J. Cell Biol., 1989, 109: 2589-2601
[88] Ferris P J. Genetics, 1995, 141: 543-549
[89] Nelson J A E, Savereide P B, Lefebvre P A. Mol. Cell. Biol., 1994, 14: 4011-4019
[90] Randolph-Anderson B L, Sato R, Johnson A M, Harris E H, Hauser C R, Oeda K, Ishige F, Nishio S, Gillham N W, Boynton J E. Plant Mol. Biol., 1998, 38: 839-859
[91] Steinbrenner J, Sandmann G. Appl. Environ. Microbiol., 2006, 72: 7477-7484
[92] Huang J, Liu J, Li Y, Chen F. J. Phycol., 2008, 44: 684-690
[93] Chang M, Li F, Odom O W, Lee J, Herrin D L. Plasmid, 2003, 49: 75-78
[94] Zaslavskaia L A, Lippmeier J C, Shih C, Ehrhardt D, Grossman A R, Apt K E. Science, 2001, 292: 2073-2075
[95] Fischer H, Robl I, Sumper M, Krger N. J. Phycol., 1999, 35, 113-120
[96] 卞曙光(Bian S G), 姜鹏(Jiang P), 崔玉琳(Cui Y L), 刘兆普(Liu Z P), 秦松(Qin S). 海洋科学(Marine Sciences), 2010, 34(5): 62-66
[97] Hall L M, Taylor K B, Jones D D. Gene, 1993, 124: 75-81
[98] Zaslavskaia L A, Lippmeier J C, Kroth P G, Grossman A R, Apt K E. J. Pycol., 2000, 36: 379-386
[99] Lohuis M R, Miller D J. Plant J., 1998, 13: 427-435
[100] Dunahay T G, Jarvis E E, Roessler P G. J. Phycol., 1995, 31: 1004-1012
[101] Stevens D R, Rochaix J D, Purton S. Mol. Gen. Genet., 1996, 251: 23-30
[102] Lumbreras V, Stevens D R, Purton S. Plant J., 1998, 14: 441-447
[103] Goldschmidt-Clermont M. Nucleic Acids Res., 1991, 19: 4083-4089
[104] Cerutti H, Johnson A M, Gillham N W, Boynton J E. Genetics, 1997, 145: 97-110
[105] Bateman J M, Purton S. Mol. Gen. Genet., 2000, 263: 404-410
[106] Berthold P, Schmitt R, Mages W. Protist, 2002, 153: 401-412
[107] Sizova I, Fuhrmann M, Hegemann P. Gene, 2001, 277: 221-229
[108] 王义琴(Wang Y Q), 陈颖(Chen Y), 白琴华(Bai Q H), 赵世民(Zhao S M), 李文彬(Li W B), 孙勇如(Sun Y R). 高技术通讯(High Technology Letters), 2001(9): 1-5
[109] 安洋(An Y), 赵世民(Zhao S M), 孙勇如(Sun Y R), 白丽莉(Bai L L), 尹维波(Yin W B), 陈宇红(Chen Y H), 李元广(Li Y G), 胡军(Hu J), 胡赞民(Hu Z M). 农业生物技术学报(Journal of Agricultural Biotechnology), 2008, 16(4): 635-641
[110] Davies J P, Weeks D P, Grossman A R. Nucleic Acids Res., 1992, 20: 2959-2965
[111] Eichler-Stahlberg A, Weisheit W, Ruecker O, Heitzer M. Planta, 2009, 229: 873-883
[112] Schroda M, Blocker D, Beck C F. Plant J., 2000, 21: 121-131
[113] Schroda M, Beck C F, Vallon O. Plant J., 2002, 31: 445-455
[114] Quinn J M, Kropat J, Merchant S. Eukaryot. Cell, 2003, 2: 995-1002
[115] Loppes R, Radoux M, Ohresser M C, Matagne R F. Plant Mol. Biol., 1999, 41: 701-711
[116] 王鹏(Wang P). 中国科学院博士论文(Doctoral Dissertation of Chinese Academy of Sciences), 2004
[117] 张小宇(Zhang X Y), 王鹏(Wang P), 赵世民(Zhao S M), 李霞(Li X), 沈昕(Shen X), 孙勇如(Sun Y R), 储成才(Chu C C), 王义琴(Wang Y Q). 遗传(Hereditas), 2006, 28(12): 1580-1584
[118] Heitzer M, Eckert A, Fuhrmann M, Griesbeck C. Adv. Exp. Med. Biol., 2007, 616: 46-53
[119] Fuhrmann M, Oertel W, Hegemann P. Plant J., 1999, 19: 353-361
[120] Fuhrmann M. Protist, 2002, 153: 357-364
[121] Wu S X, Huang R, Xu L L, Yan G Y, Wang Q X. J. Biotechnol., 2010, 146(3): 120-125
[122] Wu S X, Xu L L, Huang R, Wang Q X. Bioresource Technol., 2011, 102(3): 2610-2616
[123] Welch M, Villalobos A, Gustafsson C, Minshull J. J. R. Soc. Interface, 2009, 6(Suppl.4): 467-476
[124] Manuell A L, Beligni M V, Elder J H, Siefker D T, Tran M, Weber A, McDonald T L, Mayfield S P. Plant Biotechnol. J., 2007, 5: 402-412
[125] Surzycki R, Greenham K, Kitayama K, Dibal F, Wagner R, Rochaix J D, Ajam T, Surzycki S. Biologicals, 2009, 37: 133-138
[126] Faye L, Daniell H. Plant Biotechnol. J., 2006, 4: 275-279
[127] Neupert J, Karcher D, Bock R. Plant J., 2009, 57: 1140-1150
[128] Yao Q, Cong L, Chang J L, Li K X, Yang G X, He G Y. J. Exp. Bot., 2006, 57: 3737-3746
[129] Jayaraj J, Liang G H, Muthukrishnan S, Punja Z K. Biol. Plantarum, 2008, 52: 215-221
[130] Lowe B A, Prakash N S, Way M, Mann M T, Spencer T M, Boddupalli R S. Transgenic Res., 2009, 18: 831-840
[131] Sorokin A P, Ke X Y, Chen D F, Elliott M C. Plant Sci., 2000, 156: 227-233
[132] Butaye K M J, Cammue B P A, Delaure S L, De Bolle M F C. Mol. Breeding, 2005, 16: 79-91
[133] Wu S J, Wang H H, Li F F, Chen T Z, Zhang J, Jiang Y J, Ding Y, Guo W Z, Zhang T Z. Plant Mol. Biol. Rep., 2008, 26: 174-185
[134] Zale J M, Agarwal S, Loar S, Steber C M. Plant Cell Rep., 2009, 28: 903-913
[135] Oltmanns H, Frame B, Lee L Y, Johnson S, Li B, Wang K, Gelvin S B. Plant Physiol., 2010, 152: 1158-1166
[136] Doran P M. Trends Biotechnol., 2006, 24: 426-432
[137] Lü J, Sheahan C, Fu P C. Energy Environ. Sci., 2011, 4: 2451-2466
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微藻高油脂化基因工程研究策略