NADH再生系统和乙醇突变株的构建及其生产1，3-丙二醇初步研究
|
摘要
还原力NADH是微生物产生1,3-丙二醇途径必需的物质。微生物在生成1,3-丙二醇的途径中伴随着很多副产物,如乙醇、乳酸、琥珀酸、2,3-丁二醇(2,3-BD)等,这些副产物的生成与1,3-丙二醇产生途径竞争NADH。本工作一方面在野生型K. oxytoca M5al中引入NADH再生系统,另一方面将K. oxytoca M5al中产生乙醇的途径阻断掉,以使更多的NADH流向1,3-丙二醇产生途径。
在M5al中构建了NADH再生系统(pDK7-fdh),为了重组质粒pDK7-fdh能够在细胞繁殖过程中稳定遗传,在重组质粒pDK7-fdh中引入了质粒平均分配基因parDE得到重组菌株F6。F6在传代160代后质粒仅丢失3 %,甲酸脱氢酶(FDH)酶活与传代前相比只降低了1.77 %;而不含parDE的重组菌FY和F-1传代160代后质粒分别丢失了78 %和93 %,FDH比酶活分别降低了25 %和49 %。上罐检测甲酸脱氢酶酶活表明F6最高比酶活为26.32 U/mg,是M5al最高比酶活10.79 U/mg的2.43倍。
F6摇瓶实验表明,加入甲酸脱氢酶的底物甲酸钠后,1,3-丙二醇的生产强度明显加强,发酵47 h达到0.3 g.l-1h-1,而没有加入甲酸钠的F6只有0.19 g.l-1h-1;且添加甲酸钠后副产物乙酸提高了7.2倍,乙醇产量提高了1.2倍。另外,F6和M5al发酵58 h的1,3-丙二醇产量并没有明显提高,分别是13.75 g/l和13.16 g/l,但是乙醇和乙酸的产量分别提高了24 %和30 %,且F6的生长加快。
另外,利用自杀载体pGPCm和pGPKm成功构建了aldH缺失突变株(MH)和adhE缺失突变株(ME)。比酶活测定结果显示,MH的ADH与M5al相差不大,分别为11.44 U/mg和11.86 U/mg,而ALDH比酶活为0.796 U/mg,是M5al的22 %;ME的ADH比酶活只有4.74 U/mg,是M5al的40 %,ALDH比酶活是0.934 U/mg,为M5al的26 %。在有氧和厌氧条件下,ME都几乎没有乙醇产生,而M5al乙醇最高产量分别为3.29 g/l和1.1 g/l。摇瓶发酵表明,在阻断了菌体的乙醇生成途径后,ME的生长速率明显降低,由此造成甘油消耗率降低,1,3-丙二醇的生成速率也随之降低。但是另一种化工和食品中十分有用的产物——2,3-丁二醇的生成却明显升高,发酵48 h 2,3-丁二醇的产量是10.23 g/l,比M5al提高了51 %;延长发酵时间至72 h后1,3-丙二醇的产量最高为15.89 g/l,2,3-丁二醇最高产量却达到了13.81 g/l,M5al 1,3-丙二醇最高是17.38 g/l,2,3-丁二醇只有6.58 g/l,即ME 2,3-丁二醇的产量比对照提高了约1.1倍。与M5al相比,MH的乙醇产量并没有明显降低,1,3-丙二醇产量也无明显变化,而乙酸的产量却升高了。
NADH is very important to the 1,3-propanediol pathway in bacteria. Many byproducts could be produced coupled with 1,3-propanediol pathway, such as acetic acid, ethanol, lactate, 2,3-butanediol(2,3-BD), et al, which will compete NADH with 1,3-propanediol pathway. We constructed NADH regeneration system in wide type strain Klebsilla oxytoca M5al; and on the other hand, knocked out the genes produced ethanol.
F6 is a recombinant strain contained fdh gene and parDE gene. After 160 generations, the recombinant plasmids pDK7-fdh-parDE of F6 only lost 3%; and FDH specific enzyme activity decreased 1.77% while FY and F-1 lost 78 % and 93 % plasmids, respectively and FDH specific enzyme activity decreased 25 % and 49 %, respectively. FDH specific enzyme activity is 26.32 U/mg which is 2.43 fold of K. oxytoca M5al in 5 L fermentor filled with 2.5 L broth.
Shaking flask experiments indicated that 1.3-PD productivity of F6 adding sodium formate was 0.3 g.l-1h-1 increasing about 58 % compared with M5al at 47 h, and ethanol and acetate concetrations increased 1.2 fold and 7.2 fold, respectively. F6 didn’t improve yield of 1,3-propanediol as expected but promoted the growth and increased 24 % of ethanol and 30 % acetate at 58 h.
aldH and adhE gene deficient mutants were constructed and named K.oxytoca MH, K.oxytoca ME repectively using suicide vector. The ALDH and ADH specific activity of MH and ME were determinated and the results indicated that ADH sepecific activity of ME was only 4.74 U/mg, accounted for 40% of M5al, ALDH sepecific activity of ME was 0.934 U/mg, accounted for 26% of M5al; ADH specific activity of MH was 11.44 U/mg which didn’t decrease compared with M5al while ALDH sepecific activity was 0.796 U/mg , only 22% of M5al. ME did not produce ethonal in either anaerobic or aerobic conditions, and M5al produced 3.29 g/l and 1.1 g/l of ethanol respectively under aerobic and anaerobic conditions. The growth rate of ME was slower than K.oxytoca M5al, as well as the productivity of 1,3-propanediol and comsumption rate of glycerol. It was interesting that the yield of 2,3-butanediol increased very quickly, and the concentration was 15.89 g/l at 72 h, while the K.oxytoca M5al was only 6.58 g/l. The yield of ethanol by K.oxytoca MH didn’t decreased and the yield of 1,3-propanediol didn’t increased significantly while the yield of acetic acid increased significantly.
在M5al中构建了NADH再生系统(pDK7-fdh),为了重组质粒pDK7-fdh能够在细胞繁殖过程中稳定遗传,在重组质粒pDK7-fdh中引入了质粒平均分配基因parDE得到重组菌株F6。F6在传代160代后质粒仅丢失3 %,甲酸脱氢酶(FDH)酶活与传代前相比只降低了1.77 %;而不含parDE的重组菌FY和F-1传代160代后质粒分别丢失了78 %和93 %,FDH比酶活分别降低了25 %和49 %。上罐检测甲酸脱氢酶酶活表明F6最高比酶活为26.32 U/mg,是M5al最高比酶活10.79 U/mg的2.43倍。
F6摇瓶实验表明,加入甲酸脱氢酶的底物甲酸钠后,1,3-丙二醇的生产强度明显加强,发酵47 h达到0.3 g.l-1h-1,而没有加入甲酸钠的F6只有0.19 g.l-1h-1;且添加甲酸钠后副产物乙酸提高了7.2倍,乙醇产量提高了1.2倍。另外,F6和M5al发酵58 h的1,3-丙二醇产量并没有明显提高,分别是13.75 g/l和13.16 g/l,但是乙醇和乙酸的产量分别提高了24 %和30 %,且F6的生长加快。
另外,利用自杀载体pGPCm和pGPKm成功构建了aldH缺失突变株(MH)和adhE缺失突变株(ME)。比酶活测定结果显示,MH的ADH与M5al相差不大,分别为11.44 U/mg和11.86 U/mg,而ALDH比酶活为0.796 U/mg,是M5al的22 %;ME的ADH比酶活只有4.74 U/mg,是M5al的40 %,ALDH比酶活是0.934 U/mg,为M5al的26 %。在有氧和厌氧条件下,ME都几乎没有乙醇产生,而M5al乙醇最高产量分别为3.29 g/l和1.1 g/l。摇瓶发酵表明,在阻断了菌体的乙醇生成途径后,ME的生长速率明显降低,由此造成甘油消耗率降低,1,3-丙二醇的生成速率也随之降低。但是另一种化工和食品中十分有用的产物——2,3-丁二醇的生成却明显升高,发酵48 h 2,3-丁二醇的产量是10.23 g/l,比M5al提高了51 %;延长发酵时间至72 h后1,3-丙二醇的产量最高为15.89 g/l,2,3-丁二醇最高产量却达到了13.81 g/l,M5al 1,3-丙二醇最高是17.38 g/l,2,3-丁二醇只有6.58 g/l,即ME 2,3-丁二醇的产量比对照提高了约1.1倍。与M5al相比,MH的乙醇产量并没有明显降低,1,3-丙二醇产量也无明显变化,而乙酸的产量却升高了。
NADH is very important to the 1,3-propanediol pathway in bacteria. Many byproducts could be produced coupled with 1,3-propanediol pathway, such as acetic acid, ethanol, lactate, 2,3-butanediol(2,3-BD), et al, which will compete NADH with 1,3-propanediol pathway. We constructed NADH regeneration system in wide type strain Klebsilla oxytoca M5al; and on the other hand, knocked out the genes produced ethanol.
F6 is a recombinant strain contained fdh gene and parDE gene. After 160 generations, the recombinant plasmids pDK7-fdh-parDE of F6 only lost 3%; and FDH specific enzyme activity decreased 1.77% while FY and F-1 lost 78 % and 93 % plasmids, respectively and FDH specific enzyme activity decreased 25 % and 49 %, respectively. FDH specific enzyme activity is 26.32 U/mg which is 2.43 fold of K. oxytoca M5al in 5 L fermentor filled with 2.5 L broth.
Shaking flask experiments indicated that 1.3-PD productivity of F6 adding sodium formate was 0.3 g.l-1h-1 increasing about 58 % compared with M5al at 47 h, and ethanol and acetate concetrations increased 1.2 fold and 7.2 fold, respectively. F6 didn’t improve yield of 1,3-propanediol as expected but promoted the growth and increased 24 % of ethanol and 30 % acetate at 58 h.
aldH and adhE gene deficient mutants were constructed and named K.oxytoca MH, K.oxytoca ME repectively using suicide vector. The ALDH and ADH specific activity of MH and ME were determinated and the results indicated that ADH sepecific activity of ME was only 4.74 U/mg, accounted for 40% of M5al, ALDH sepecific activity of ME was 0.934 U/mg, accounted for 26% of M5al; ADH specific activity of MH was 11.44 U/mg which didn’t decrease compared with M5al while ALDH sepecific activity was 0.796 U/mg , only 22% of M5al. ME did not produce ethonal in either anaerobic or aerobic conditions, and M5al produced 3.29 g/l and 1.1 g/l of ethanol respectively under aerobic and anaerobic conditions. The growth rate of ME was slower than K.oxytoca M5al, as well as the productivity of 1,3-propanediol and comsumption rate of glycerol. It was interesting that the yield of 2,3-butanediol increased very quickly, and the concentration was 15.89 g/l at 72 h, while the K.oxytoca M5al was only 6.58 g/l. The yield of ethanol by K.oxytoca MH didn’t decreased and the yield of 1,3-propanediol didn’t increased significantly while the yield of acetic acid increased significantly.
引文
[1] 修志龙. 微生物发酵法生产 1,3-丙二醇的研究进展. 微生物学通报. 2000,4: 300-302
[2] 刘艳杰, 赵庆国, 蒋巍, 等. 1,3-丙二醇的生产技术分析. 化学工程师. 2002, 88(1): 45~46
[3] 杨菊群, 王幸宜. 1,3-丙二醇的合成工艺进展. 化学工业与工程技术. 2002, 23: 11~15
[4] 薛丽梅, 王琲, 韩大维, 等. 1,3-丙二醇的化学合成方法. 化学与粘合. 2000, 1: 38~40
[5] 杨佳庆, 黄象安, 顾利霞. 1,3-丙二醇合成新进展. 合成纤维工业. 1999, 22(1): 26-28
[6] Biebl H, Menzel H, Zeng A P, et al. Microbial production of 1,3-propanediol. Appl. Microbiol. Biotechnol. 1999, 52: 289~297
[7] Abbad-Andaloussi S, Guedon E, Spiesser E, et al. Glycerol dehydratase activity: the limiting step for 1,3-propanediol production by Clostridium butyricum DSM 5431. Lett Appl Microbiol. 1996, 22: 311~314
[8] Papanikolaou S, Fick M, Aggelis G. The effect of raw glycerol concentration on the production of 1,3-propanediol by Clostridium butyricum. J Chem Technol Biotechnol. 2004, 79: 1189~1196
[9] Boenigk R, Bowien S, Gottschalk G. Fermentation of glycerol to 1,3-propanediol in continuous cultures of Citrobacter freundii. Appl Microbiol Biotechnol. 1993, 38: 453~457
[10] Barbirato F, Astruc S, Soucaille P, et al. Anaerobic pathways of glycerol dissimilation by Enterobacter agglomerans CNCM 1210: limitations and regulations. Microbiology. 1997, 143: 2423~2432
[11] Menzel K, Zeng A P, Deckwer W D, et al. Enzymatic evidence for an involvement of pyruvate dehydrogenase in the anaerobic glycerol metabolism of Klebsiella pneumoniae. J Biotechnol. 1997, 56: 135-142
[12] Deckwer W D. Microbial conversion of glycerol to 1,3-propanediol. FEMS Microbiology Reviews.1995(16): 143~149
[13] Menzel K, Zeng A P, Deckwer W D. High concentration and productivity of 1,3-propanediol from continuous fermentation of glycerol by Klebsiella pneumoniae. Enzyme Microb Technol, 1997, 20: 82~86
[14] 王宝光,刘铭,等.微生物法生产1,3-丙二醇过程的代谢工程研究进展.过程工程学报. 2006,6(1): 144~148
[15] Gopal C H, Tim D, Amy H. The Commercial Production of Chemicals Using Pathway Engineering. Biochim Biophys Acta. 2000, 1543: 434?455
[16] 邵敬伟. 微生物源甘油脱水酶的研究进展. 郑州工程学院学报. 2004, 25 (4): 85-88
[17] Daniel R, Bobik T A, Gottschalk G. Biochemistry of coenzyme B12-dependent glycerol and dioldehydratase and organization of the encoding gene. FEMS Microbiol reviews. 1999, 22: 553-566
[18] Macis L, Daniel R, Gottschalk G. Properties and Sequence of the Coenzyme B12-dependent Glycerol Dehydratase of Clostridium pasteurianum. FEMS Microbiol Lett. 1998, 164: 21?28
[19] 綦文涛,修志龙. 甘油歧化生产1,3?丙二醇过程的代谢和基因调控机理研究进展. 中国生物工程杂志, 2003, 23(2): 64?68
[20] Kajiura H, Mori K, Tobimatsu T, et al. Characterization and Mechanism of Action of a Reactivating Factor for Adenosylcobalamin-dependent Glycerol Dehydratase. J Biol Chem. 2001, 276: 36514?36519
[21] Seifert C, Bowien S, Gottschalk G, et al. Identification and Expression of the Genes and Purification and Characterization of the Gene Products Involved in Reactivation of Coenzyme B12-dependent Glycerol Dehydratase of Citrobacter freundii. Eur J Biochem. 2001, 268: 2369?2378
[22] Claisse O, Lonvaud-Funel A. Detection of Lactic Acid Bacteria Producing 3-Hydroxypropionaldehyde (Acrolein Precursor) from Glycerol by Molecular Tests. Lait. 2001, 81: 173?181
[23] Biebl H, Sproeer C. Taxonomy of the Glycerol Fermenting Clostridia and Description of Clostridium diolis sp. nov.. Syst Appl Microbiol. 2002, 25: 491?497
[24] Seyfried M, Lyon D, Rainey F A, et al. Caloramator viterbensis sp.nov., a Novel Thermophilic, Glycerol-fermenting Bacterium Isolated from a Hot Spring in Italy. Int J Syst Evol Microbiol. 2002, 52: 1177?1184
[25] Knietsch A, Bowien S, Whited G, et al. Identification and Characterization of Coenzyme B12-dependent Glycerol Dehydratase and Diol Dehydratase Encoding Genes from metagenomic DNA Libraries Derived from Enrichment Cultures. Appl Environ Microbiol. 2003, 69: 3048?3060
[26] Tobimatsu T, Azuma M, Matsubara H, et al. Cloning, Sequencing, and High Level Expression of the Genes Encoding Adenosylcobalamin-dependent Glycerol Dehydrase of Klebsiella pneumoniae. J Biol Chem. 1996, 271: 22352?22357
[27] Daniel R, Boenigk R, Gottschalk G. Purification of 1,3-Propanediol Dehydrogenase from Citrobacter freundii and Cloning, Sequencing, and Overexpression of the Corresponding Gene in Escherichia coli. J Bacteriol. 1995, 177: 2151?2156
[28] Seyfried M, Daniel R, Gottschalk G. Cloning, Sequencing, and Overexpression of the Genes Encoding Coenzyme B12-dependent Glycerol Dehydratase of Citrobacter freundii. J. Bacteriol. 1996, 178: 5793?5796
[29] Luers F, Seyfried M, Daniel R, et al. Glycerol Conversion to 1,3-Propanediol by Clostridium pasteurianum: Cloning and Expression of the Gene Encoding 1,3-Propanediol Dehydrogenase. FEMSMicrobiol Lett. 1997, 154: 337?345
[30] Skraly F A, Lytle B L, Cameron D C. Construction and Characterization of a 1,3-Propanediol Operon. Appl Environ Microbiol. 1998, 64: 98?105
[31] Zeng A-P, Biebl H. Bulk Chemicals from Biotechnology: The Case of 1,3-Propanediol Production and the New Trends. Adv Biochem Eng Biotechnol. 2002, 74: 239?259
[32] 周文广,黄日波. 克雷伯氏菌1,3?丙二醇氧化还原酶基因在大肠杆菌中的克隆与表达. 广西农业生物科学. 2004, 23(2):145?148
[33] 迟乃玉,刘长江,刘英昊,等. 编码1,3?丙二醇氧化还原酶基因的克隆和表达.微生物学报. 2003, 43(6): 717?721
[34] 金平,张建刚,佟明友,等. 甘油发酵制取1,3?丙二醇菌株筛选. 精细与专用化学品. 2004, 12(16): 13?15
[35] 张延平,饶治,杜晨宇,等. 能量驱动对Klebsiella pneumoniae发酵甘油合成1,3?丙二醇的影响. 过程工程学报. 2004, 4(6):567?571
[36] Guang Yang, Jiesheng Tian, Jilun Li. Fermentation of 1,3-propanediol by a lactate deficient mutant of Klebsiella oxytoca under microaerobic conditions. Appl Microbiol Biotechnol. 2007, 73, 1017-1024
[37] 黄志华,杜晨宇,王宝光, 等. 微生物法生产 1,3-丙二醇的基因工程研究进展. 生物加工过程, 2006, 4(2): 1-6
[38] Ruch FE, Lin ECC. Independent constitutive expression of theaerobic and anaerobic pathway of glycerol catabolismin Klebsiellaa erogenes. J Bacteriol. 1975, 124: 3482-3521
[39] Johnson EA, Levine RL, Lin ECC. Inactivation of glycerol dehydrogenase of Klebsiella pneumoniae and the role of divalentcations. J Bacteriol. 1985, 164: 4792-4831
[40] Sprenger GA, Hammer BA, Johnson EA, et al. Anaerobic growth of E.coli on glycerol by importing genes of the dha regulon from Klebsiella pneumoniae. J general Microbiol. 1989, 135: 1255~1262
[41] Tong I T, Cameron D C. Enhancement of 1,3-propanediol production by cofermentation in Escherichia coli expressing Klebsiella pneumoniae dha regulon genes. Appl Biochem Biotechnol. 1992, 34(35):149~159
[42] Skraly FA, Lytle BL, Cameron DC. Construction and characterization of a 1,3-propanediol operon. Appl Environ Microbiol. 1998, 64:98~105
[43] Zhu MM, Skraly FA, Cameron DC. Accumulation of methylglyoxal in anaerobically grown Escherichia coli and its detoxifacation by expression of the Pseudomonas putida glyoxalase Ⅰ gene. Metabolic Engineering. 2001, 3:218~225.
[44] Zhu MM, Lawman PD, Cameron DC. Improving 1,3-propanediol productions from glycerol in ametabolically engineered Escherichia coli by reducing accumulation of sn-glucerol-3-phosphate. Biotechnol Prog. 2002, 18(4): 694~699
[45] Cameron DC, Altartas NE, Hoffman ML, et al. Metabolic engineering of propandiol pathways. Biotechnol Prog. 1998, 14: 1162-1251
[46] Laffend LA, NagarajanV, Nakamura CE. Bioconversion of a fermentable carbon source to 1,3-propanediol by a single microorganism.United States Patent: 6025184, 2000-02-15
[47] Skraly FA, Cameron DC. Purification and Characterization of a Bacillus licheniformis Phosphatase Specific for D-α-Glycerophosphate. Arc Biochem Biophys. 1998, 349(1): 27~35
[48] Norbeck J, Pahlman AK, Akhtr N, et al. Purification and characterization of two isoenzymes of DL-glycerol-3-phosphatase from Saccharomyces cerevisiae. J Biol Chem. 1996, 271: 13875-13881
[49] Emptage M, Haynie S, Laffend L, et al. Process for the biological production of 1,3-propanediol with high titer. USA, Patent, US 07067300, June 27, 2006
[50] Chen X, Zhang D J, Qi W T, et al. Microbial Fed-batch Production of 1,3-Propanediol by Klebsiella pneumoniae under Micro-aerobic Conditions. Appl Microbiol Biotechnol. 2003, 63: 143?146
[51] Cheng K K, Liu D H, Sun Y, et al. 1,3-Propanediol Production by Klebsiella pneumoniae under Different Aeration Strategies. Biotechnol Lett. 2004, 26: 911?915
[52] 林日辉,刘宏娟,等. 氧对Klebsiella pneumoniae产1,3-丙二醇代谢的影响. 过程工程学报. 2006, 6(1): 96-99
[53] Schmid A, Dordick JS, Hauer B, et al. Industrial biocatalysis today and tomorrow. Nature, 2001, 409(11): 258~268
[54] Koeller K, Wong C. Enzymes for chemical synthesis. Nature, 2001, 409: 232~240
[55] Schoemaker H, Mink D, Wubbolts M. Dispelling the myths-biocatalysis in industrial synthesis. Science, 2003, 299: 1694~1697
[56] David RK. Biotechnology : Biotransformations I, ed. 2nd. Berlin: WILEY2VCH, 1998
[57] Delecoul S K, Basseguy R, Bergel A. Membrane electrochemical reactor (MER): application to NADH regeneration for ADH-catalysed synthesis. Chemical Engineering Science, 2002, 57(21): 4633~4642
[58] Schroder I, Steckhan E, Liese A. In situ NAD regeneration using 2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonate) as an electron transfer mediator. Journal of Electroanalytical Chemistry, 2003, 541: 109~115
[59] Tayhas G, Palmore R, Bertsehy H, et al. Methanol Pdioxygen biofuel cell that uses NAD+-dependent dehydrogenases as catalysts: application of an electroenzymatic method to regenerate nicotinamideadenine dinucletiode at low overpotentials. Journal of Electroanalytical Chemistry, 1998, 443(1): 155~161
[60] Long Y T, Chen H Y. Electrochemical regeneration of coenzyme NADH on a histidine modified silver electrode. Journal of Electroanalytical Chemistry, 1997, 440: 239~242
[61] Leonida M D, Fry A J, Sobolov S B, et al. Co-Electorpolymerization of a Viologen Oligomer and Lipoamide Dehydrogenase on an Electrode Surface: Application to Cofactor Regeneration. Bioorganic and Medicinal Chemistry Letters, 1996, 14(6): 1663~1666
[62] 张玉彬. 生物催化的手性合成. 北京: 化学工业出版社, 2001
[63] Wichmann R, Wandrey C, Buckmann A F, et al. Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration. Biotechnol Bioeng, 2000, 67: 791~804
[64] Seelbach K, Riabel B, Hummel W. A novel, efficient regeneration method of NADPH using a new formate dehydrogenase. Tetrahedron Letters, 1996, 37(9): 1377~1380
[65] May SW. Applications of oxidoreductases. Current Opinion in Biotechnology, 1999, 10(4): 370~375
[66] Wong C, Drueckhammer D, Sweers H. Enzymatic vs fermentative synthesis - thermostable glucose-dehydrogenase catalyzed regeneration of NAD(P)H for use in enzymatic synthesis. J Am Chem Soc, 1985, 107: 4028~4031
[67] Bastos F M, Franca T K, Machado G D. Kinetic modeling of coupled redox enzymatic systems for in situ regeneration of NADPH. Journal of Molecular Catalysis B: Enzymatic, 2002, 19/20: 459~465
[68] Cantet J, Bergel A, Comtat M, et al. Coupling of the electroenzymatic reduction of NAD+ with a synthesis reaction. Enzyme and Microbial Technology, 1996, 18(1): 72~79
[69] Lee L, Whitesides G. Preparation of opitically-active 1,2-diols and α-hydroxy ketones using glycerol dehydrogenase as catalyst- limits to enzyme-catalyzed synthesis due to noncompetitive and mixed inhibition by product. J. Org. Chem, 1986, 51: 25~36
[70] Geueke B, Riebel B, Hummel W. NADH oxidase from Lactobacillus brevis: a new catalyst for the regeneration of NAD. Enzyme and Microbial Technology, 2003, 32(2): 205~211
[71] Felix L, Michiel K, Willem M, et al. Cofactor engineering: a novel approach to metabolic engineering in Lactococcus lactis by controlled expression of NADH oxidase. Journal of Bacteriology, 1998, 180(15): 3804~3808
[72] Neves A R, Ventura R, Mansour N, et al. Is the Glycolytic Flux in Lactococcus lactis Primarily Controlled by the Redox Charge? The Journal of Biological Chemistry, 2002, 277(31): 28088~28098
[73] Berrios-Rivera S J, et al. Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD+-dependent formate dehydrogenase. Metabolic Eng, 2002, 4: 217~229
[74] Berrios-Rivera S J, Bennett G N, San K Y. The effect of increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures. Metabolic Engineering, 2002, 4: 230~237
[75] 李凡锋, 周玉杰, 刘德华. 1,3-丙二醇发酵液的絮凝预处理研究. 微生物学通报. 2004, 31(2): 30~35
[76] Gong Y, Tang Y, Wang XL, et al. The possibility of the desalination of actual 1,3-propanediol fermentation broth by electrodialysis. Desalination. 2004, 161(2): 169-178
[77] 龚燕, 唐宇, 王晓琳, 等. 电渗析用于 1,3-丙二醇发酵液脱盐的实验研究. 膜科学与技术, 2004, 5: 33~36
[78] 郝健, 刘德华. 1,3-丙二醇发酵液电渗析法脱盐. 过程工程学报. 2005, 5: 36-39
[79] Janusz J M. Reactive extraction for downstream separation of 1,3-propanediol. Biotechnol Prog. 2000, 16(1): 76-79
[80] Malinowski J J. Evaluation of liquid extraction potentials for downstream separation of 1,3-propanediol. Biotechnol Techniq. 1999, 13(2): 127-130
[81] 周鹏, 方云进. 发酵液中低浓度1,3-丙二醇浓缩提纯工艺研究进展. 化学与生物工程. 2005,2: 4-6
[82] 黄志华,张延平,刘铭,等. 甲酸脱氢酶在Klebsiella pneumoniae中的表达和功能分析. 微生物学报. 2007, 47(1): 64?68
[83] Miller VL and Mekalanos JJ. A novel suicide vector and its use in construction of insertion mutations: Osmoregulation of outer membrane proteins and virulence determinants in Vibrio Cholerae requires toxR. J Bacteriol. 1988, 170(6): 2575~2583
[84] 田杰生, 宋海琛, 吴伯和, 等. 高产稳产聚羟基烷酸的重组大肠杆菌的构建. 微生物学报. 2000, 40(1): 26?30
[85] Kleiner D, Paul W, MErrick M J. Construction of Multicopy Expression Vectors for Regulated Overproduction of Proteins in Klebsiella pneumoniae and Other Enteric Bacteria. J Gen Microbiol. 1988, 134(7): 1779?1784
[86] Lorenzo VD, Herrero M, Jakubzik U. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol. 1990, 172(11): 6568~6572
[87] Schutte H, Flossdorf J, Sahm H, et al. Purification and Properties of Formaldehyde Dehydrogenase and Formate Dehydrogenase from Candida boidinii. Eur J Biochem. 1976, 62, 151?160
[88] 余冰宾. 生物化学实验指南. 北京:清华大学出版社, 2004. 136?138
[89] Gerlitz M, Hrabak O, Schwab H. Partitioning of Broad Host Range Plasmid RP4 Is a Complex System Involving Site-specific Recombination. J Bacteriol. 1990, 172(11): 6194?6203
[90] 萨姆布鲁克 J., 弗里奇 E.F., 曼尼阿蒂斯 T. 分子克隆实验指南.(第三版)北京: 科学出版社. 2002
[91] Lidya B, Sanchez. Aldehyde dehydrogenase (CoA-Acetylating)and the mechanism of ethanol formation in the Amitochondriate Protist, Giardia Lamblia. Archives of biochemistry and biopHysics. 1998, 354(1): 57-64
[92] Andreas L, Murillo V F. Production of Fine Chemicals Using Biocatalysis. Curr Opin Biotechnol. 1999, 10(6): 595?603
[93] 赵立平,张丽,李艳琴,等. 利用 RK2 的 parDE 片段提高重组质粒在生防菌成团泛菌中的稳定性. 中国生物防治. 1999, 15(2): 49?53
[94] Weinstein M, Roberts R C, Helinske D R. A Region of the Broad-host-range Plasmid RK2 Cause Stable in Planta Inheritance of Plasmids in Rhizobium Meliloti Cells Isolated from Alfalfa Root Nodules. J Bacteriol.1992 ,174(22): 7486?7489
[95] Rasmussen P N, Gerdes K, Molin S. Genetic Analysis of the parB Locus of Plasmid R1. Mol. Gen. Genet. 1987, 209: 122?128
[96] Zeng A P, Biebl H, Deckwer W D. Pathway analysis of glycerol fermentation by Klebsiella pneumoniae: regulation of reducing equivalent balance and product formation. Enzyme Microb Technol. 1993, 15: 770~779
[97] Gregory N Stephanopoulos, Aristos A Aristidou, Jens Nielsen.代谢工程:原理与方法(赵学明,白冬梅等译). 北京: 化学工业出版社, 2003, 72
[98] Ma L C, Lee S L, Lee C Z, et al. Molecular cloning and kinetic characterization of an aldehyde dehydrogenase gene in Klebsiella pneumoniae. J Formos Med Assoc, 2005, 104(4): 221~230
[99] Miller VL,Mekalanos JJ.A novel suicide vector and its use in construction of insertion mutations:osmoregulation of outer Membrane proteins and virulence deter minants in Vibro cholerae requires toxR. J Bacteriol. 1988, 170(6):2575
[100] Venkatesan M, Fernandez-Prada C, Buysse JM, et al. Virulence phenotype and genetic characteristic of the T32-ISTRATI Shigella flexneri 2a vaccine strain.Vaccine. 1991, 9(5): 358-363
[101] 芮贤良,徐永强,王红,等. 稳定、无抗药的痢疾弗氏 2a 和宋内双价菌苗候选株的构建. 生物工程学报. 1996,12(2):134
[102] Clark D P. The fermentation pathways of Escherichia coli. FEMS Microbiol.Rev. 1989, 63: 223-234
[103] Holland-staley CA, Lee K, Clark DP, and Cunningham PR. Aerobic activity of Escherichia colialcohol dehydrogenase is determined by a single amino acid. J Bacteriol. 2000, 18(2): 6049-6054
[104] Nakamura CE,Whited G. Metabolic engineering for the microbial production of 1,3-propanediol. Curr Opin Biotechnol. 2003, 14: 4542-4591
[105] Takamasa T, Muneaki A , Hirokazu M, et al .Cloning , sequencing , and high level expressionof the genes encoding adenosylcobalamin-dependent glycerol dehydrase of Klebsiella pneumoniae. J Biol Chem. 1996, 271 (37) : 22352-22357
[106] Liao DI, Dotson G, Turner I, et al . Crystal structure of substrate free form of glycerol dehydratase. J of Inorganic Biochemistry. 2003, 93: 84-91
[107] Toraya T, Fukui S. Diol dehydratase. In B12: Biochemistry and medicine. New York: NYJohn Wily &Sons, 1982
[108] Johnson EA, Lin ECC. Klebsiella pneumoniae 1,3-propanrdiol: NAD+ oxidoredutase. J Bateriol. 1987, 169: 2050-2054
[109] Johnson EA, Burke SK, Forage RG, et al. Purification and properties of dihydroxyacetone kinase from Klebsiella pneumoniae. J Bacteriol. 1984, 160: 55-60
[2] 刘艳杰, 赵庆国, 蒋巍, 等. 1,3-丙二醇的生产技术分析. 化学工程师. 2002, 88(1): 45~46
[3] 杨菊群, 王幸宜. 1,3-丙二醇的合成工艺进展. 化学工业与工程技术. 2002, 23: 11~15
[4] 薛丽梅, 王琲, 韩大维, 等. 1,3-丙二醇的化学合成方法. 化学与粘合. 2000, 1: 38~40
[5] 杨佳庆, 黄象安, 顾利霞. 1,3-丙二醇合成新进展. 合成纤维工业. 1999, 22(1): 26-28
[6] Biebl H, Menzel H, Zeng A P, et al. Microbial production of 1,3-propanediol. Appl. Microbiol. Biotechnol. 1999, 52: 289~297
[7] Abbad-Andaloussi S, Guedon E, Spiesser E, et al. Glycerol dehydratase activity: the limiting step for 1,3-propanediol production by Clostridium butyricum DSM 5431. Lett Appl Microbiol. 1996, 22: 311~314
[8] Papanikolaou S, Fick M, Aggelis G. The effect of raw glycerol concentration on the production of 1,3-propanediol by Clostridium butyricum. J Chem Technol Biotechnol. 2004, 79: 1189~1196
[9] Boenigk R, Bowien S, Gottschalk G. Fermentation of glycerol to 1,3-propanediol in continuous cultures of Citrobacter freundii. Appl Microbiol Biotechnol. 1993, 38: 453~457
[10] Barbirato F, Astruc S, Soucaille P, et al. Anaerobic pathways of glycerol dissimilation by Enterobacter agglomerans CNCM 1210: limitations and regulations. Microbiology. 1997, 143: 2423~2432
[11] Menzel K, Zeng A P, Deckwer W D, et al. Enzymatic evidence for an involvement of pyruvate dehydrogenase in the anaerobic glycerol metabolism of Klebsiella pneumoniae. J Biotechnol. 1997, 56: 135-142
[12] Deckwer W D. Microbial conversion of glycerol to 1,3-propanediol. FEMS Microbiology Reviews.1995(16): 143~149
[13] Menzel K, Zeng A P, Deckwer W D. High concentration and productivity of 1,3-propanediol from continuous fermentation of glycerol by Klebsiella pneumoniae. Enzyme Microb Technol, 1997, 20: 82~86
[14] 王宝光,刘铭,等.微生物法生产1,3-丙二醇过程的代谢工程研究进展.过程工程学报. 2006,6(1): 144~148
[15] Gopal C H, Tim D, Amy H. The Commercial Production of Chemicals Using Pathway Engineering. Biochim Biophys Acta. 2000, 1543: 434?455
[16] 邵敬伟. 微生物源甘油脱水酶的研究进展. 郑州工程学院学报. 2004, 25 (4): 85-88
[17] Daniel R, Bobik T A, Gottschalk G. Biochemistry of coenzyme B12-dependent glycerol and dioldehydratase and organization of the encoding gene. FEMS Microbiol reviews. 1999, 22: 553-566
[18] Macis L, Daniel R, Gottschalk G. Properties and Sequence of the Coenzyme B12-dependent Glycerol Dehydratase of Clostridium pasteurianum. FEMS Microbiol Lett. 1998, 164: 21?28
[19] 綦文涛,修志龙. 甘油歧化生产1,3?丙二醇过程的代谢和基因调控机理研究进展. 中国生物工程杂志, 2003, 23(2): 64?68
[20] Kajiura H, Mori K, Tobimatsu T, et al. Characterization and Mechanism of Action of a Reactivating Factor for Adenosylcobalamin-dependent Glycerol Dehydratase. J Biol Chem. 2001, 276: 36514?36519
[21] Seifert C, Bowien S, Gottschalk G, et al. Identification and Expression of the Genes and Purification and Characterization of the Gene Products Involved in Reactivation of Coenzyme B12-dependent Glycerol Dehydratase of Citrobacter freundii. Eur J Biochem. 2001, 268: 2369?2378
[22] Claisse O, Lonvaud-Funel A. Detection of Lactic Acid Bacteria Producing 3-Hydroxypropionaldehyde (Acrolein Precursor) from Glycerol by Molecular Tests. Lait. 2001, 81: 173?181
[23] Biebl H, Sproeer C. Taxonomy of the Glycerol Fermenting Clostridia and Description of Clostridium diolis sp. nov.. Syst Appl Microbiol. 2002, 25: 491?497
[24] Seyfried M, Lyon D, Rainey F A, et al. Caloramator viterbensis sp.nov., a Novel Thermophilic, Glycerol-fermenting Bacterium Isolated from a Hot Spring in Italy. Int J Syst Evol Microbiol. 2002, 52: 1177?1184
[25] Knietsch A, Bowien S, Whited G, et al. Identification and Characterization of Coenzyme B12-dependent Glycerol Dehydratase and Diol Dehydratase Encoding Genes from metagenomic DNA Libraries Derived from Enrichment Cultures. Appl Environ Microbiol. 2003, 69: 3048?3060
[26] Tobimatsu T, Azuma M, Matsubara H, et al. Cloning, Sequencing, and High Level Expression of the Genes Encoding Adenosylcobalamin-dependent Glycerol Dehydrase of Klebsiella pneumoniae. J Biol Chem. 1996, 271: 22352?22357
[27] Daniel R, Boenigk R, Gottschalk G. Purification of 1,3-Propanediol Dehydrogenase from Citrobacter freundii and Cloning, Sequencing, and Overexpression of the Corresponding Gene in Escherichia coli. J Bacteriol. 1995, 177: 2151?2156
[28] Seyfried M, Daniel R, Gottschalk G. Cloning, Sequencing, and Overexpression of the Genes Encoding Coenzyme B12-dependent Glycerol Dehydratase of Citrobacter freundii. J. Bacteriol. 1996, 178: 5793?5796
[29] Luers F, Seyfried M, Daniel R, et al. Glycerol Conversion to 1,3-Propanediol by Clostridium pasteurianum: Cloning and Expression of the Gene Encoding 1,3-Propanediol Dehydrogenase. FEMSMicrobiol Lett. 1997, 154: 337?345
[30] Skraly F A, Lytle B L, Cameron D C. Construction and Characterization of a 1,3-Propanediol Operon. Appl Environ Microbiol. 1998, 64: 98?105
[31] Zeng A-P, Biebl H. Bulk Chemicals from Biotechnology: The Case of 1,3-Propanediol Production and the New Trends. Adv Biochem Eng Biotechnol. 2002, 74: 239?259
[32] 周文广,黄日波. 克雷伯氏菌1,3?丙二醇氧化还原酶基因在大肠杆菌中的克隆与表达. 广西农业生物科学. 2004, 23(2):145?148
[33] 迟乃玉,刘长江,刘英昊,等. 编码1,3?丙二醇氧化还原酶基因的克隆和表达.微生物学报. 2003, 43(6): 717?721
[34] 金平,张建刚,佟明友,等. 甘油发酵制取1,3?丙二醇菌株筛选. 精细与专用化学品. 2004, 12(16): 13?15
[35] 张延平,饶治,杜晨宇,等. 能量驱动对Klebsiella pneumoniae发酵甘油合成1,3?丙二醇的影响. 过程工程学报. 2004, 4(6):567?571
[36] Guang Yang, Jiesheng Tian, Jilun Li. Fermentation of 1,3-propanediol by a lactate deficient mutant of Klebsiella oxytoca under microaerobic conditions. Appl Microbiol Biotechnol. 2007, 73, 1017-1024
[37] 黄志华,杜晨宇,王宝光, 等. 微生物法生产 1,3-丙二醇的基因工程研究进展. 生物加工过程, 2006, 4(2): 1-6
[38] Ruch FE, Lin ECC. Independent constitutive expression of theaerobic and anaerobic pathway of glycerol catabolismin Klebsiellaa erogenes. J Bacteriol. 1975, 124: 3482-3521
[39] Johnson EA, Levine RL, Lin ECC. Inactivation of glycerol dehydrogenase of Klebsiella pneumoniae and the role of divalentcations. J Bacteriol. 1985, 164: 4792-4831
[40] Sprenger GA, Hammer BA, Johnson EA, et al. Anaerobic growth of E.coli on glycerol by importing genes of the dha regulon from Klebsiella pneumoniae. J general Microbiol. 1989, 135: 1255~1262
[41] Tong I T, Cameron D C. Enhancement of 1,3-propanediol production by cofermentation in Escherichia coli expressing Klebsiella pneumoniae dha regulon genes. Appl Biochem Biotechnol. 1992, 34(35):149~159
[42] Skraly FA, Lytle BL, Cameron DC. Construction and characterization of a 1,3-propanediol operon. Appl Environ Microbiol. 1998, 64:98~105
[43] Zhu MM, Skraly FA, Cameron DC. Accumulation of methylglyoxal in anaerobically grown Escherichia coli and its detoxifacation by expression of the Pseudomonas putida glyoxalase Ⅰ gene. Metabolic Engineering. 2001, 3:218~225.
[44] Zhu MM, Lawman PD, Cameron DC. Improving 1,3-propanediol productions from glycerol in ametabolically engineered Escherichia coli by reducing accumulation of sn-glucerol-3-phosphate. Biotechnol Prog. 2002, 18(4): 694~699
[45] Cameron DC, Altartas NE, Hoffman ML, et al. Metabolic engineering of propandiol pathways. Biotechnol Prog. 1998, 14: 1162-1251
[46] Laffend LA, NagarajanV, Nakamura CE. Bioconversion of a fermentable carbon source to 1,3-propanediol by a single microorganism.United States Patent: 6025184, 2000-02-15
[47] Skraly FA, Cameron DC. Purification and Characterization of a Bacillus licheniformis Phosphatase Specific for D-α-Glycerophosphate. Arc Biochem Biophys. 1998, 349(1): 27~35
[48] Norbeck J, Pahlman AK, Akhtr N, et al. Purification and characterization of two isoenzymes of DL-glycerol-3-phosphatase from Saccharomyces cerevisiae. J Biol Chem. 1996, 271: 13875-13881
[49] Emptage M, Haynie S, Laffend L, et al. Process for the biological production of 1,3-propanediol with high titer. USA, Patent, US 07067300, June 27, 2006
[50] Chen X, Zhang D J, Qi W T, et al. Microbial Fed-batch Production of 1,3-Propanediol by Klebsiella pneumoniae under Micro-aerobic Conditions. Appl Microbiol Biotechnol. 2003, 63: 143?146
[51] Cheng K K, Liu D H, Sun Y, et al. 1,3-Propanediol Production by Klebsiella pneumoniae under Different Aeration Strategies. Biotechnol Lett. 2004, 26: 911?915
[52] 林日辉,刘宏娟,等. 氧对Klebsiella pneumoniae产1,3-丙二醇代谢的影响. 过程工程学报. 2006, 6(1): 96-99
[53] Schmid A, Dordick JS, Hauer B, et al. Industrial biocatalysis today and tomorrow. Nature, 2001, 409(11): 258~268
[54] Koeller K, Wong C. Enzymes for chemical synthesis. Nature, 2001, 409: 232~240
[55] Schoemaker H, Mink D, Wubbolts M. Dispelling the myths-biocatalysis in industrial synthesis. Science, 2003, 299: 1694~1697
[56] David RK. Biotechnology : Biotransformations I, ed. 2nd. Berlin: WILEY2VCH, 1998
[57] Delecoul S K, Basseguy R, Bergel A. Membrane electrochemical reactor (MER): application to NADH regeneration for ADH-catalysed synthesis. Chemical Engineering Science, 2002, 57(21): 4633~4642
[58] Schroder I, Steckhan E, Liese A. In situ NAD regeneration using 2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonate) as an electron transfer mediator. Journal of Electroanalytical Chemistry, 2003, 541: 109~115
[59] Tayhas G, Palmore R, Bertsehy H, et al. Methanol Pdioxygen biofuel cell that uses NAD+-dependent dehydrogenases as catalysts: application of an electroenzymatic method to regenerate nicotinamideadenine dinucletiode at low overpotentials. Journal of Electroanalytical Chemistry, 1998, 443(1): 155~161
[60] Long Y T, Chen H Y. Electrochemical regeneration of coenzyme NADH on a histidine modified silver electrode. Journal of Electroanalytical Chemistry, 1997, 440: 239~242
[61] Leonida M D, Fry A J, Sobolov S B, et al. Co-Electorpolymerization of a Viologen Oligomer and Lipoamide Dehydrogenase on an Electrode Surface: Application to Cofactor Regeneration. Bioorganic and Medicinal Chemistry Letters, 1996, 14(6): 1663~1666
[62] 张玉彬. 生物催化的手性合成. 北京: 化学工业出版社, 2001
[63] Wichmann R, Wandrey C, Buckmann A F, et al. Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration. Biotechnol Bioeng, 2000, 67: 791~804
[64] Seelbach K, Riabel B, Hummel W. A novel, efficient regeneration method of NADPH using a new formate dehydrogenase. Tetrahedron Letters, 1996, 37(9): 1377~1380
[65] May SW. Applications of oxidoreductases. Current Opinion in Biotechnology, 1999, 10(4): 370~375
[66] Wong C, Drueckhammer D, Sweers H. Enzymatic vs fermentative synthesis - thermostable glucose-dehydrogenase catalyzed regeneration of NAD(P)H for use in enzymatic synthesis. J Am Chem Soc, 1985, 107: 4028~4031
[67] Bastos F M, Franca T K, Machado G D. Kinetic modeling of coupled redox enzymatic systems for in situ regeneration of NADPH. Journal of Molecular Catalysis B: Enzymatic, 2002, 19/20: 459~465
[68] Cantet J, Bergel A, Comtat M, et al. Coupling of the electroenzymatic reduction of NAD+ with a synthesis reaction. Enzyme and Microbial Technology, 1996, 18(1): 72~79
[69] Lee L, Whitesides G. Preparation of opitically-active 1,2-diols and α-hydroxy ketones using glycerol dehydrogenase as catalyst- limits to enzyme-catalyzed synthesis due to noncompetitive and mixed inhibition by product. J. Org. Chem, 1986, 51: 25~36
[70] Geueke B, Riebel B, Hummel W. NADH oxidase from Lactobacillus brevis: a new catalyst for the regeneration of NAD. Enzyme and Microbial Technology, 2003, 32(2): 205~211
[71] Felix L, Michiel K, Willem M, et al. Cofactor engineering: a novel approach to metabolic engineering in Lactococcus lactis by controlled expression of NADH oxidase. Journal of Bacteriology, 1998, 180(15): 3804~3808
[72] Neves A R, Ventura R, Mansour N, et al. Is the Glycolytic Flux in Lactococcus lactis Primarily Controlled by the Redox Charge? The Journal of Biological Chemistry, 2002, 277(31): 28088~28098
[73] Berrios-Rivera S J, et al. Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD+-dependent formate dehydrogenase. Metabolic Eng, 2002, 4: 217~229
[74] Berrios-Rivera S J, Bennett G N, San K Y. The effect of increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures. Metabolic Engineering, 2002, 4: 230~237
[75] 李凡锋, 周玉杰, 刘德华. 1,3-丙二醇发酵液的絮凝预处理研究. 微生物学通报. 2004, 31(2): 30~35
[76] Gong Y, Tang Y, Wang XL, et al. The possibility of the desalination of actual 1,3-propanediol fermentation broth by electrodialysis. Desalination. 2004, 161(2): 169-178
[77] 龚燕, 唐宇, 王晓琳, 等. 电渗析用于 1,3-丙二醇发酵液脱盐的实验研究. 膜科学与技术, 2004, 5: 33~36
[78] 郝健, 刘德华. 1,3-丙二醇发酵液电渗析法脱盐. 过程工程学报. 2005, 5: 36-39
[79] Janusz J M. Reactive extraction for downstream separation of 1,3-propanediol. Biotechnol Prog. 2000, 16(1): 76-79
[80] Malinowski J J. Evaluation of liquid extraction potentials for downstream separation of 1,3-propanediol. Biotechnol Techniq. 1999, 13(2): 127-130
[81] 周鹏, 方云进. 发酵液中低浓度1,3-丙二醇浓缩提纯工艺研究进展. 化学与生物工程. 2005,2: 4-6
[82] 黄志华,张延平,刘铭,等. 甲酸脱氢酶在Klebsiella pneumoniae中的表达和功能分析. 微生物学报. 2007, 47(1): 64?68
[83] Miller VL and Mekalanos JJ. A novel suicide vector and its use in construction of insertion mutations: Osmoregulation of outer membrane proteins and virulence determinants in Vibrio Cholerae requires toxR. J Bacteriol. 1988, 170(6): 2575~2583
[84] 田杰生, 宋海琛, 吴伯和, 等. 高产稳产聚羟基烷酸的重组大肠杆菌的构建. 微生物学报. 2000, 40(1): 26?30
[85] Kleiner D, Paul W, MErrick M J. Construction of Multicopy Expression Vectors for Regulated Overproduction of Proteins in Klebsiella pneumoniae and Other Enteric Bacteria. J Gen Microbiol. 1988, 134(7): 1779?1784
[86] Lorenzo VD, Herrero M, Jakubzik U. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol. 1990, 172(11): 6568~6572
[87] Schutte H, Flossdorf J, Sahm H, et al. Purification and Properties of Formaldehyde Dehydrogenase and Formate Dehydrogenase from Candida boidinii. Eur J Biochem. 1976, 62, 151?160
[88] 余冰宾. 生物化学实验指南. 北京:清华大学出版社, 2004. 136?138
[89] Gerlitz M, Hrabak O, Schwab H. Partitioning of Broad Host Range Plasmid RP4 Is a Complex System Involving Site-specific Recombination. J Bacteriol. 1990, 172(11): 6194?6203
[90] 萨姆布鲁克 J., 弗里奇 E.F., 曼尼阿蒂斯 T. 分子克隆实验指南.(第三版)北京: 科学出版社. 2002
[91] Lidya B, Sanchez. Aldehyde dehydrogenase (CoA-Acetylating)and the mechanism of ethanol formation in the Amitochondriate Protist, Giardia Lamblia. Archives of biochemistry and biopHysics. 1998, 354(1): 57-64
[92] Andreas L, Murillo V F. Production of Fine Chemicals Using Biocatalysis. Curr Opin Biotechnol. 1999, 10(6): 595?603
[93] 赵立平,张丽,李艳琴,等. 利用 RK2 的 parDE 片段提高重组质粒在生防菌成团泛菌中的稳定性. 中国生物防治. 1999, 15(2): 49?53
[94] Weinstein M, Roberts R C, Helinske D R. A Region of the Broad-host-range Plasmid RK2 Cause Stable in Planta Inheritance of Plasmids in Rhizobium Meliloti Cells Isolated from Alfalfa Root Nodules. J Bacteriol.1992 ,174(22): 7486?7489
[95] Rasmussen P N, Gerdes K, Molin S. Genetic Analysis of the parB Locus of Plasmid R1. Mol. Gen. Genet. 1987, 209: 122?128
[96] Zeng A P, Biebl H, Deckwer W D. Pathway analysis of glycerol fermentation by Klebsiella pneumoniae: regulation of reducing equivalent balance and product formation. Enzyme Microb Technol. 1993, 15: 770~779
[97] Gregory N Stephanopoulos, Aristos A Aristidou, Jens Nielsen.代谢工程:原理与方法(赵学明,白冬梅等译). 北京: 化学工业出版社, 2003, 72
[98] Ma L C, Lee S L, Lee C Z, et al. Molecular cloning and kinetic characterization of an aldehyde dehydrogenase gene in Klebsiella pneumoniae. J Formos Med Assoc, 2005, 104(4): 221~230
[99] Miller VL,Mekalanos JJ.A novel suicide vector and its use in construction of insertion mutations:osmoregulation of outer Membrane proteins and virulence deter minants in Vibro cholerae requires toxR. J Bacteriol. 1988, 170(6):2575
[100] Venkatesan M, Fernandez-Prada C, Buysse JM, et al. Virulence phenotype and genetic characteristic of the T32-ISTRATI Shigella flexneri 2a vaccine strain.Vaccine. 1991, 9(5): 358-363
[101] 芮贤良,徐永强,王红,等. 稳定、无抗药的痢疾弗氏 2a 和宋内双价菌苗候选株的构建. 生物工程学报. 1996,12(2):134
[102] Clark D P. The fermentation pathways of Escherichia coli. FEMS Microbiol.Rev. 1989, 63: 223-234
[103] Holland-staley CA, Lee K, Clark DP, and Cunningham PR. Aerobic activity of Escherichia colialcohol dehydrogenase is determined by a single amino acid. J Bacteriol. 2000, 18(2): 6049-6054
[104] Nakamura CE,Whited G. Metabolic engineering for the microbial production of 1,3-propanediol. Curr Opin Biotechnol. 2003, 14: 4542-4591
[105] Takamasa T, Muneaki A , Hirokazu M, et al .Cloning , sequencing , and high level expressionof the genes encoding adenosylcobalamin-dependent glycerol dehydrase of Klebsiella pneumoniae. J Biol Chem. 1996, 271 (37) : 22352-22357
[106] Liao DI, Dotson G, Turner I, et al . Crystal structure of substrate free form of glycerol dehydratase. J of Inorganic Biochemistry. 2003, 93: 84-91
[107] Toraya T, Fukui S. Diol dehydratase. In B12: Biochemistry and medicine. New York: NYJohn Wily &Sons, 1982
[108] Johnson EA, Lin ECC. Klebsiella pneumoniae 1,3-propanrdiol: NAD+ oxidoredutase. J Bateriol. 1987, 169: 2050-2054
[109] Johnson EA, Burke SK, Forage RG, et al. Purification and properties of dihydroxyacetone kinase from Klebsiella pneumoniae. J Bacteriol. 1984, 160: 55-60