青蒿素前体合成酵母工程菌构建及发酵产物生物转化研究
|
摘要
青蒿素的化学本质是倍半萜内酯过氧化物,它是由我国科技工作者于20世纪70年代从传统中药青蒿中分离纯化的抗疟单体。作为一种次生代谢产物,青蒿素在青蒿中的含量极低。同时,野生青蒿资源非常有限,人工种植青蒿要占用大量耕地,化学合成青蒿素的反应复杂、成本高、毒性大,无法实现工业化生产,于是国内外兴起了一股利用基因工程微生物生产青蒿素的热潮。可是,到目前为止,还没有一种基因工程微生物能实现青蒿素的全合成,而只能合成青蒿素的前体。在成功构建表达青蒿紫穗槐二烯合酶基因(ADS)的酵母工程菌并获得青蒿素前体紫穗槐二烯的基础上,本研究进一步将青蒿素合成所需的细胞色素P450单加氧酶基因(CYP71AV1)和青蒿醛双键还原酶基因(DBR2)与ADS一起导入酵母菌中,通过PCR、双酶切和重测序确认了携带上述青蒿素合成基因的重组质粒在酵母工程菌中的存在,并利用表型鉴定和RT-PCR证实了重组青蒿素合成基因的功能表达。酸化水相GC-MS分析表明,酵母工程菌的发酵产物比野生型酵母明显增加,其中三基因转化酵母(ADS+CYP71AV1+DBR2)的产物多于双基因转化酵母(ADS+CYP71AV1),而双基因转化酵母(ADS+CYP71AV1)的产物又多于单基因转化酵母(ADS),表明不同青蒿合成酶基因在酵母工程菌中的表达导致了不同青蒿素前体的合成。有机相GC-MS分析测得紫穗槐二烯的生成,其含量为1.7μg/mL。为了将酵母合成的青蒿素前体通过酶促反应转变成青蒿素,本研究将上述不同酵母工程菌的正己烷抽提物与冷处理青蒿无细胞酶混合液保温进行生物转化,并通过HPLC测定其青蒿素含量。结果表明,转基因酵母生物转化产物中的青蒿素含量显著提高,最高达到3.44mg/mL,这是迄今为止青蒿素含量提高幅度最大的青蒿素高产代谢工程研究之一。本研究的创新之处在于：(1)成功培育能同时表达3个青蒿素合成酶基因的新型酵母工程菌；(2)将酵母工程菌生产的青蒿素前体通过生物转化技术转变成青蒿素；(3)建立了利用微生物(酵母)和植物(青蒿)相互合作生产青蒿素的“二步法”。本研究为充分发挥工业发酵规模性与农业种植经济性的双重优势大幅度提高青蒿素产量提供了理论依据,同时为初步解决目前青蒿素原料供给紧张局面满足全球抗疟药市场需求提出了一个可行的解决办法。
The chemical nature of artemisinin is the sesquiterpene lactone peroxide, which is an antimalarial monomer isolated and purified from the traditional Chinese medicinal herb Artemisia annua by Chinese scientists in 1970's. As a kind of secondary metabolites, artemisinin scarcely accumulates in A. annua. Additionally, the natural resource of A. annua is extremely rare; plantation of A. annua occupys a large area of cultivated fields; and chemical biosynthesis of artemisinin cannot be readily industrialized due to involving a complicated, costly and toxic process. In consequence, production of artemisinin in engineered microorganisms has been attempted worldwide although no artemisinin per se rather than artemisinin precursors has been synthesized in any engineered microorganisms up to the present. With success in construction of the engineered yeast that expresses amorphadiene synthase gene (ADS) of A. annua and production of the artemisinin precursor amorphadiene, we further introduce two other necessary genes for artemisinin biosynthesis, cytochrome P450 monooxygenase gene (CYP71AV1) and artemisinic aldehyde double-bond reductase gene (DBR2), into the yeast with ADS. The presence of the recombinant plasmid containing A. annua genes in the engineered yeast was verified by PCR, double digestion and re-sequencing, while the functional expression of recombinant artemisinin biosynthetic genes was validated by phenotyping and RT-PCR. GC-MS analysis of the acidic water phase indicated that fermtative products in three-type of engineered yeast strains were more than those in the wild-type yeast, in which products in the three-gene-transferred yeast were more than those in the two-gene-transferred yeast, whereas products in the two-gene-transferred yeast were more than those in the one-gene-transferred yeast, demonstrating that the expression of different artemisinin biosynthetic genes in engineered yeast cells led to the biosynthesis of distinct artemisinin precursors. GC-MS analysis of the organic phase confirmed the presence of amorphadiene with 1.7μg/mL. To enzymatically convert the yeast-produced artemisinin precursors into artemisinin, biotransformation by incubation of the hexane extract from each engineered yeast with the cell-free enzyme mixture of cold-acclimed A. annua was carried out and artemisinin content was monitored by HPLC. As results, artemisinin content in biotransformation products of transgenic yeast strains was 3.28mg/mL, accounting for approximately 10 folds higher than the control, which may represent one of the most enhanced artemisinin production projects of metabolic engineering aiming at artemisinin overproduction. The creative outcomes of the present study are that:(1) a novel engineered yeast that simultaneously express three artemisinin biosynthetic genes has been available; (2) artemisinin precursors produced by engineered yeast strains have been converted to artemisinin by the biotransformation procedure; (3) a "two-step method" for production of artemisinin by the interplay between the microorganism (yeast) and the plant(A. annua) has been established. The prospective applications of these achievements are providing thereotic supports for harnessing dual merits of the scaled industrial fermentation and the economic agricultural plantation to realize enhanced artemisinin production and to resolving the predicment in the insufficient artemisinin supply.
The chemical nature of artemisinin is the sesquiterpene lactone peroxide, which is an antimalarial monomer isolated and purified from the traditional Chinese medicinal herb Artemisia annua by Chinese scientists in 1970's. As a kind of secondary metabolites, artemisinin scarcely accumulates in A. annua. Additionally, the natural resource of A. annua is extremely rare; plantation of A. annua occupys a large area of cultivated fields; and chemical biosynthesis of artemisinin cannot be readily industrialized due to involving a complicated, costly and toxic process. In consequence, production of artemisinin in engineered microorganisms has been attempted worldwide although no artemisinin per se rather than artemisinin precursors has been synthesized in any engineered microorganisms up to the present. With success in construction of the engineered yeast that expresses amorphadiene synthase gene (ADS) of A. annua and production of the artemisinin precursor amorphadiene, we further introduce two other necessary genes for artemisinin biosynthesis, cytochrome P450 monooxygenase gene (CYP71AV1) and artemisinic aldehyde double-bond reductase gene (DBR2), into the yeast with ADS. The presence of the recombinant plasmid containing A. annua genes in the engineered yeast was verified by PCR, double digestion and re-sequencing, while the functional expression of recombinant artemisinin biosynthetic genes was validated by phenotyping and RT-PCR. GC-MS analysis of the acidic water phase indicated that fermtative products in three-type of engineered yeast strains were more than those in the wild-type yeast, in which products in the three-gene-transferred yeast were more than those in the two-gene-transferred yeast, whereas products in the two-gene-transferred yeast were more than those in the one-gene-transferred yeast, demonstrating that the expression of different artemisinin biosynthetic genes in engineered yeast cells led to the biosynthesis of distinct artemisinin precursors. GC-MS analysis of the organic phase confirmed the presence of amorphadiene with 1.7μg/mL. To enzymatically convert the yeast-produced artemisinin precursors into artemisinin, biotransformation by incubation of the hexane extract from each engineered yeast with the cell-free enzyme mixture of cold-acclimed A. annua was carried out and artemisinin content was monitored by HPLC. As results, artemisinin content in biotransformation products of transgenic yeast strains was 3.28mg/mL, accounting for approximately 10 folds higher than the control, which may represent one of the most enhanced artemisinin production projects of metabolic engineering aiming at artemisinin overproduction. The creative outcomes of the present study are that:(1) a novel engineered yeast that simultaneously express three artemisinin biosynthetic genes has been available; (2) artemisinin precursors produced by engineered yeast strains have been converted to artemisinin by the biotransformation procedure; (3) a "two-step method" for production of artemisinin by the interplay between the microorganism (yeast) and the plant(A. annua) has been established. The prospective applications of these achievements are providing thereotic supports for harnessing dual merits of the scaled industrial fermentation and the economic agricultural plantation to realize enhanced artemisinin production and to resolving the predicment in the insufficient artemisinin supply.
引文
1. WHO. Antimalarial drug combination therapy, report of a WHO technical consultation. April 2001.
2. Yamachika E, Habte T, Oda D, et al. Artemisinin:an alternative treatment for oral squamous cell carcinoma. Anticancer Res,2004,24:2153-2160.
3. Bouwmeester HJ, Wallaart TE, Janssen MHA, et al. Amorpha-4-11-diene synthase catalyses the first probable step in artemisinin biosynthesis. Phytochemistry,1999,52:843-854
4. Wallaart TE, Van UW, Lubberink HG, et al. Isolation and identification of dihydroartemisinic acid from Artemisia annua and its possible role in the biosynthesis of artemisinin. J Nat Prod,1999,62: 430-433.
5. Bertea CM, Freije JR, Woudeh VD, et al. Identification of intermediates and enzymes invoved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med,2005,71:40-47.
6. 王红,叶和春,刘本叶等.青蒿素生物合成分子调控研究进展.生物工程学报,2003,19(6)：646-650
7. Bouwmeester HJ, Wallaart TE, Janssen MH, et al. Amorpha-4,11-diene synthase catalyses the first probable step in artemisinin biosynthesis. Phytochemistry,1999; 52:843-854
8. Teoh KH, Polichuk DR, Reed DW, et al. Artemisia annua L. (Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin. FEBS Lett,2006,580:1411-1416
9. Ro DK, Paradise EM, Ouellet M, et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature,2006,440:940-943
10. Zhang Y, Teoch KH, Reed DW, et al. The molecular cloning of artemisinic aldehyde △11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua. J Biol Chem,2008,283:21501-21508
11. Zeng QP, Frank Q, Yuan L. Production of artemisinin by genetically-modified microbes. Biotechnol Lett,2008,30:581-592
12. El-Feraly FS, AI-Meshal LA, Alyahya MA, et al. On the possible role of Qinghao acid in the biosynthesis of artemisinin. Phytochemistry,1986,25:2777-2778.
13. Nair MS, Basile DV. Bioconversion of arteannuin B to artemisinin. J Nat Prod,1993,56: 1559-1566.
14.汪猷,夏志强,周凤仪等.青蒿素生物合成的研究：青蒿素和青蒿素B生物合成中的关键性中间体青蒿酸.化学学报,1988;46：1152-1153
15. Sangwan RS, Agarwal K, Luthra R, et al. Biotransformation of arteannuic acid into arteannuin B and artemisinin in Artemisia annua. Phytochemistry,1993,34:1301-1302
16. Wallaart TE, Van UW, Lubber IHG, et al. Isolation and identification of dihydroartemisinic acid from Artemisia annua and its possible role in the biosynthesis of artemisinin. J Nat Prod,1999,62: 430-433.
17. Sy LK, Brown GD. Themechanism of the spontaneous autoxidation of dihydroartemisinic acid. Tetrahedron,2002,58:897-908.
18. Brown GD, Sy LK. In vivo transformations of dihydroartemisinic acid in Artemisia annua plants. Tetrahedron,2004,60:1139-1159.
19. Wallaart TE, Pras N, Quax WJ, et al. Seasonal variation of artemisinin and its biosynthetic precursors in plants of Artemisia annua of different geographicalorigin:proof for the existence of chemotypes. Planta Med,2000,66:57-62
20. Chappell J, Wolf F, Proulx J, et al. Is the rcaction catalyzed by 3-hydroxyl--3-methylglutaryl coenzyme A reductase a rate-limiting step for isoprenoid biosynthesis in plants? Plant Physiol,1995, 109:1337-1343
21. Matsushit AY, Kang WK, Charlwood BV. Cloning and analysis of a cDNA encoding farnesyl diphosphate synthase from Artemisia annua L. Gene,1996,172:207-209.
22. Zhao YJ, Ye HC, Li GF, et al. Cloning and characterization of AaFPS cDNA in Artemisia annua L. Chin Sci Bull,2003,48:162-166.
23. Wallaart TE, Bouwmeester HJ, Hille J, et al. Amorpha-4,11-diene synthase:cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin. Planta,2001,212:460-465
24. Yin LL, Zhao C, Huang Y, et al. Abiotic streee-induced expression of artemisinin biosynthesis genes in Artemisia annua L. Chin App Environ Biol,2008,14:1-5
25. Bertea CM, Freije JR, Woude HVD, et al. Identification of intermediates and enzymes invoved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med,2005,71:40-47.
26. Keat H. Teoh, Devin R, et al.Covello Molecular cloning of an aldehyde dehydrogenase implicated in artemisinin biosynthesis in Artemisia annua.Botany,2009,87:635-642.
27. Covello P S,Teoh K H,Polichuk D R,et al.Functional genomics and the biosynthesis of artemisinin.Phytochem,2007,68:1864-1871
28. Martin V J J, P iterad J, Withers S T, et al. Engineering a mevalonate pathway in Escherich coli for production of terpenoids. Nat Biothchnol,2003,21:796-803.
29. Lindahl AL, Olsson ME, Mercke P, et al. Production of the artemisinin precursor amorpha-4, 11-diene by engineered Saccharomyces cerevisiae. Biotechnol Lett,2006,28:571-580.
30.潘维锋,李师鹏.酵母表达系统在植物功能基因组学研究中应用的局限性.植物生理学通讯,2006,42(6)：1168-1172
31. Gellissen G. Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol, 2000,54:741-750
32. Giga-Hama Y, Kumagai H. Expression system for foreign genes using the fission yeast Schizosaccharomyces pombe. Biotechnol Appl Biochem,1999,30:235-244
33. Valenzuela P, Medina R. Synthesis and assemble of hepatitis B virussurface antigen particles in yeast. Nature,298:347-350
34. Chemler JA,Yan Y, Koffas MA. Biosynthesis of isoprenoids, polyunsaturated fatty acids and flavonoids in Saccharomyces cerevisiae. Microb Cell Fact,2006,5:20
35.陈玉梅.酿酒酵母工程菌研究的进展.生物工程进展,1993,13(1)：11-16
36. Clare JJ, Rayment FB, Ballantine SP. High level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Biotechnology,1991,9: 455-460.
37. Paul M Sharp. Condon usage in yeast :cluster analysis clearly differentiates highly and lowly expressed genes. Nucleic Acids Res,1986,14:5125-5143.
38. Hoekema A. Codon replacement in the PGK1 gene:Experimental approach to study the role of codon usage in gene exp ression. Mol Cell Biol,1987:2914-2924.
39. Mellor J, Dobson MJ, Roberts NA, et al. Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae. Gene,1983,24:1-14
40. Zealey GR, Goodey AR, Piggott JR, et al. Amplification of plasmid copy number by thymidine kinase expression in Saccharomyces cerevisiae. Mol Gen Genet,1988,211:155-159
41. Lopes TS, De Wijs IJ, Steenhauer SI. Factor affecting the miotic stability of high-cope-number
integration into ribosomal DNA of Saccharomyces cerevisiae. Yeast,1996,12:467-477.
42. Cregg JM, Tschopp JF, Stillman C. High-level expression and efficient assembly of hepatitis B surface antigen in the methylitrophic yeast Pichia pastoris. Biotechnology,1987,5:479.
43. Thill GP, Davis GR, Stillman C. Positive and negative effect s of multicopy integrated expression vectors on protein expression in Pichia pastoris. Proceedings of the 6th international symposium on genetics of microorganisms. Societe Franscaise de Microbiologie. Paris,1990,477-490.
44. Shen YY, Wang XF, Wu FQ, et al. The Mg-chelatase H subunit is an abscisic acid receptor. Nature, 2006,443:823-826.
45.扈飞凡.植物生物转化技术在生物药物中的应用.赤峰学院学报,2008,24(6)：89-92
46. Dai JG, Ye M, Guo HZ, et al. Substrate specificity for the hydroxylation of polyoxygenated 4(20),11-taxadienes by Ginkgo cell suspension cultures. Bioorg Chem,2003,31:345-356.
47.刘洋洋,时杰,刘平怀.中药活性成分的生物转化研究进展.化学与生物工程,2009,26(7)：8-10
48. Surodjo S, Salim AA, Syahrani A, et al. Biotransformation of mefenamic acid by cell suspension cultures of Solanum mammosum. Nat Prod Comm,2008,3:257-262.
49. Hartanti L, Widjaja I, Syahrani A, et al. High yield formation of o-aminobenzoie acid-7-O-beta-D-(beta-1,6-O-D-glucopyranosyl)-glucopyranosyl ester in cell suspension cultures of Solanum mammosum. J Asian Nat Prod Res,2002,4:63-67.
50.冯冰,马百平.天然产物的生物转化研究进展.中草药,2005,36(6)：941-945.
51.严春艳,于荣敏.植物生物转化技术与中药活性化合物研究.食品与药品,2005,7(10)：4-8.
52.张羽飞,吴洪,姜忠义.生物转化中药化学成分的研究进展.生物加工过程,2005,3(3)：29-32.
53.吴薛明,何冰芳.耐有机溶剂极端微生物及酶类与天然产物的生物转化.中国天然药物,2007,5(4)：245-250.
54.王延亮,张庆林.epothilone类构效关系及生物合成、转化的研究进展.国外医学(药学分册),2007,34(1)：31-34.
55.汤亚杰,徐小玲,李艳,等.中药全成分生物转化.中国天然药物,2007,5(4)：241-244.
56.马骁驰,果德安.中药活性成分生物转化的研究思路与方法.中国天然药物,2007,5(3)：162-168.
57.欧阳平凯.加强生物催化与生物转化技术在我国药物源头创新中的应用.中国天然药物,2007,5(3)： 161.
58.王文兰,黄贤荣,张丽萍.药用植物细胞发酵培养的研究进展.实用医药杂志,2007,24(7)：867-869.
59. Bhushan B, Paguet L, Spain JC, et al. Biotransformation of 2,4,6,8,10,12-hexanitro-2,4,6,8,10, 12-hexaazaisowurtzitane (CL-20) by denitrifying pseudomonas sp. strain FA1. Appl Environ Microbiol,2003,69:5216-5221.
60. Bhushan B, Trott S, Spain JC, et al. Biotransformation of hexahydro-1,3,5-trinitm-1,3,5-triazine (RDX) by a rabbit liver cytochrome P450:insight into the mechanism of RDX biodegradation by rhodococcus sp. strain DN22. Appl Environ Microbiol,2003,69:1347-1351.
61. Jj Y, Bennett BM. Biotransformation of glyceryl trinitrate by rat hepatic mierosomal glutathione S-transferase 1. J Pharmacol Exp Ther,2006,318:1050-1056.
62. Berdikova Bohne VJ, Hamre K, Arukwe A. Hepatic biotransformation and metabolite profile during a 2-week depuration period in atlantic salmon fed graded levels of the synthetic antioxidant, ethoxyquin. Toxicol Sci,2006,93:11-21.
63. Lefebvre DD, Kelly D, Budd K. Biotransformation of Hg (Ⅱ) by eyanobacteria. Appl Environ Microbiol,2007,73(1):243-249.
64.李莉欣,苏艳芳,刘晓峰,等.大黄毛状根对青蒿素的生物转化研究.中国药学,2002,11(4)：122-124.
65.占纪勋,郭洪祝,韩健,等.华根霉和雅致小克银汉霉对青蒿素的生物转化研究.中草药,2002,33(10)：869-872.
66.韩健,戴均贵,崔亚君,等.长春花及银杏植物细胞悬浮培养对青蒿素的生物转化研究.中草药,2003,34(2)：166-168.
67.章焰生,叶和春,李国凤.辣根过氧化物酶在体外条件下对青蒿素生物合成的影响.应用与环境生物学报,2003,9(6)：616-618.
68.卢文婕.紫穗槐二烯合成酵母工程菌构建及鲨烯合酶基因表达阻遏代谢工程研究.学位论文,2009.
69.黄瑛,尹录录,冯丽玲,等.青蒿紫穗槐二烯合酶基因的克隆与表达.广州中医药大学学报,2008,25(1)：68-73
70. Kong JQ, Wang W, Wang LN, et al. The improvement of amorpha-4,11-diene production by a yeast-conform variant. J App Microbiol,2009,106:941-951
71.谭骏,刘昕.基因工程原理及实验操作技术,北京,科学技术文献出版社,1994,3：27-31.
72.J.萨母布鲁克,D.W.拉塞尔.分子克隆实验指南,北京,科学出版社,2002,3：1443-1447.
73. Keasling JD. Synthetic biology for synthetic chemistry. ACS Chem Biol 2008,3:64-76.
74. Feng LL, Yang RY, Yang XQ, et al. Synergistic re-channeling of mevalonate pathway for artemisinin overproduction in transgenic Artemisia annua. Plant Sci,2009,177:57-67
75. Mannan A, Liu C, Arsenault PR, et al. DMSO triggers the generation of ROS leading to an increase in artemisinin and dihydroartemisinic acid in Artemisia annua shoot clutures. Plant Cell Rep,2010, 29:143-152.
2. Yamachika E, Habte T, Oda D, et al. Artemisinin:an alternative treatment for oral squamous cell carcinoma. Anticancer Res,2004,24:2153-2160.
3. Bouwmeester HJ, Wallaart TE, Janssen MHA, et al. Amorpha-4-11-diene synthase catalyses the first probable step in artemisinin biosynthesis. Phytochemistry,1999,52:843-854
4. Wallaart TE, Van UW, Lubberink HG, et al. Isolation and identification of dihydroartemisinic acid from Artemisia annua and its possible role in the biosynthesis of artemisinin. J Nat Prod,1999,62: 430-433.
5. Bertea CM, Freije JR, Woudeh VD, et al. Identification of intermediates and enzymes invoved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med,2005,71:40-47.
6. 王红,叶和春,刘本叶等.青蒿素生物合成分子调控研究进展.生物工程学报,2003,19(6)：646-650
7. Bouwmeester HJ, Wallaart TE, Janssen MH, et al. Amorpha-4,11-diene synthase catalyses the first probable step in artemisinin biosynthesis. Phytochemistry,1999; 52:843-854
8. Teoh KH, Polichuk DR, Reed DW, et al. Artemisia annua L. (Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin. FEBS Lett,2006,580:1411-1416
9. Ro DK, Paradise EM, Ouellet M, et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature,2006,440:940-943
10. Zhang Y, Teoch KH, Reed DW, et al. The molecular cloning of artemisinic aldehyde △11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua. J Biol Chem,2008,283:21501-21508
11. Zeng QP, Frank Q, Yuan L. Production of artemisinin by genetically-modified microbes. Biotechnol Lett,2008,30:581-592
12. El-Feraly FS, AI-Meshal LA, Alyahya MA, et al. On the possible role of Qinghao acid in the biosynthesis of artemisinin. Phytochemistry,1986,25:2777-2778.
13. Nair MS, Basile DV. Bioconversion of arteannuin B to artemisinin. J Nat Prod,1993,56: 1559-1566.
14.汪猷,夏志强,周凤仪等.青蒿素生物合成的研究：青蒿素和青蒿素B生物合成中的关键性中间体青蒿酸.化学学报,1988;46：1152-1153
15. Sangwan RS, Agarwal K, Luthra R, et al. Biotransformation of arteannuic acid into arteannuin B and artemisinin in Artemisia annua. Phytochemistry,1993,34:1301-1302
16. Wallaart TE, Van UW, Lubber IHG, et al. Isolation and identification of dihydroartemisinic acid from Artemisia annua and its possible role in the biosynthesis of artemisinin. J Nat Prod,1999,62: 430-433.
17. Sy LK, Brown GD. Themechanism of the spontaneous autoxidation of dihydroartemisinic acid. Tetrahedron,2002,58:897-908.
18. Brown GD, Sy LK. In vivo transformations of dihydroartemisinic acid in Artemisia annua plants. Tetrahedron,2004,60:1139-1159.
19. Wallaart TE, Pras N, Quax WJ, et al. Seasonal variation of artemisinin and its biosynthetic precursors in plants of Artemisia annua of different geographicalorigin:proof for the existence of chemotypes. Planta Med,2000,66:57-62
20. Chappell J, Wolf F, Proulx J, et al. Is the rcaction catalyzed by 3-hydroxyl--3-methylglutaryl coenzyme A reductase a rate-limiting step for isoprenoid biosynthesis in plants? Plant Physiol,1995, 109:1337-1343
21. Matsushit AY, Kang WK, Charlwood BV. Cloning and analysis of a cDNA encoding farnesyl diphosphate synthase from Artemisia annua L. Gene,1996,172:207-209.
22. Zhao YJ, Ye HC, Li GF, et al. Cloning and characterization of AaFPS cDNA in Artemisia annua L. Chin Sci Bull,2003,48:162-166.
23. Wallaart TE, Bouwmeester HJ, Hille J, et al. Amorpha-4,11-diene synthase:cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin. Planta,2001,212:460-465
24. Yin LL, Zhao C, Huang Y, et al. Abiotic streee-induced expression of artemisinin biosynthesis genes in Artemisia annua L. Chin App Environ Biol,2008,14:1-5
25. Bertea CM, Freije JR, Woude HVD, et al. Identification of intermediates and enzymes invoved in the early steps of artemisinin biosynthesis in Artemisia annua. Planta Med,2005,71:40-47.
26. Keat H. Teoh, Devin R, et al.Covello Molecular cloning of an aldehyde dehydrogenase implicated in artemisinin biosynthesis in Artemisia annua.Botany,2009,87:635-642.
27. Covello P S,Teoh K H,Polichuk D R,et al.Functional genomics and the biosynthesis of artemisinin.Phytochem,2007,68:1864-1871
28. Martin V J J, P iterad J, Withers S T, et al. Engineering a mevalonate pathway in Escherich coli for production of terpenoids. Nat Biothchnol,2003,21:796-803.
29. Lindahl AL, Olsson ME, Mercke P, et al. Production of the artemisinin precursor amorpha-4, 11-diene by engineered Saccharomyces cerevisiae. Biotechnol Lett,2006,28:571-580.
30.潘维锋,李师鹏.酵母表达系统在植物功能基因组学研究中应用的局限性.植物生理学通讯,2006,42(6)：1168-1172
31. Gellissen G. Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol, 2000,54:741-750
32. Giga-Hama Y, Kumagai H. Expression system for foreign genes using the fission yeast Schizosaccharomyces pombe. Biotechnol Appl Biochem,1999,30:235-244
33. Valenzuela P, Medina R. Synthesis and assemble of hepatitis B virussurface antigen particles in yeast. Nature,298:347-350
34. Chemler JA,Yan Y, Koffas MA. Biosynthesis of isoprenoids, polyunsaturated fatty acids and flavonoids in Saccharomyces cerevisiae. Microb Cell Fact,2006,5:20
35.陈玉梅.酿酒酵母工程菌研究的进展.生物工程进展,1993,13(1)：11-16
36. Clare JJ, Rayment FB, Ballantine SP. High level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Biotechnology,1991,9: 455-460.
37. Paul M Sharp. Condon usage in yeast :cluster analysis clearly differentiates highly and lowly expressed genes. Nucleic Acids Res,1986,14:5125-5143.
38. Hoekema A. Codon replacement in the PGK1 gene:Experimental approach to study the role of codon usage in gene exp ression. Mol Cell Biol,1987:2914-2924.
39. Mellor J, Dobson MJ, Roberts NA, et al. Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae. Gene,1983,24:1-14
40. Zealey GR, Goodey AR, Piggott JR, et al. Amplification of plasmid copy number by thymidine kinase expression in Saccharomyces cerevisiae. Mol Gen Genet,1988,211:155-159
41. Lopes TS, De Wijs IJ, Steenhauer SI. Factor affecting the miotic stability of high-cope-number
integration into ribosomal DNA of Saccharomyces cerevisiae. Yeast,1996,12:467-477.
42. Cregg JM, Tschopp JF, Stillman C. High-level expression and efficient assembly of hepatitis B surface antigen in the methylitrophic yeast Pichia pastoris. Biotechnology,1987,5:479.
43. Thill GP, Davis GR, Stillman C. Positive and negative effect s of multicopy integrated expression vectors on protein expression in Pichia pastoris. Proceedings of the 6th international symposium on genetics of microorganisms. Societe Franscaise de Microbiologie. Paris,1990,477-490.
44. Shen YY, Wang XF, Wu FQ, et al. The Mg-chelatase H subunit is an abscisic acid receptor. Nature, 2006,443:823-826.
45.扈飞凡.植物生物转化技术在生物药物中的应用.赤峰学院学报,2008,24(6)：89-92
46. Dai JG, Ye M, Guo HZ, et al. Substrate specificity for the hydroxylation of polyoxygenated 4(20),11-taxadienes by Ginkgo cell suspension cultures. Bioorg Chem,2003,31:345-356.
47.刘洋洋,时杰,刘平怀.中药活性成分的生物转化研究进展.化学与生物工程,2009,26(7)：8-10
48. Surodjo S, Salim AA, Syahrani A, et al. Biotransformation of mefenamic acid by cell suspension cultures of Solanum mammosum. Nat Prod Comm,2008,3:257-262.
49. Hartanti L, Widjaja I, Syahrani A, et al. High yield formation of o-aminobenzoie acid-7-O-beta-D-(beta-1,6-O-D-glucopyranosyl)-glucopyranosyl ester in cell suspension cultures of Solanum mammosum. J Asian Nat Prod Res,2002,4:63-67.
50.冯冰,马百平.天然产物的生物转化研究进展.中草药,2005,36(6)：941-945.
51.严春艳,于荣敏.植物生物转化技术与中药活性化合物研究.食品与药品,2005,7(10)：4-8.
52.张羽飞,吴洪,姜忠义.生物转化中药化学成分的研究进展.生物加工过程,2005,3(3)：29-32.
53.吴薛明,何冰芳.耐有机溶剂极端微生物及酶类与天然产物的生物转化.中国天然药物,2007,5(4)：245-250.
54.王延亮,张庆林.epothilone类构效关系及生物合成、转化的研究进展.国外医学(药学分册),2007,34(1)：31-34.
55.汤亚杰,徐小玲,李艳,等.中药全成分生物转化.中国天然药物,2007,5(4)：241-244.
56.马骁驰,果德安.中药活性成分生物转化的研究思路与方法.中国天然药物,2007,5(3)：162-168.
57.欧阳平凯.加强生物催化与生物转化技术在我国药物源头创新中的应用.中国天然药物,2007,5(3)： 161.
58.王文兰,黄贤荣,张丽萍.药用植物细胞发酵培养的研究进展.实用医药杂志,2007,24(7)：867-869.
59. Bhushan B, Paguet L, Spain JC, et al. Biotransformation of 2,4,6,8,10,12-hexanitro-2,4,6,8,10, 12-hexaazaisowurtzitane (CL-20) by denitrifying pseudomonas sp. strain FA1. Appl Environ Microbiol,2003,69:5216-5221.
60. Bhushan B, Trott S, Spain JC, et al. Biotransformation of hexahydro-1,3,5-trinitm-1,3,5-triazine (RDX) by a rabbit liver cytochrome P450:insight into the mechanism of RDX biodegradation by rhodococcus sp. strain DN22. Appl Environ Microbiol,2003,69:1347-1351.
61. Jj Y, Bennett BM. Biotransformation of glyceryl trinitrate by rat hepatic mierosomal glutathione S-transferase 1. J Pharmacol Exp Ther,2006,318:1050-1056.
62. Berdikova Bohne VJ, Hamre K, Arukwe A. Hepatic biotransformation and metabolite profile during a 2-week depuration period in atlantic salmon fed graded levels of the synthetic antioxidant, ethoxyquin. Toxicol Sci,2006,93:11-21.
63. Lefebvre DD, Kelly D, Budd K. Biotransformation of Hg (Ⅱ) by eyanobacteria. Appl Environ Microbiol,2007,73(1):243-249.
64.李莉欣,苏艳芳,刘晓峰,等.大黄毛状根对青蒿素的生物转化研究.中国药学,2002,11(4)：122-124.
65.占纪勋,郭洪祝,韩健,等.华根霉和雅致小克银汉霉对青蒿素的生物转化研究.中草药,2002,33(10)：869-872.
66.韩健,戴均贵,崔亚君,等.长春花及银杏植物细胞悬浮培养对青蒿素的生物转化研究.中草药,2003,34(2)：166-168.
67.章焰生,叶和春,李国凤.辣根过氧化物酶在体外条件下对青蒿素生物合成的影响.应用与环境生物学报,2003,9(6)：616-618.
68.卢文婕.紫穗槐二烯合成酵母工程菌构建及鲨烯合酶基因表达阻遏代谢工程研究.学位论文,2009.
69.黄瑛,尹录录,冯丽玲,等.青蒿紫穗槐二烯合酶基因的克隆与表达.广州中医药大学学报,2008,25(1)：68-73
70. Kong JQ, Wang W, Wang LN, et al. The improvement of amorpha-4,11-diene production by a yeast-conform variant. J App Microbiol,2009,106:941-951
71.谭骏,刘昕.基因工程原理及实验操作技术,北京,科学技术文献出版社,1994,3：27-31.
72.J.萨母布鲁克,D.W.拉塞尔.分子克隆实验指南,北京,科学出版社,2002,3：1443-1447.
73. Keasling JD. Synthetic biology for synthetic chemistry. ACS Chem Biol 2008,3:64-76.
74. Feng LL, Yang RY, Yang XQ, et al. Synergistic re-channeling of mevalonate pathway for artemisinin overproduction in transgenic Artemisia annua. Plant Sci,2009,177:57-67
75. Mannan A, Liu C, Arsenault PR, et al. DMSO triggers the generation of ROS leading to an increase in artemisinin and dihydroartemisinic acid in Artemisia annua shoot clutures. Plant Cell Rep,2010, 29:143-152.