糖多孢红霉菌酮还原酶基因的克隆及其在手性醇中的应用
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摘要
具有特定功能基团的手性醇是合成手性药物的重要中间体,从羰基化合物不对称还原合成手性醇,已成为手性合成的一个重要部分。在羰基的不对称催化还原反应研究中,生物催化以其温和的反应条件、对环境的压力较小和较高的光学选择性,在羰基不对称还原合成中占有很重要的地位。选择合适的生物催化剂对于生物催化至关重要。2006年Bali等报道了短链脱氢酶家族(short-chain dehydrogenase superfamily, SDR)成员之一的糖多孢红霉菌聚酮合成酶中的酮还原酶对很多的底物,都有比较好的生物选择性还原能力,特别是对结构中含有环己酮的底物。
本实验以糖多孢红霉菌基因组作为DNA供体,并根据2005年Alexandros的报道设计特异性引物,扩增野生型的糖多孢红霉菌聚酮合成酶中的酮还原酶域基因(eryKR1)。再根据Genbank中报道的eryKR1基因和放线菌素聚酮合成酶中的酮还原酶基因(ActKR),设计了用于定点突变的特异性引物,利用重叠PCR技术将KR1中决定其底物特异性的位点定点突变为ActKR中决定其底物专一性的那段基因位点,得到突变的KR1片段命为eryKR1M。将其克隆到表达载体pET-28a上,从中挑选阳性克隆菌株pET-eryKR1和pET-eryKR1M进行测序。用同样的方法构建重组质粒pET-GDH作为辅酶再生。将重组质粒pET-GDH,pET-eryKR1和pET-eryKR1M转化到大肠杆菌BL21中进行表达。加定量的IPTG诱导6小时后,通过SDS-PAGE检测重组蛋白的表达。最后通过发酵检验野生型和突变后的酮还原酶对四种底物(4-氯乙酰乙酸乙酯,苯乙酮,2-辛酮和环己酮)还原作用。
结果表明,扩增eryKR1,eryKR1M基因的最佳退火温度为68℃,GDH基因的最佳退火温度为54℃。扩增的eryKR1,eryKR1M和GDH基因所对应的蛋白序列与报道的同源性高达98%。pET-eryKR1M中的目标基因与Genbank中报道的KR1基因仅在控制底物专一性的位点处不同,被放线菌素聚酮合成酶中的酮还原酶基因(ActKR)上的这段位点所取代,这也与我们设想一致。另外,SDS-PAGE检测出eryKR1,eryKR1M,GDH目标蛋白带。最后,发酵实验结果表明野生型的eryKR1对环己酮有很好的还原作用,而eryKR1M对环己酮的作用降低。这为今后该工程菌应用于手性醇的生物催化奠定了一定的基础。
Chiral alcohols with special functional groups are important building blocks for synthesizing chiral drug, and synthesizing chiral alcohols by the asymmetric reduction of carbonyl compounds has become an important part of chiral synthesis. Biocatalysis plays a very important role in carbonyl asymmetric reduction because of its mild reaction conditions, less stressful environment and efficient stereoselectivity. Screening the most suitable biocatalyst is critical. In 2006, Bali reported that ketoreductase of polyketides synthase in Saccharopolyspora erythraea as one of the members of short-chain dehydrogenase superfamily existed optimistic ability of biological selective reduction, especially to the substrates whose structures contain cyclohexanone.
In this work, DNA of S.erythraea was extracted.The primer sequences were designed according to the result Alexandros reported.We cloned the mild eryKR1 gene. Based on the nucleotide sequences of the ketoreductase from the first extension module of the erythromycin polyketide synthase, and the nucleotide sequences of ketoreductase of polyketide synthase in Actinorhodin (ActKR) in the Genbank, gene specific primers were designed. Through the overlapping PCR manner, the gene sites determining its substrate specificity in the ketoreductase (KR1) domain is replaced by that determining its specificity in ActKR, and we get the mutated KR1 domain DNA fragment eryKR1M. We cloned eryKR1 and eryKR1M into vector pET-28a, built the plasmid pET-eryKR1M and introduced the plasmid pET-eryKR1M and pET-eryKR1 into Escherichia coli BL21.Then we built the plasmid pET-GDH with the same method as the coenzyme regeneration. After 6 hours of inducing by IPTG, we detected the expression of the protein eryKR1M ,eryKR1,GDH by SDS-PAGE. At last, we examined its effect on four kinds of different substrates (ethyl 4-chloro-3-oxobutanoate, acetophenone, 2-octanone and cyclohexanone) using the fermentation technology.
The results showed that the best annealing temperature was 68℃.The protein sequences of the cloned genes had the high homology compared with the reported protein sequences(reached 98%). The target gene of pET-eryKR1M differented from the gene of the ketoreductase from the first extension module of the erythromycin polyketide synthase only in the sites determing the specificity of the substrates, and was replaced by the sites of ketoreductase of polyketide synthase in Actinorhodin(ActKR), which is accordant with our initial presumption. In addition, through SDS-PAGE, all the target protein eryKR1M,eryKR1,GDH were detected. At last, the results of fermentation showed the wild gene existed optimic effects on Cyclohexanone ,which suggests that the engineering bacteria would be used for the biocatalysis of chiral alcohols the in the future.
本实验以糖多孢红霉菌基因组作为DNA供体,并根据2005年Alexandros的报道设计特异性引物,扩增野生型的糖多孢红霉菌聚酮合成酶中的酮还原酶域基因(eryKR1)。再根据Genbank中报道的eryKR1基因和放线菌素聚酮合成酶中的酮还原酶基因(ActKR),设计了用于定点突变的特异性引物,利用重叠PCR技术将KR1中决定其底物特异性的位点定点突变为ActKR中决定其底物专一性的那段基因位点,得到突变的KR1片段命为eryKR1M。将其克隆到表达载体pET-28a上,从中挑选阳性克隆菌株pET-eryKR1和pET-eryKR1M进行测序。用同样的方法构建重组质粒pET-GDH作为辅酶再生。将重组质粒pET-GDH,pET-eryKR1和pET-eryKR1M转化到大肠杆菌BL21中进行表达。加定量的IPTG诱导6小时后,通过SDS-PAGE检测重组蛋白的表达。最后通过发酵检验野生型和突变后的酮还原酶对四种底物(4-氯乙酰乙酸乙酯,苯乙酮,2-辛酮和环己酮)还原作用。
结果表明,扩增eryKR1,eryKR1M基因的最佳退火温度为68℃,GDH基因的最佳退火温度为54℃。扩增的eryKR1,eryKR1M和GDH基因所对应的蛋白序列与报道的同源性高达98%。pET-eryKR1M中的目标基因与Genbank中报道的KR1基因仅在控制底物专一性的位点处不同,被放线菌素聚酮合成酶中的酮还原酶基因(ActKR)上的这段位点所取代,这也与我们设想一致。另外,SDS-PAGE检测出eryKR1,eryKR1M,GDH目标蛋白带。最后,发酵实验结果表明野生型的eryKR1对环己酮有很好的还原作用,而eryKR1M对环己酮的作用降低。这为今后该工程菌应用于手性醇的生物催化奠定了一定的基础。
Chiral alcohols with special functional groups are important building blocks for synthesizing chiral drug, and synthesizing chiral alcohols by the asymmetric reduction of carbonyl compounds has become an important part of chiral synthesis. Biocatalysis plays a very important role in carbonyl asymmetric reduction because of its mild reaction conditions, less stressful environment and efficient stereoselectivity. Screening the most suitable biocatalyst is critical. In 2006, Bali reported that ketoreductase of polyketides synthase in Saccharopolyspora erythraea as one of the members of short-chain dehydrogenase superfamily existed optimistic ability of biological selective reduction, especially to the substrates whose structures contain cyclohexanone.
In this work, DNA of S.erythraea was extracted.The primer sequences were designed according to the result Alexandros reported.We cloned the mild eryKR1 gene. Based on the nucleotide sequences of the ketoreductase from the first extension module of the erythromycin polyketide synthase, and the nucleotide sequences of ketoreductase of polyketide synthase in Actinorhodin (ActKR) in the Genbank, gene specific primers were designed. Through the overlapping PCR manner, the gene sites determining its substrate specificity in the ketoreductase (KR1) domain is replaced by that determining its specificity in ActKR, and we get the mutated KR1 domain DNA fragment eryKR1M. We cloned eryKR1 and eryKR1M into vector pET-28a, built the plasmid pET-eryKR1M and introduced the plasmid pET-eryKR1M and pET-eryKR1 into Escherichia coli BL21.Then we built the plasmid pET-GDH with the same method as the coenzyme regeneration. After 6 hours of inducing by IPTG, we detected the expression of the protein eryKR1M ,eryKR1,GDH by SDS-PAGE. At last, we examined its effect on four kinds of different substrates (ethyl 4-chloro-3-oxobutanoate, acetophenone, 2-octanone and cyclohexanone) using the fermentation technology.
The results showed that the best annealing temperature was 68℃.The protein sequences of the cloned genes had the high homology compared with the reported protein sequences(reached 98%). The target gene of pET-eryKR1M differented from the gene of the ketoreductase from the first extension module of the erythromycin polyketide synthase only in the sites determing the specificity of the substrates, and was replaced by the sites of ketoreductase of polyketide synthase in Actinorhodin(ActKR), which is accordant with our initial presumption. In addition, through SDS-PAGE, all the target protein eryKR1M,eryKR1,GDH were detected. At last, the results of fermentation showed the wild gene existed optimic effects on Cyclohexanone ,which suggests that the engineering bacteria would be used for the biocatalysis of chiral alcohols the in the future.
引文
[1]尤启东,林国强.手性药物[M].北京:化学工业出版社, 2004, 26-47
[2]许关煜,刘玲玲.手性药物[J].上海医药, 1999, 20(10): 9-11
[3]王普善.手性药物的生物合成[J].精细与专用化学品, 1999, 7(5): 9-11
[4]曾嵘,杨忠华,姚善泾.生物催化羰基不对称还原合成手性醇的研究及应用进展[J].化工进展, 2004, 23(11): 1169-1172
[5]陶文沂,李江华.生物催化剂在制药工业的应用[J].无锡轻工大学学报, 2002, 21(5): 538-544
[6]林国强,陈耀全,陈新滋,等.手性合成[M].北京:北京出版社, 2000
[7]欧阳平凯,卢定强,资源生态化利用中的生物加工过程[J].生物加工过程, 2003,1: 1-6
[8]孙志浩.手性技术中的微生物或酶拆分方法.生物加工技术研讨会集, 2000, 25-33
[9] Nakamura K, Yamanaka R, Matsuda T, et al. Recent developments in asymmetric reduction of ketones with biocatalyst [J]. Tetrahedron: Asymmetry, 2003, 14: 2659-2681
[10]欧志敏,吴坚平,杨立荣,等.微生物法还原羰基化合物生产手性醇的研究[J].山东大学学报, 2003, 34 (3): 459-462
[11]魏志亮,李祖义,林国强.生物还原反应在手性药物不对称合成中的应用[J].有机化学, 2001, 6: 403- 412
[12]娄文勇,宗敏华,王菊芳,等.马肝醇脱氢酶催化有机硅酮不对称还原反应动力学[J].生物化学与生物物理进展, 2003, 30(3): 431-434
[13] Chartrain M, Lynch J, Choi W B, et al. Asymmetric bioreduction of a bisaryl ketone to its corresponding (S)-bisaryl alcohol by the yeast Rhodotorula pilimanae ATCC 32762[J] . Journal of Molecular Catalysis B: Enzymatic, 2000, 8(4-6): 285-288
[14] Groger H,Hummel W,Rollman C,et al. Preparative asymmetric reduction of ketones in a biphasic medium with an (S)-alcohol dehydrogenase under in situ-cofactor-recycling with formate dehydrogenase[J]. Tetrahedron, 2004, 60: 633-640
[15] Nakamura K, Yamanaka R, Tohi K, et al. Cyanobacterium-catalyzed asymmetric reduction of ketones [J]. Tetrahedron Lett, 2000, 41: 6799-6802
[16] Nakamtlra K, Yamanaka R. Light mediated cofactor recycling system in biocatalytic asymmetric reduction of ketone[J]. Chem .Commun, 2002, 16: 1782-1783
[17] Nakamura K, Yamanaka R. Light-mediated regulation of asymmetric reduction of ketones by a cyanobacterium[J]. Tetrahedron: Asymmetry, 2002, 13: 2529-2533
[18] Yuan R, Watanabe S, Kuwabata S, et al. Asymmetric electroreduction of ketone and aldehyde derivatives to the corresponding alcohols using alcohol dehydrogenase as an electrocatalyst [J]. J .Org.Chem, 1997, 62: 2494-2499
[19] Leonida M D, Sobolov S B, Fry A J. FAD-mediated enzymatic conversion of NAD+ toNADH: Application to chiral synthesis of L-lactate [J]. Bioorg Med Chem Lett, 1998, 8: 2819-2824.
[20] Yamada H, Shimizu S, Kataoka M, et al. A novel NADPH-dependent aldehyde reductase, catalyzing asymmetric reduction ofβ-keto acid esters, from Sporobolomyces salmonicolor: purification and characterization[J] . FEMS Microbiol Lett, 1990, 70(1): 45-48
[21] Wada M, Kataoka M, Kawabata H, et al. Purification and characterization of NADPH-dependent carbonyl reductase, involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate from Candida magnoliae[J] . Biosci Biotechol Biochem, 1998, 62(2): 280-285
[22] Wada M, Kawabata H, Kataoka M, et al. Purification and characterization of an aldehyde reductase from Candida magnoliae [J] . Journal of Molecular Catalysis B: Enzymatic, 1999, 6(3): 333-339
[23] Itoh N, Matsuda M, Mabuchi M, et al. Chiral alcohol production by NADH- dependent phenylacetaldehyde reductase coupled with in situ regeneration of NADH [J] . Eur J Biochem, 2002, 269(9): 2394-2402
[24] Kataoka M, Yamamoto K, Kawabata H, et al. Stereoselective reduction of ethyl 4-chloro-3-oxobutanoate by Escherichia coli transformant cells coexpressing the aldehyde reductase and glucose dehydrogenase genes[J] . Appl Microbiol Biotechnol, 1999, 51(4): 486-490
[25] Kita K, Matsuzaki K, Hashimoto T, et al. Cloning of the aldehyde reductase gene from a red yeast, Sporobolomyces salmonicolor, and characterization of the gene and its product [J] . Applied and environmental microbiology, 1996, 62(7): 2303-2310
[26] Wang J C, Sakakibara M, Liu J Q, et al. Cloning sequence analysis and expression in Escherichia coli of the gene encoding phenylacetaldehyde reductase from styrene- assimilating Corynebacterium sp. strain ST-10[J]. Appl Microbiol Biotechnol, 1999, 52(3): 386-392
[27] Wada M, Kawabata H, Yoshizumi A, et al. Occurrence of multiple ethyl 4-chloro-3-oxobutanoate-reducing enzymes in Candida magnoliae[J]. Journal of Bioscience and Bioengineering, 1999, 87(2): 144-148
[28] Siskos A P, Baerga-O A, Bali S, et al. Molecular basis of celmer’s rules: stereochemistry of catalysis by isolated ketoreductase domains from modular polyketide synthases [J]. Chem Biol, 2005, 12(10): 1145-1153
[29] Korman T P, Hill J A, Vu T N, et al. Structural analysis of actinordin ketoreductase:cofactor binding and substrate specificity[J]. Biochemistry, 2004, 43(46): 14529-14538
[30] Bali S O, Hare H M, Weissman K J. Broad substrate specificity of ketoreductases derived from modular polyketide synthases[J]. Chem Biochem, 2006, 7(3): 478-484
[31] Allen S J, Holbrook J. Production of an activated form of Bacillus stearothermophilus L-2-hydroxyacid dehydrogenase by directed evolution[J].Protein Eng, 2000, 13:5-7
[32] Wendhausen J R, Moran P J S, Joekes l, et al.Continuous process for large-scale preparation of chiral alcohols with bakers’yeast immobilized on chrysotile fibers[J]. J. Mol. Catal B:Enzymatic, 1998, 5: 69-73
[33] Vansonsbeek H M, Beeftink H H, Tramper J.Two liquid-phase bioreactors[J]. Enzyme Microb Technol, 1993, 15: 722-728
[34] Buque E M, Chin-Joel, Strathof A J J, et al. Immobilization affects the rate and enantioselectivity of 3-oxo ester reduction by bakers’yeast[J]. Enzyme Microb Technol,2002, 31: 656-664
[35] Lou W, Zong M, Zhang Y Y, et al. Efficient synthesis of optically active organosilyl alcohol via Asymmetric reduction of acyl silane with immobilized yeast[J]. Enzyme Microb Technol, 2004, 35: 190-196
[36] Green K D, Gill l S, Khan J A, et al.Microencapsulation of yeast cells and their use as a biocata1yst in ogranic solvents[J]. Biotechnol Bioeng, 1996, 49(5): 535-543.
[37] Liu X, Zhu T, Sun P,et al.Asymmetric reduction of aromatic ketones by the bakers’yeast in ogranic solvent system[J]. Synthetic commun, 2001, 31(10): 1521-1526.
[38] Rothaus O, Kruger D, Demuth M, et al. Reduction of ketoesters with Baker’s yeast in organic solvents–A comparision with the results in water[J]. Tetrahedro, 1997, 53(3): 935-938
[39] Cui J N, Teraoka R, Ema T, et al. lighly Regio-and Enantioseleclive reduction on 1-Chloro-2,4-alkanediones using BakercYeast:Effect of organic solvents as Additives[J]. Trahedron Lett, 1997, 38(17) : 3021-3024
[40] Cui J N, EmaT , Sakai T, et al.Control of enantiose1ectivity in the bakers yeast asymmetric reduction ofγ-chloro-β-diketones toγ-chloro-β-hydroxyketones[J].Tetrabedron:Asymmetry, 1998, 9: 2681-2692
[41] Matsuda T, Harada T, Nakamura K. Alcohol dehydrogenase is active in supercritical carbondioxide[J]. Chem commun, 2000, 15: 1167-1368
[42] Matsuda T, Watanabe K, Kamitanaka T, et al. Biocatalytic reduction of ketones by a semi-continuous flow process using supercritical cabrondioxide[J]. Chem Commun, 2003, 10: 1198-1199
[43] Howarth J, James P, Dai J. Immobilized bakers yeast reduction of ketones in an ionicliquic [bmim] P F6 and water mix[J]. Tetrahedron Let, 2001, 42: 7517-7519
[44] Nakamura K, Inoue K, Ushio K, et al.Sterochemical control on yeast reduction ofα-keto easter:Reduction by immobilized bakers yeast in hexane[J]. Org Chem l988, 53(11): 2589-2593
[45] Zhou B N, Gopalan A S, VanMiddlesworth F, et al. Stereochemical control of yeast reduction Asymmetric synthesis of L-carntine[J]. J Am Chem soc, 1983, 105: 5925-5926
[46] Ferraboschi P, Grisenti P, Manzocchi A, et al. Bakers yeast rnediated preparation of optically activaryl alcohols and diols for the synthesis of chiral hydroxyacids[J]. Chem Soc Perkin Transl, 1990, 9: 2469-2474.
[47] Dao D H, Okamura M, Akasaka T, et al.Stereochemical control in microbial reduction: Reduction of alkyl 2-oxo-4-arylbutyrates by bakers yeast under selected reaction conditions [J]. Tetrahedron:Asmmetry, 1998, 9(15): 2725-2737
[48] Yasohara Y, Kizaki N, Hasegawa J, et al.Synthesis of optically active ethyl-4-chloro-3-hydroxybutanoate by microbial reduction[J]. Appl.Microbiol Biotechnol, 1999, 51: 847-851
[49]. Dahl A, Fjeldberg M, Madsen J O. Bakers yeast:improving the D-stereoselectivity in reduction on 3-oxo ester[J]. Tetrahedron:Asymmetry, 1999, 10: 551-559.
[50] Naoshima Y, Maeda J, Munakata Y. Bioreduction with immobilized bakers yeast in hexane using alcohols as an energy source[J]. J Chem Soc Chem Commun, 1990, 14: 964-965
[51] Nakamura K, Inoue Y, Ohono A. Improvement of enantioselectivity of microbial reduction by using ograic solvent redox coupler system[J].Tetarhedron Lett, 1995, 36(2): 265-266
[52] Dahl A C, Madsen J O. Bakers yeast: Production of D-andL-3-hydoxy esters[J]. Tetrahedron.Asymmetry, 1998, 9: 4395-4417.
[53] Nakamura K, Yamanaka R, Matsuda T, et al. Recent deve1opments in asymmetric reduction of ketones with biocatalysist[J]. Tetrahedron: Asymmetry,2003,14: 2659-2681
[54] Nakamura K, Kawai Y, Ohno A. A novel method to synthesize (L)-β-hydroxyl esters by the reduction with bakers yeast[J]. Tetrahedron Lett, 1990, 31: 267-270
[55] Nakamura K, Kawai Y, Oka S, Ohno A. A new method for stereochemical control of microbiral reduction.Reduction ofβ-ketoesters with bakers yeast immobilized by magnesium alginate[J]. Tetarhedron lett, 1989, 30: 2245-2246
[56]张部昌.新酮内酯类抗生素组合生物合成的研究[D].军事医学科学院博士学位论文, 2002: 3-103
[57]张部昌,赵志虎,马清钧.红霉素生物合成的分子生物学[J].生物技术通讯, 2001, 12(2): 151-160
[58]张部昌,赵志虎,马清钧.红霉素基因工程研究进展[J].中国生物工程杂志, 2002, 22(3):40-44
[59]李凌凌.糖多孢红霉菌λC3-SRR突变体构建及其产物鉴定[D].安徽大学硕士学位论文. 2005: 3-48
[60] Weissman K J. Polyketide biosynthesis: understanding and exploiting modularity[J] . Philos Transact A Math Phys Eng Sci, 2004, 362(1825): 2671-2690
[61] Kim C Y, Alekseyev V Y, Chen A Y, et al. Reconstituting modular activity from separated domains of 6-deoxyerythronolide B synthase[J] . Biochemistry, 2004, 43(44): 13892-13898
[62] Korman T P, Hill J A, Vu T N, et al. Structural analysis of actinordin ketoreductase:cofactor binding and substrate specificity[J] . Biochemistry, 2004, 43(46): 14529-14538
[63] Sambrook J, Frisch E F, Maniatis T.分子克隆实验指南[M].金冬雁译,第二版.北京:科学出版社, 1992: 1-600
[64]杨建雄.生物化学与分子生物学实验技术教程[M].北京:科学出版社, 1996:1~226
[65] Alexandros P S, Abel B O, Shilpa, et al. Molecular basis of calmers rules: Stereochemistry of catalysis by isolated ketoreductase Domains from modular polyketide synthase[J]. Chemistry and Biology, 2005, 12:1145-1153
[66] Adrian T, Keatinge C, Robert M. The structure of a ketoreductase determines the organization of theβ-carbon processing enzymes of modular polyketide synthase[J]. Structure, 2006, 14: 737-748
[67] Tyler P K, Jason A H, Thanh N V, et al. Structurl analysis of Actinor hodin polyketide ketoreductase: cofactor binding and substrate specificity[J]. Biochemistry, 2004, 43: 14529-14538
[2]许关煜,刘玲玲.手性药物[J].上海医药, 1999, 20(10): 9-11
[3]王普善.手性药物的生物合成[J].精细与专用化学品, 1999, 7(5): 9-11
[4]曾嵘,杨忠华,姚善泾.生物催化羰基不对称还原合成手性醇的研究及应用进展[J].化工进展, 2004, 23(11): 1169-1172
[5]陶文沂,李江华.生物催化剂在制药工业的应用[J].无锡轻工大学学报, 2002, 21(5): 538-544
[6]林国强,陈耀全,陈新滋,等.手性合成[M].北京:北京出版社, 2000
[7]欧阳平凯,卢定强,资源生态化利用中的生物加工过程[J].生物加工过程, 2003,1: 1-6
[8]孙志浩.手性技术中的微生物或酶拆分方法.生物加工技术研讨会集, 2000, 25-33
[9] Nakamura K, Yamanaka R, Matsuda T, et al. Recent developments in asymmetric reduction of ketones with biocatalyst [J]. Tetrahedron: Asymmetry, 2003, 14: 2659-2681
[10]欧志敏,吴坚平,杨立荣,等.微生物法还原羰基化合物生产手性醇的研究[J].山东大学学报, 2003, 34 (3): 459-462
[11]魏志亮,李祖义,林国强.生物还原反应在手性药物不对称合成中的应用[J].有机化学, 2001, 6: 403- 412
[12]娄文勇,宗敏华,王菊芳,等.马肝醇脱氢酶催化有机硅酮不对称还原反应动力学[J].生物化学与生物物理进展, 2003, 30(3): 431-434
[13] Chartrain M, Lynch J, Choi W B, et al. Asymmetric bioreduction of a bisaryl ketone to its corresponding (S)-bisaryl alcohol by the yeast Rhodotorula pilimanae ATCC 32762[J] . Journal of Molecular Catalysis B: Enzymatic, 2000, 8(4-6): 285-288
[14] Groger H,Hummel W,Rollman C,et al. Preparative asymmetric reduction of ketones in a biphasic medium with an (S)-alcohol dehydrogenase under in situ-cofactor-recycling with formate dehydrogenase[J]. Tetrahedron, 2004, 60: 633-640
[15] Nakamura K, Yamanaka R, Tohi K, et al. Cyanobacterium-catalyzed asymmetric reduction of ketones [J]. Tetrahedron Lett, 2000, 41: 6799-6802
[16] Nakamtlra K, Yamanaka R. Light mediated cofactor recycling system in biocatalytic asymmetric reduction of ketone[J]. Chem .Commun, 2002, 16: 1782-1783
[17] Nakamura K, Yamanaka R. Light-mediated regulation of asymmetric reduction of ketones by a cyanobacterium[J]. Tetrahedron: Asymmetry, 2002, 13: 2529-2533
[18] Yuan R, Watanabe S, Kuwabata S, et al. Asymmetric electroreduction of ketone and aldehyde derivatives to the corresponding alcohols using alcohol dehydrogenase as an electrocatalyst [J]. J .Org.Chem, 1997, 62: 2494-2499
[19] Leonida M D, Sobolov S B, Fry A J. FAD-mediated enzymatic conversion of NAD+ toNADH: Application to chiral synthesis of L-lactate [J]. Bioorg Med Chem Lett, 1998, 8: 2819-2824.
[20] Yamada H, Shimizu S, Kataoka M, et al. A novel NADPH-dependent aldehyde reductase, catalyzing asymmetric reduction ofβ-keto acid esters, from Sporobolomyces salmonicolor: purification and characterization[J] . FEMS Microbiol Lett, 1990, 70(1): 45-48
[21] Wada M, Kataoka M, Kawabata H, et al. Purification and characterization of NADPH-dependent carbonyl reductase, involved in stereoselective reduction of ethyl 4-chloro-3-oxobutanoate from Candida magnoliae[J] . Biosci Biotechol Biochem, 1998, 62(2): 280-285
[22] Wada M, Kawabata H, Kataoka M, et al. Purification and characterization of an aldehyde reductase from Candida magnoliae [J] . Journal of Molecular Catalysis B: Enzymatic, 1999, 6(3): 333-339
[23] Itoh N, Matsuda M, Mabuchi M, et al. Chiral alcohol production by NADH- dependent phenylacetaldehyde reductase coupled with in situ regeneration of NADH [J] . Eur J Biochem, 2002, 269(9): 2394-2402
[24] Kataoka M, Yamamoto K, Kawabata H, et al. Stereoselective reduction of ethyl 4-chloro-3-oxobutanoate by Escherichia coli transformant cells coexpressing the aldehyde reductase and glucose dehydrogenase genes[J] . Appl Microbiol Biotechnol, 1999, 51(4): 486-490
[25] Kita K, Matsuzaki K, Hashimoto T, et al. Cloning of the aldehyde reductase gene from a red yeast, Sporobolomyces salmonicolor, and characterization of the gene and its product [J] . Applied and environmental microbiology, 1996, 62(7): 2303-2310
[26] Wang J C, Sakakibara M, Liu J Q, et al. Cloning sequence analysis and expression in Escherichia coli of the gene encoding phenylacetaldehyde reductase from styrene- assimilating Corynebacterium sp. strain ST-10[J]. Appl Microbiol Biotechnol, 1999, 52(3): 386-392
[27] Wada M, Kawabata H, Yoshizumi A, et al. Occurrence of multiple ethyl 4-chloro-3-oxobutanoate-reducing enzymes in Candida magnoliae[J]. Journal of Bioscience and Bioengineering, 1999, 87(2): 144-148
[28] Siskos A P, Baerga-O A, Bali S, et al. Molecular basis of celmer’s rules: stereochemistry of catalysis by isolated ketoreductase domains from modular polyketide synthases [J]. Chem Biol, 2005, 12(10): 1145-1153
[29] Korman T P, Hill J A, Vu T N, et al. Structural analysis of actinordin ketoreductase:cofactor binding and substrate specificity[J]. Biochemistry, 2004, 43(46): 14529-14538
[30] Bali S O, Hare H M, Weissman K J. Broad substrate specificity of ketoreductases derived from modular polyketide synthases[J]. Chem Biochem, 2006, 7(3): 478-484
[31] Allen S J, Holbrook J. Production of an activated form of Bacillus stearothermophilus L-2-hydroxyacid dehydrogenase by directed evolution[J].Protein Eng, 2000, 13:5-7
[32] Wendhausen J R, Moran P J S, Joekes l, et al.Continuous process for large-scale preparation of chiral alcohols with bakers’yeast immobilized on chrysotile fibers[J]. J. Mol. Catal B:Enzymatic, 1998, 5: 69-73
[33] Vansonsbeek H M, Beeftink H H, Tramper J.Two liquid-phase bioreactors[J]. Enzyme Microb Technol, 1993, 15: 722-728
[34] Buque E M, Chin-Joel, Strathof A J J, et al. Immobilization affects the rate and enantioselectivity of 3-oxo ester reduction by bakers’yeast[J]. Enzyme Microb Technol,2002, 31: 656-664
[35] Lou W, Zong M, Zhang Y Y, et al. Efficient synthesis of optically active organosilyl alcohol via Asymmetric reduction of acyl silane with immobilized yeast[J]. Enzyme Microb Technol, 2004, 35: 190-196
[36] Green K D, Gill l S, Khan J A, et al.Microencapsulation of yeast cells and their use as a biocata1yst in ogranic solvents[J]. Biotechnol Bioeng, 1996, 49(5): 535-543.
[37] Liu X, Zhu T, Sun P,et al.Asymmetric reduction of aromatic ketones by the bakers’yeast in ogranic solvent system[J]. Synthetic commun, 2001, 31(10): 1521-1526.
[38] Rothaus O, Kruger D, Demuth M, et al. Reduction of ketoesters with Baker’s yeast in organic solvents–A comparision with the results in water[J]. Tetrahedro, 1997, 53(3): 935-938
[39] Cui J N, Teraoka R, Ema T, et al. lighly Regio-and Enantioseleclive reduction on 1-Chloro-2,4-alkanediones using BakercYeast:Effect of organic solvents as Additives[J]. Trahedron Lett, 1997, 38(17) : 3021-3024
[40] Cui J N, EmaT , Sakai T, et al.Control of enantiose1ectivity in the bakers yeast asymmetric reduction ofγ-chloro-β-diketones toγ-chloro-β-hydroxyketones[J].Tetrabedron:Asymmetry, 1998, 9: 2681-2692
[41] Matsuda T, Harada T, Nakamura K. Alcohol dehydrogenase is active in supercritical carbondioxide[J]. Chem commun, 2000, 15: 1167-1368
[42] Matsuda T, Watanabe K, Kamitanaka T, et al. Biocatalytic reduction of ketones by a semi-continuous flow process using supercritical cabrondioxide[J]. Chem Commun, 2003, 10: 1198-1199
[43] Howarth J, James P, Dai J. Immobilized bakers yeast reduction of ketones in an ionicliquic [bmim] P F6 and water mix[J]. Tetrahedron Let, 2001, 42: 7517-7519
[44] Nakamura K, Inoue K, Ushio K, et al.Sterochemical control on yeast reduction ofα-keto easter:Reduction by immobilized bakers yeast in hexane[J]. Org Chem l988, 53(11): 2589-2593
[45] Zhou B N, Gopalan A S, VanMiddlesworth F, et al. Stereochemical control of yeast reduction Asymmetric synthesis of L-carntine[J]. J Am Chem soc, 1983, 105: 5925-5926
[46] Ferraboschi P, Grisenti P, Manzocchi A, et al. Bakers yeast rnediated preparation of optically activaryl alcohols and diols for the synthesis of chiral hydroxyacids[J]. Chem Soc Perkin Transl, 1990, 9: 2469-2474.
[47] Dao D H, Okamura M, Akasaka T, et al.Stereochemical control in microbial reduction: Reduction of alkyl 2-oxo-4-arylbutyrates by bakers yeast under selected reaction conditions [J]. Tetrahedron:Asmmetry, 1998, 9(15): 2725-2737
[48] Yasohara Y, Kizaki N, Hasegawa J, et al.Synthesis of optically active ethyl-4-chloro-3-hydroxybutanoate by microbial reduction[J]. Appl.Microbiol Biotechnol, 1999, 51: 847-851
[49]. Dahl A, Fjeldberg M, Madsen J O. Bakers yeast:improving the D-stereoselectivity in reduction on 3-oxo ester[J]. Tetrahedron:Asymmetry, 1999, 10: 551-559.
[50] Naoshima Y, Maeda J, Munakata Y. Bioreduction with immobilized bakers yeast in hexane using alcohols as an energy source[J]. J Chem Soc Chem Commun, 1990, 14: 964-965
[51] Nakamura K, Inoue Y, Ohono A. Improvement of enantioselectivity of microbial reduction by using ograic solvent redox coupler system[J].Tetarhedron Lett, 1995, 36(2): 265-266
[52] Dahl A C, Madsen J O. Bakers yeast: Production of D-andL-3-hydoxy esters[J]. Tetrahedron.Asymmetry, 1998, 9: 4395-4417.
[53] Nakamura K, Yamanaka R, Matsuda T, et al. Recent deve1opments in asymmetric reduction of ketones with biocatalysist[J]. Tetrahedron: Asymmetry,2003,14: 2659-2681
[54] Nakamura K, Kawai Y, Ohno A. A novel method to synthesize (L)-β-hydroxyl esters by the reduction with bakers yeast[J]. Tetrahedron Lett, 1990, 31: 267-270
[55] Nakamura K, Kawai Y, Oka S, Ohno A. A new method for stereochemical control of microbiral reduction.Reduction ofβ-ketoesters with bakers yeast immobilized by magnesium alginate[J]. Tetarhedron lett, 1989, 30: 2245-2246
[56]张部昌.新酮内酯类抗生素组合生物合成的研究[D].军事医学科学院博士学位论文, 2002: 3-103
[57]张部昌,赵志虎,马清钧.红霉素生物合成的分子生物学[J].生物技术通讯, 2001, 12(2): 151-160
[58]张部昌,赵志虎,马清钧.红霉素基因工程研究进展[J].中国生物工程杂志, 2002, 22(3):40-44
[59]李凌凌.糖多孢红霉菌λC3-SRR突变体构建及其产物鉴定[D].安徽大学硕士学位论文. 2005: 3-48
[60] Weissman K J. Polyketide biosynthesis: understanding and exploiting modularity[J] . Philos Transact A Math Phys Eng Sci, 2004, 362(1825): 2671-2690
[61] Kim C Y, Alekseyev V Y, Chen A Y, et al. Reconstituting modular activity from separated domains of 6-deoxyerythronolide B synthase[J] . Biochemistry, 2004, 43(44): 13892-13898
[62] Korman T P, Hill J A, Vu T N, et al. Structural analysis of actinordin ketoreductase:cofactor binding and substrate specificity[J] . Biochemistry, 2004, 43(46): 14529-14538
[63] Sambrook J, Frisch E F, Maniatis T.分子克隆实验指南[M].金冬雁译,第二版.北京:科学出版社, 1992: 1-600
[64]杨建雄.生物化学与分子生物学实验技术教程[M].北京:科学出版社, 1996:1~226
[65] Alexandros P S, Abel B O, Shilpa, et al. Molecular basis of calmers rules: Stereochemistry of catalysis by isolated ketoreductase Domains from modular polyketide synthase[J]. Chemistry and Biology, 2005, 12:1145-1153
[66] Adrian T, Keatinge C, Robert M. The structure of a ketoreductase determines the organization of theβ-carbon processing enzymes of modular polyketide synthase[J]. Structure, 2006, 14: 737-748
[67] Tyler P K, Jason A H, Thanh N V, et al. Structurl analysis of Actinor hodin polyketide ketoreductase: cofactor binding and substrate specificity[J]. Biochemistry, 2004, 43: 14529-14538