摘要
生物柴油是一种重要的可再生能源,其生产方法是将原料油脂和短链醇在催化剂的催化下,转化合成生物柴油(如脂肪酸甲酯/脂肪酸乙酯)。生物柴油已在各国广泛生产和应用,2008年全球生物柴油产量已达到1400万吨,近几年生物柴油产量正以较快的速度增长。目前,原料成本高、生产工艺较复杂是生物柴油生产工艺中主要问题。因此,如何利用微生物直接发酵生产生物柴油,建立一种高效的、可替代石化柴油和现有生物柴油生产的新技术、新路线显得日益迫切。
本论文分别从长链生物柴油基因工程菌的构建及发酵条件优化,中链脂肪酸乙酯的合成、中链脂肪酸及其乙酯基因工程菌的构建等方面进行了研究。
1.构建了产长链生物柴油的酿酒酵母基因工程菌。通过重组质粒的构建、转化和重组子筛选,导入质粒YEp352-PLC的重组酿酒酵母表达了大小约为42KDa的重组蛋白,脂肪酶的比活力为12.12U/mg,胞内油脂中长链脂肪酸含量超过92%。发酵培养96h,提取的重组酿酒酵母油脂中脂肪酸乙酯产量约为3.7mg/g (DCW)。
2.为了提高重组酿酒酵母脂肪酸乙酯产量,利用响应面法优化了酿酒酵母产油脂发酵条件。在优化的发酵条件下,重组酿酒酵母油脂产量比对照提高了2倍左右,达到14.55%。
3.通过均匀设计优化了酿酒酵母联产油脂和乙醇的发酵条件。考察了通气量、搅拌速率和葡萄糖流加对酿酒酵母产油脂和乙醇的影响,通过非线性拟合得到两个相关性在90%的方程。分析表明,搅拌速率对酿酒酵母产油脂和乙醇的影响大于其它两个因素。通过计算得到产油脂、产乙醇、联产油脂和乙醇的最优条件,并得以实验验证。
4.优化了酿酒酵母工程菌产脂肪酸乙酯的发酵条件。在酿酒酵母产油脂发酵条件的基础上,3次添加反应体系体积4%的乙醇效果最佳,所得到的FAEEs量达到11.4mg/g (DCW)。
5.开发了利用樟树籽油合成中链生物柴油的合成工艺。首先利用正交实验优化了微波辅助萃取法提取樟树籽油的提取条件,油脂提取率达到90%以上,樟树籽油中脂肪酸组成以癸酸(53.4%)和月桂酸(38.7%)为主,中链脂肪酸含量占脂肪酸总含量的94%。其次采用固定化脂肪酶Candida sp.99-125催化樟树籽油合成生物柴油(脂肪酸乙酯),优化后的合成工艺条件为：水含量10%(wt),酶用量15%(wt),油／醇摩尔比1：3.2,9次流加乙醇(间隔时间2.67h),反应温度40-C,摇床转速170rpm,反应24h,脂肪酸乙酯产率达93.5%。
6.构建了产中链脂肪酸的基因工程菌。克隆樟树硫酯酶基因ccFatB并转入大肠杆菌表达。重组大肠杆菌发酵培养产物中,肉豆蔻酸产量为52mg/L,占总脂肪酸含量的10.6%。
7.构建了产中链生物柴油的基因工程菌。克隆不动杆菌酰基转移基因atfA和ccFatB,构建了双基因表达载体pETDuet-ccFatB-atfA,转入大肠杆菌表达。基因工程菌利用外源流加4%的乙醇在胞内合成脂肪酸乙酯,肉豆蔻酸乙酯产量约为2.1mg/L,占乙酯总量的10%。
Biodiesel is an important renewable energy, which synthesized by catalytic conversion of oils and short chain alcohols (such as fatty acid methyl ester, FAME/fatty acid ethyl ester, FAEE). Biodiesel have been widely applied in many countries. About14millions ton biodiesel was produced in the world in2008, and the worldwide demand for biodiesel is growing at a fast speed in recent years. Currently, high cost of raw materials and complex processes are main constraints on biodiesel production. Therefore, how to produced biodiesel directly by fermentation and built a new biological routine alternative to petroleum diesel and existing biodiesel production technology are very urgent.
In this thesis, engineered yeast strain capable of direct production of long-chain biodiesel was constructed and its fermentation conditions were optimized. Medium-chain fatty acid ethyl esters synthesized by immobilized lipase and directly by engineered bacteria were studied.
1. A strain of Saccharomyces cerevisiae was genetically engineered to produce long-chain biodiesel directly. The recombinant plasmid YEp352-PLC was transformed into S.cerevisiae and the transformants were screened by nutritional supplement. The results showed that a42kDa protein was expressed in recombinant S.cerevisiae and the specific activity of the lipase was12.12U/mg. The content of long-chain fatty acids exceeded92%in extracted lipid. After96h fermentation culture, the yield of FAEEs reached3.7mg/g (DCW).
2. Response surface method was applied to optimize the fermentation conditions for lipid production by S.cerevisiae. Under the optimized fermentation conditions, a lipid production of14.55%was achieved which was about2-fold of the control.
3. Uniform design was used to optimize the cofermentation conditions of lipid and ethanol production by S. cerevisiae. Effects of aeration rate, agitation rate, and glucose feeding on lipid and ethanol production were investigated. Two equations with90%correlation were obtained by nonlinear fitting. By analysis, the stirring speed was observed to exert more influence than other factors on lipid and ethanol production. The optimized fermentation conditions for lipid and ethanol production was achieved by calculation and verified by experiments.
4. Fermentation conditions for FAEEs production by engineered S. cerevisiae were optimized. On the basis of the favored fermentation condition for lipid production, a high FAEEs yield of11.4mg/g (DCW) was attained with three-stepwise4%(V/V) ethanol addition.
5. A new process for medium-chain biodiesel synthesis with camphor tree seed oil was provided. Microwave assisted extraction was implied to extract camphor tree seed oil and the extraction conditions was optimized by orthogonal experiment. The results showed that the rate of oil extraction exceeded90%and the predominant fatty acid composition were capric acid (53.4%) and lauric acid (38.7%), which content of medium-chain fatty acids reached94%of the total fatty acids. Secondly, the immobilized Candida sp.99-125lipase was used to catalyze camphor tree seed oil for biodiesel synthesis. A FAEEs yield of93.5%was obtained after optimization of the synthetic process conditions, the water content of10%(wt), enzyme loading15%(wt), oil/ethanol molar ratio1:3.2,9stepwise ethanol addition (intervals2.67h), reaction temperature40℃, shaking speed170rpm, reaction time24h.
6. An engineered bacteria capable of medium-chain fatty acid production was constructed. ccFatB from Cinnamonum camphora was cloned and transformed into E. coli. By fermentation, the yield of myristic acid was approximately52mg/L, which is10.6%of the total fatty acid content.
7. An engineered bacteria capable of medium-chain biodiesel production was constructed. atfA was cloned from Acinetobacter baylyi. A dual-gene expression vector pETDuet-ccFatB-atfA was constructed and transformed into E. coli. FAEEs were intracellularly synthesized and the maximum yield of myristic acid ethyl ester reached2.1mg/L by adding4%(V/V) ethanol, which is10%of the total ester content.
引文
[1]谭天伟,王芳,邓利,徐家立,于丽娟.生物柴油的生产和莹用[J].现代化工.2002,22(2)：4-6
[2]韩毅,邓宇.生物柴油的发展现状及新技术[J].化工技术与开发.2007,36(3)：19-25
[3]http://www.chinaim.com[Z].2012年5月份我国柴油消费量统计分析.2012-6-26
[4]王一平,翟怡,张金利,李铧,韩振亭.生物柴油制备方法研究进展[J].化工进展.2003,22：8-12
[5]宁守俭.生物柴油产业步入跨越发展——2011年回顾与2012年展望[J].中国生物柴油.2012,1：1-4
[6]Haifeng Liu, Chia-fon Lee, Ming Huo, Mingfa Yao. Comparison of ethanol and butanol as additives in soybean biodiesel using a constant volume combustion chamber[J]. Energy Fuels.2011,25:1837-1846
[7]Adams C, Peters J F, Rand, et al. Investigation ofsoybean oil as diesel fuel extender. Endurance tests[J]. JAOCS.1983,60(8):1574-1579
[8]Michael J. Haas. Improving the economics of biediesel production through the use of low value lipids as feedstocks:vegetable oil soapstock[J]. Fuel Processing Technology.2005, 86:1087-1096
[9]Satoshi Furuta, Hiromi Matsuhashi, Kazushi Arata. Biodiesel fuel production with solid amorphous-zirconia catalysis in fixed bed reactor[J]. Biomass and Bioenergy.2006, 30(10):870-873
[10]Lara Pizarro A V, Park E Y. Lipase-catalyzed production of biodiesel fuel from vegetable oils contained in waste activated bleaching earth[J]. Process Biochemistry.2003,38(7): 1077-1082
[11]李香春,甄宗园.脂肪酶特性及其应用[J].粮食与油脂.2003,3：19-20
[12]Esper Boel, Birgitte Huge-Jensen, Mogens Christensen, Lars Thim, Niels P. Fiil. Rhizomucor miehei triglyceride lipase is synthesized as a precursor[J]. Lipids.1988,23(7): 701-706
[13]Villeneuve P, Muderhwa J M, Graille J, Haas M J. Customizing lipases for biocatalysis:a survey of chemical, physical and molecular biological approaches [J]. Journal of Molecular Catalyze B:Enzymatic.2000,9(4-6):113-148
[14]Jegannathan K R, Abang S, Poncelet D, Chan E S, Ravindra P. Production of biodiesel using immobilized lipase-a critical review[J]. Critical Review Biotechnology.2008,28(4): 253-264
[15]Du W, Xu Y, Liu D, Zeng J. Comparative study on lipase-catalyzed transformation of soybean oil for biodiesel production with different acyl acceptors[J]. Journal of Molecular Catalyze B:Enzymatic.2004,30(3-4):125-129
[16]Wang J, Huang Q, Huang F, Wang J, Huang Q. Lipase-catalyzed production of biodiesel from high acid value waste oil using ultrasonic assistant J]. Chinese Journal of Biotechnology.2007,23(6):1121-1128
[17]Lu J, Deng L, Zhao R, Zhang R, Wang F, Tan T. Pretreatment of immobilized Candida sp.99-125 lipase to improve its methanol tolerance for biodiesel production[J]. Journal of Molecular Catalyze B:Enzymatic.2010,62(1):15-18
[18]Tan T, Nie K, Wang F. Production of biodiesel by immobilized Candida sp.99-125 lipase at high water content[J]. Applied Biochemistry and Biotechnology.2006,128(2):109-116
[19]Iso M, Chen B, Eguchi M, Kudo T, Shrestha S. Production of biodiesel fuel from triglycerides and alcohol using immobilized lipase[J]. Journal of Molecular Catalyze B: Enzymatic.2001,16(1):53-58
[20]Salis A, Pinna M, Monduzzi M, Solinas V. Comparison among immobilised Upases on macroporous polypropylene toward biodiesel synthesis[J]. Journal of Molecular Catalyze B:Enzymatic.2008,54(1-2):19-26
[21]Salis A, Pinna M, Monduzzi M, Solinas V. Biodiesel production from triolein and short chain alcohols through biocatalysis[J]. Journal of Biotechnology.2005,119(3):291-299
[22]Shah S, Gupta M N. Lipase catalyzed preparation of biodiesel from Jatropha oil in a solvent free system[J]. Process Biochemistry.2007,42(3):409-414
[23]Yesiloglu Y. Immobilized lipase-catalyzed ethanolysis of sunflower oil[J]. Journal of American Oil Chemistry Society.2004,81(2):157-160
[24]Shah S, Sharma S, Gupta M N. Biodiesel preparation by lipase-catalyzed transesterification of jatropha oil[J]. Energy Fuels.2004,18(1):154-159
[25]Shieh C J, Liao H F, Lee C C. Optimization of lipase-catalyzed biodiesel by response surface methodology[J]. Bioresource Technology.2003,88(2):103-106
[26]贺芹,徐岩,滕云,王栋.华根霉全细胞脂肪酶催化合成生物柴油[J].催化学报.2008,29(1)：41-46
[27]李治林,李迅,王飞,蒋剑春.全细胞生物催化制备生物柴油研究[J].林产化学与工业.2009,29(2)：1-5
[28]Yamaguchi S, Takeuchi K, Mase T, Matsuura A. Efficient expression of mono-and diacylglycerol lipase gene from Penicillium camembertii U-150 in Aspergillus oryzae under the control of its own promoter [J]. Bioscience Biotechnology Biochemistry.1997, 61(5):800-805
[29]何耀强,王炳武,谭天伟.假丝酵母99-125脂肪酶的发酵工艺研究[J].生物工程学报.2004,20(6)：918-921
[30]Takahashi, S., Ueda, M., Tanaka, A. Independent production of two molecular froms of a recombinant Rhizopus oryzae lipase by KEX2-engineered strains of Saccharomyces cerevisiae[J]. Applied Microbiology and Biotechnology.1999,52(4):534-540
[31]Beer H D, McCarthy J E G, Bornscheuer U T, Schmid R D. Cloning, expression, characterization and role of the leader sequence of a lipase from Rhizopus oryzae[J]. Biochimica et Biophysica Acta.1998,1399(2-3):173-180
[32]张慧慧,梁艺福.超临界法制备生物柴油的研究进展[J].云南化工.2008,35(5)：77-80
[33]Ayhan Demirbas. Biodiesel production via non-catalytic SCF method and biodiesel fiael characteristics[J]. Energy Conversion and Management.2006,47 (15-16):2271-2282
[34]谢明霞,张敏华,姜浩锡.超临界甲醇制备生物柴油的研究[J].化学与生物工程.2007,24(9)：40-42
[35]Demirbas A. Biodiesel production from vegetable oils via catalytic and non-catalytic supercritical methanol transesterification methods [J]. Progress in Energy and Combustion Science.2005,31(5-6):466-487
[36]王成,刘忠义,陈于陇,徐玉娟,吴继军.生物柴油制备技术研究进展[J].广东农业科学.2012,1：107-112
[37]张爱华,张玉军,肖志红,李培旺,李昌珠.吡啶丁烷磺酸硫酸氢盐离子液体催化制备生物柴油[J].石油化工.2009,38(4)：389-393
[38]胡震,于海莲.超声波法制备生物柴油的新工艺研究[J].粮油加工.2009,9：86-88
[39]周彩荣,石晓华,程春燕.微波法由棉籽油制备生物柴油[J].郑州大学学报(工学版).2008,29(4)：51-53
[40]王云,俞云良,陆向红,徐之超,计建炳.水力空化技术强化酯交换反应合成生物柴油的研究[J].浙江工业大学学报.2008,36(1)：12-15
[41]Naik S N, Vaibhav V Goud, Prasant K Rout, Ajay K Dalai. Production of first and second generation biofuels:A comprehensive review[J]. Renewable and Sustainable Energy Reviews.2010,14(2):578-597
[42]Tianwei Tan, Jike Lu, Kaili Nie, Li Deng, Fang Wang. Biodiesel production with immobilized lipase:A review[J]. Biotechnology Advances.2010,28(5):628-634
[43]K. Shaine Tyson, Joseph Bozell, Robert Wallace, Eugene Petersen, and Luc Moens. Biomass oil analysis:research needs and recommendations[R]. National Renewable Energy Laboratory.2004,6(1):68-73
[44]Ivana B. Bankovic -Ilie, Olivera S. Stamenkovic, Vlada B. Veljkovi'c. Biodiesel production from non-edible plant oils. Renewable and Sustainable Energy Reviews.2012, 16:3621-3647
[45]解庆龙,孔丝纺,刘阳生,曾辉.生物质气化制合成气技术研究进展[J].现代化工.2011,31(7)：16-20
[46]Papanikolaou S, Komaitis M, Aggelis G Single cell oil(SCO) production by Mortierella isabellin grown on high-sugar content media [J]. Bioresource Technology.2004,95: 287-291
[47]Gransson Kristina, Sderlind U, He Jie, Wennan Zhang. Review of syngas production via biomass DFBGs[J]. Renewable and Sustainable Energy Reviews.2011,15(1):482-492
[48]Karmakar M K, Datta A B. Generation of hydrogen rich gas through fluidized bed gasification of biomass[J]. Bioresource Technology.2011,102(2):1907-1913
[49]杜丽娟,李建芬,肖波,易仁金,许小荣.生物质催化裂解制合成气的研究[J].化学工程师.2008,153(6)：3-5
[50]Eric J S, Kang Y, Gregory B, Hu Z, Andreas S, Amy M, Stephen B del C, Jay D K. Microbial production of fatty-acid-derived fuels and chemicals from plant biomass[J]. Nature.2010,463:559-562
[51]缪晓玲.藻类可再生能源的利用及藻细胞抗环境胁迫的研究[D].清华大学,2004.
[52]赵宗保.加快微生物油脂研究为生物柴油产业提供廉价原料[J].中国生物工程杂志.2005,25(2)：8-11
[53]薛飞燕,张栩,谭天伟.微生物油脂的研究进展及展望[J].生物加工过程.2005,3(1)：23-27
[54]Ratledge C, Wynn J P. The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms[J]. Advances in Applied Microbiology.2002,51:1-51
[55]徐华顺,罗玉萍,李思光.微生物发酵产油脂的研究进展[J].中国油脂.1999,24(2)：34-37
[56]Certik M, Shimizu S. Biosynthesis and regulation of microbial polyunsaturated fatty acid production[J]. Journal of Bioscience and Bioengineering.1999,87(1):1-14
[57]Somashekar D, Venkateshwaran G, Sambaiah K, B R Lokesh. Effect of culture conditions on lipid and gamma-linolenic acid production by mucoraceous fungi[J]. Process Biochemistry.2003,38(12):1719-1724
[58]朱法科,林炜铁,鲍时翔.花生四烯酸高产菌株的选育[J].工业微生物.1999,2：14
[59]Bajpai P, Bajpai P K, Ward O P. Eicosapentaenoic acid(EPA) production by Mortierella alpina ATCC 32222[J]. Applied Biochemistry and Biotechnology.1991,31(3):267-272
[60]Singh A, Ward O. Microbial production of docosahexaenoic acid(DHA.C22:6) [J]. Advances in Applied Microbiology.1997,45:271-312
[61]施安辉,周波.粘红酵母GLR513生产油脂最佳小型工艺发酵条件的探讨[J].食品科学.2003，1：48-51
[62]Rainer Kalscheuer, Heinrich Luftmann, Alexander Steinbuchel. Sythesis of novel lipid in Saccharomyces cerevisiae by heterologous expression of an unspecific bacterial acyltransferase[J]. Applied and Environmental Microbiology.2004,70 (12):7119-7125
[63]罗明典.什么是生物柴油[N].科技日报.2001-10-22
[64]Tibor Czabany, Karin Athenstaedt, Gunther Daum. Synthesis, storage and degradation of neutral lipids in yeast[J]. Biochimica et Biophysica Acta.2007,1771 (3):299-309
[65]赵宗保.加快微生物油脂研究为生物柴油产业提供廉价原料[J].中国生物工程杂志.2005,25(2)：8-11
[66]易绍金,郑义平.产油微生物的研究及其应用[J].中外能源.2006,11(2)：90-94
[67]许鑫,段仰凯,董晓宇,闫飞,杨秀山.斯达氏油脂酵母产油脂培养基及产油条件的优化[J].太阳能学报.2007,8：857-860
[68]刘波,孙艳,刘永红,赵宗保.产油微生物油脂生物合成与代谢调控研究进展[J].微生物学报.2005,45(1)：153-155
[69]李永红,刘波,赵宗保.圆红冬孢酵母菌发酵产油脂培养基及发酵条件的优化研究[J].生物工程学报.2006,22(4)：650-655
[70]孔祥莉,刘波,赵宗保.斯达氏油脂酵母利用混合糖发酵产油脂[J].生物加工过程.2007,5(2)：36-41
[71]Oksana Tehlivets, Kim Scheuringer, Sepp D Kohlwein. Fatty acid synthesis and elongation in yeast[J]. Biochimica et Biophysica Acta,2007,1771(3):255-270
[72]Ohlrogge J, Browse J. Lipid biosynthesis[J]. Plant Cell.1995,7(7):957-970
[73]Sorger D, Daum G Triacylglycerol biosynthesis in yeast[J]. Applied Microbiological Biotechnology.2003,61(4):289-299
[74]Zheng Z, Zou J. The initial step of the glycerolipid pathway:identification of glycerol 3-phosphate/dihydroxyacetone phosphatedual substrate aeyltransferases in Saccharomyces cerevisiae[J]. Journal of Biological Chemistry.2001,276(45):41710-41716
[75]M McDermott, M J Wakelam, A J Morris. Phospholipase D[J]. Biochemistry and Cell Biology.2004,82(1):225-253
[76]Anders Dahlqvist, Ulf Stahl, Marit Lenman, Antoni Banas, Michael Lee, Line Sandager, Hans Ronne, and Sten Stymne. Phospholipid:diacylglycerol acyltransferase:an enzyme that catalyzes the acyl-CoA-independent formation of triaeylglyeerol in yeast and plants[J]. Proceedings of the National Academy of Sciences of the United States of America.2000,97(12):6487-6492
[77]Oelkers P, Cromley D, Padamsee M, Billheimer JT, Sturley SL. The DAG1 gene determines a second triglyeeride synthetic pathway in yeast[J]. Journal of Biological Chemistry.2002,277:8877-8881
[78]Buhman K. K, Chen H C, Farese R V. The enzymes of neutral lipid synthesis[J]. Journal of Biological Chemistry.2001,276:40369-40372
[79]Sorger D, Daum G. Synthesis of triaeylglyeerols by the acyleoenzyme A:diacyl-glycerol aeyltransferase DgalP in lipid particles of the yeast Saccharomyces cerevisiae[J]. Journal of Bacteriology.2002,184(2):519-524
[80]Athenstaedt K, Jolivet P, Boulard C, Zivy M, Negroni L, Nicaud JM, Chardot T. Lipid particle composition of the yeast Yarrowia lipolytica depends on the carbon source[J]. Proteomics.2006,6(5):1450-1459
[81]Botham P A, Ratledge C. A biochemical explanation for lipid accumulation in Candida 107 and other oleaginous microorganisms[J]. Journal of Genetic Microbiology.1979, 114(2):361-375
[82]Wynn J P, Hamid A A, Li Y, Ratledge C. Biochemical events leading to the diversion of carbon into storage lipids in the Oleaginous fungi Mucor circinelloides and Mortierella alpina[J]. Microbiology.2001,147(10):2857-2864
[83]Papanikolaou S, Sarantou S, Komaitis M, Aggelis G. Repression of reserve lipid turnover in Cunninghamella echinulata and Mortierella isabellina cultivated in multiple-limited media[J]. Journal of Applied Microbiology.2004,97(4):867-875
[84]Adams P, Dack S, Dickinson F M, Ratledge C. The distinctiveness of ATP:citrate lyase from Aspergillus nidulans[J]. Biochimica et Biophysica Acta.2002,1597(1):36-41
[85]Ratledge C. Regulation of lipid accumulation in oleaginous microorganisms[J]. Biochemical Society Transactions.2002,30(6):1047-1050
[86]Santos M M M, Vijayendran R, Kotter P, Olsson L, and Nielsen J. Manipulation of malie enzyme in Saceharomyces cerevisiae for increasing NADPH production capacity aerobically in different cellular compartments[J]. Metabolic Engineering.2004,6:352-363
[87]Tibor Czabany, Karin Athenstaedt, GUnther Daum. Synthesis, storage and degradation of neutral lipids in yeast[J]. Biochimica et Biophysica Acta.2007,1771 (3):299-309
[88]Daum G, Wagner A, Czabany T, Athenstaedt K. Dynamics of neutral lipid storage and mobilization in yeast[J]. Biochimie.2007,89(2):243-248
[89]Athenstaedt K, Daum G. YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae[J]. Journal of Biological Chemistry.2003, 278(26):23317-23323
[90]里伟,杜伟,李永红,刘德华,赵宗保.生物酶法转化酵母油脂合成生物柴油[J].生物加工过程.2007,7(1)：137-140
[91]缪晓玲,吴庆余.微藻油脂制备生物柴油的研究[J].太阳能学报.2007,28(2)：219-222
[92]Nookaew I, Jewett M C, Meecha A, Thammarongtham C, Laoteng K, Cheevadhanarak S, Nielsen J and Bhumiratana S. The genome-scale metabolic model iIN800 of Saccharomyces cerevisiae and its validation:a scaffold to query lipid metabolism[J]. BMC Systems Biology.2008,2:71-86
[93]Davis M S, Solbiati J, Cronan J E. Over production of acetyl-CoA carboxylase activity increases the rate of fatty acid biosynthesis in Escherichia coli[J]. Journal of Biological Chemistry.2000,275(37):28593-28598
[94]Paul Handke, Sean A Lynch, Ryan T Gill. Application and engineering of fatty acid biosynthesis in Escherichia coli for advanced fuels and chemicals[J]. Metabolic Engineering.2011,13(1):28-37
[95]Lu X F, Vora H, Khosla C. Over production of free fatty acids in E. coli:Implications for biodiesel production[J]. Metabolic Engineering.2008,10(6):333-339
[96]Campbell W, Morgan-Kiss R M, Cronan J E. A new Escherichia coli metabolic competency:growth on fatty acids by anovel anaerobic beta-oxidation pathway [J]. Molecular Microbiology,2003,47(3):793-805
[97]Voelker T A, Davies H M. Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of plant medium-chain acyl-acyl carrier protein thioesterase[J]. Journal of Bacteriology.1994,176(23):7320-7327
[98]Dellomonaco C, Rivera C, Campbell P, Gonzalez R. Engineered respiro-fermentative metabolism for the production of biofuels and biochemicals from fatty acid-rich feedstocks[J]. Applied and Environmental Microbiology.2010,76(15):5067-5078
[99]Zhang Y M, Marrakchi H, Rock C O. The FabR(YijC) transcription factor regulates unsaturated fatty acid biosynthesis in Escherichia coli[J]. Journal of Biological Chemistry. 2002,277(18):15558-15565
[100]Campbell J W, Cronan J E. Escherichia coli FadR positively regulates transcription of the fabB fatty acid biosynthetic gene[J]. Journal of Bacteriology.2001,183(20):5982-5990
[101]Liu T G, Vora H, Khosla C. Quantitative analysis and engineering of fatty acid biosynthesis in E. coli[J]. Metabolic Engineering.2010,12(4):378-386
[102]Heath R J, Rock C O. Inhibition of beta-ketoacyl-acyl carrier protein synthase III (FabH) by acyl-acyl carrier protein in Escherichia coli[J]. Journal of Biological Chemistry.1996, 271(18):10996-11000
[103]Jha K, Sinha S, Maiti M K, Basu A, Mukhopadhyay U K, Sen S K. Functional expression of an acyl carrier protein(ACP) from Azospirillum brasilense alters fatty acid profiles in Escherichia coli and Brassica juncea[J]. Plant Physiology and Biochemistry.2007, 45(6-7):490-500
[104]Cho H S, Cronan J E. Defective export of a periplasmic enzyme disrupts regulation of fatty-acid synthesis[J].Journal of Biological Chemistry.1995,270(9):4216-4219
[105]Kalscheuer R, Stolting T, Steinbuchel A. Microdiesel:Escherichia coli engineered for fuel production[J]. Microbiology.2006,152:2529-2536
[106]王幼平,贾勇炯,罗鹏.某些特种植物资源的脂肪酸类型及用途[J].中国油料.1997,19(2)：72-76
[107]Dehesh K. How can we genetically engineer oilseed crops to produce high levels of medium-chain fatty acids? [J]. European Journal of Lipid Science and Technology.2001, 103:688-697
[108]Topfer R, Martini N, SCheH J. Modification of plant lipid synthesis[J]. Science.1995, 268(5211):681-686
[109]Voclk T, Kinney A J. Variations in the biosynthesis of seed storage lipids[J]. Annual Review of Plant Physiology and Plant Molecular Biology.2001,52:335-361
[110]Murphy D J. Engineering oil production in rapeseed and other oil crops[J]. Trends in Biotechnology.1996,14(6):206-213
[111]Davis H M, Anderson L, Fan C, Hawkins D J. Developmental induction, purification and further characterization of 12:0-ACP thioesterase from immature cotyledons of Umbeliularia california[J]. Archives Biochemistry Biophysics.1991,290(1):37-45
[112]李振华,温强,戴小英,江香梅.樟树资源利用现状与展望[J].江州林业科技.2007,6：30-33
[113]Dehesh K, Edwards P, Fillatti J, Slabaugh M, Byrne J. KAS Ⅳ:a 3-ketoacyl-ACP synthase from Cuphea sp. is a medium-chain specific condensing enzyme[J]. Plant Journal.1998,15(3):383-390
[114]Abbadi A, Bmmmel M, Spener F. Knockout of the regulatory site of a 3-ketoacyl-ACP synthaseⅢ enhances shortand mediuln-chain acyl-ACP synthesis[J]. Plant Journal.2000, 24(1):1-9
[115]Shintani D K, Ohlrogge J B. The characterization of a mitochondrial acyl carrier protein isoform isolated from Arabidopsis thaliana[J]. Plant Physiology.1994,104(4):1221-1229
[116]Sehutt B S, Brummel M, Schuch R, Spener F. The role of acyl carrier protein isoforms from Cuphea lanceolata seeds in the de-novo biosynthesis of medium-chain fatty acids[J]. Planta.1998,205(2):263-268
[117]Voelker T A, Davies H M. Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of a plant medium-chain acyl-acyl carrier protein thioesterase[J]. Journal of Bacteriology.1994,176(23):7320-7327
[118]Dormann P, Voelker T A, Ohlrogge J B. Accumulation of palmitate in Arabidopsis mediated by the acyl-acyl carrier protein thioesterase FATB1[J]. Plant Physiology.2000, 123(2):637-644
[119]Dehesh K, Edwards P, Hayes T R, Cranmer A M, Fillatti J. Two novel thioesterases are key determinants of the bimodal distribution of acyl chain length of Cuphea palustris seed oil[J]. Plant Physiology.1996,110(1):203-210
[120]Zhou Zh, Zhang D, Lu M. Cloning and expression analysis of PtFatB gene encoding the acyl-acyl carrier protein thioesterase in Populus tomen-tosa Carr[J]. Genetics and Genomics,2007,34(3):267-274
[121]Lu X, Vora H, Khosla C. Overproduction of free fatty acids in E. coli:implications for biodiesel production[J]. Metabolic Engineering.2008,10(6):333-339
[122]程涛,杨建明,刘辉,张英伟,宋秋华,咸漠.拟南芥硫酯酶基因(atFata)在大肠杆菌中的表达及其对游离脂肪酸合成的影响[J].应用与环境生物学报.2011,17(4)：568-571
[123]Jha J K, Maiti M K, Bhattacharjee A, Basu A, Sen PC, Sen SK. Cloning and functional expression of an acyl-ACP thioesterase FatB type from Diploknema (madhuca) butyracea seeds in Escherichia coli[J]. Plant Physiol Biochemistry.2006,44(11-12):645-655
[124]Mayer K M, Shanklin J. A structural model of the plant acyl-acyl carrier protein thioesterase FatB comprises two helix/4 stranded sheet domains, the N-terminal domain containing catalytic residues[J]. Journal of Biological Chemistry.2005,280(5):3621-3627
[125]沈奇,秦信蓉,杜才富.植物中短碳链脂肪酸的产生及其在油菜基因工程中的应用[J].种子.2008,27(11)：94-97
[126]Knutzon D S, Hayes R, Wyrick A, Hui Xiong, H Maelor Davies and Toni A Voelker. Lyso-phosphatidie acid acyhransferase from coconut endosperm mediates the insertion of laurate at the sn-2 position of triacylglycerols in laurie rapeseed oil and can increase total laurate levels[J]. Plant Physiology.1999,120(3):739-746
[127]史亚亚.樟树籽油制备轻质型生物柴油(D).南昌大学,2008.
[128]Paola D'Arrigo, Valentino Piergianni, Domenico Scarcelli, Stefano Servi. A spectrophotometric assay for phospholipase D[J]. Analytica Chimica Acta.1995,304(2): 249-254
[129]Jens Nielsen. Systems biology of lipid metabolism:From yeast to human[J]. FEBS Letters.2009,583(24):3905-3913
[130]Mingrui Yu, Shaowei Qin, Tianwei Tan. Purification and characterization of the extracellular lipase Lip2 from Yarrowia lipolytica. Process Biochemistry.2007,42(3): 384-391
[131]Song H T, Jiang Zh B, Ma L X. Expression and purification of two lipases from Yarrowia lipolytica AS 2.1216[J]. Protein Expression and Purification.2006,47(2):393-397
[132]Minning S, Serrano A, Ferrer P, Sola C, Schmid R D, Valero F. Optimization of the high-level production of Rhizopus oryzae lipase in Pichia pastoris[J]. Journal of Biotechnology.2001,86(1):59-70
[133]杨实权.生物柴油基因工程菌的构建及酿酒酵母产油脂条件研究(D).北京化工大学,2010.
[134]Valero E, Millan C, Ortega J M. Influence of oxygen addition during growth phase on the biosynthesis of lipids in Saccharomyces cerevisiae (M330-9) in enological fermentations[J]. Journal of Bioscience and Bioengineering.2001,92(6):33-38
[135]Xue F Y, Gao B, Zhu Y Q, Tan T W. Pilot-scale production of microbial lipid using starch wastewater as raw material[J]. Bioresource Technology.2010,101(15):6092-6095
[136]Zhang F Zh, Sarah R, Jay DK. Metabolic engineering of microbial pathways for advanced biofuels production[J]. Current Opinion in Biotechnology.2011,22(6):775-783
[137]Courchesne N M D, Parisien A, Wang B, Lan C Q. Enhancement of lipid production using biochemical, genetic and transcription factor engineering approaches[J]. Journal of Biotechnology.2009,141(1-2):31-41
[138]Yu J L, Zhang X, Tan T W. Optimization of media conditions for the production of ethanol from sweet sorghum juice by immobilized Saccharomyces cerevisiae[J]. Biomass and Bioenergy.2009,33(3):521-526
[139]Alexandre H, Rousseaux I, Charpentier C. Relationship between ethanol tolerance, lipid composition and plasma membrane fluidity in Saccharomyces cerevisiae and Kloeckera apiculata[J]. FEMS Microbiology Letters.1994,124(1):17-22
[140]Mannazzu I, Angelozzi D, Belviso S, Budroni M, Farris G A, Goffrini P, Lodi T, Marzona M, Bardi L. Behaviour of Saccharomyces cerevisiae wine strains during adaptation to unfavourable conditions of fermentation on synthetic medium:Cell lipid composition, membrane integrity, viability and fermentative activity[J]. International Journal of Food Microbiology.2008,121(1):84-91
[141]邓利,谭天伟,王芳.脂肪酶催化合成生物柴油研究[J].生物工程学报.2008,19(1)：45-55
[142]邓丹雯,张彬,周武.超临界CO2萃取樟树籽油脂的实验研究[J].食品科学.2003,24(9)：108-110
[143]侯继贤,鲁吉珂,聂开立,王芳,谭天伟.固定化Candida sp.99-125脂肪酶催化大豆油合成脂肪酸乙酯.过程工程学报.2008，8(2)：355-358
[144]Sun J, Yu B, Curran P, Liu S. Lipase-catalysed transesterification of coconut oil with fusel alcohols in a solvent-free system[J]. Food Chemistry.2012,134 (1):89-94
[145]Li Z, Deng L, Lu J, Guo X, Yang Z, Tan T. Enzymatic synthesis of fatty acid methyl esters from crude rice bran oil with immobilized Candida sp.99-125[J]. Chinese Journal Chemical Engineering.2010,18(5):870-875
[146]Tan T, Lu J, Nie K, Deng L, Wang F. Biodiesel production with immobilized lipase:a review[J]. Biotechnology Advances.2010,28(5):628-634
[147]Lu J, Chen Y, Wang F, Tan T. Effect of water on methanolysis of glycerol trioleate catalyzed by immobilized lipase Candida sp.99-125 in organic solvent system[J]. Journal of Molecular Catalyze B:Enzymatic.2009,56(2):122-125
[148]Hernandez-Martin E, Otero C. Different enzyme requirements for the synthesis of biodiesel:Novozym 435 and Lipozyme TL IM[J]. Bioresource Technology.2008, 99(2):277-286
[149]Lu J, Nie K, Xue F, Wang F, Tan T. Enzymatic synthesis of fatty acid methyl esters from lard with immobilized Candida sp.99-125[J]. Process Biochemistry.2007,42(9): 1367-1370
[150]Soumanou M M, Bornscheuer U T. Improvement in lipase-catalyzed synthesis of fatty acid methyl esters from sunflower oil[J]. Enzyme and Microbial Technology.2003,33(1): 97-103
[151]Guanyi Chen, Ming Ying and Weizhung Li. Enzymatic conversion of waste cooking oils into alternative fuel-Biodiesel[J]. Applied Biochemistry and Biotechnology.2006,132 (1-3):911-921
[152]Toni A Voelker, Thomas R Hayes, Ann M Cranmer, Joann C Turner, H Maelor Davies. Genetic engineering of a quantitative trait:metabolic and genetic parameters influencing the accumulation of laurate in rapeseed[J]. Plant Journal.1996,9(2):229-241