缺失丙酮酸甲酸裂解酶和乳酸脱氢酶的产乙醇重组大肠杆菌构建及特性分析
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摘要
生物乙醇的生产主要以淀粉或己糖作为发酵原料,生产成本偏高。利用分子生物学技术改造构建产乙醇工程菌,扩大菌株的底物利用范围,提高乙醇产率和高乙醇耐受性,使其能高效利用丰富、廉价的纤维素及其水解产物是目前研究的重点。本研究选用具有广泛底物降解范围(如秸秆等纤维素、半纤维素水解产物中几乎的所有糖类)、易于糖类代谢和具有优先的工业经验和理论基础的大肠杆菌(Eescherichia coli作为研究对象,从筛选具有高耐乙醇的大肠杆菌出发,构建产乙醇重组工程菌大肠杆菌,并利用分子生物学手段对其进行了进一步的改造。
本研究从10余种不同来源的大肠杆菌中筛选获得具有较高乙醇耐受性的MG1655菌株,通过连续在高乙醇浓度环境中定向筛选,获得了耐受乙醇的浓度达6.5%(w/v)的大肠杆菌菌株MG1655E。以该菌作为出发菌株,从运动运动发酵单胞菌(Zommonas Mobilis)中克隆高效乙醇代谢途径关键基因丙酮酸脱羧酶(PDC)和乙醇脱氢酶(ADHB)基因,构建含有强启动子Ptac及下游的pdc和adhB基因的表达质粒(P4),转化到耐乙醇大肠杆菌中,获得具有产乙醇能力的重组大肠杆菌(MG1655E-P4)。该重组菌在厌氧条件下,发酵葡萄糖生产的产物以乙醇为主,同时含有一定量的乙酸、乳酸等产物。其乙醇产率达到了理论值的56.8%,乙醇耐受浓度能保持在6.5%左右。
为了提高重组大肠杆菌的乙醇产率,减少副产物乙酸、乳酸产生。通过对大肠杆菌的糖代谢以及碳代谢流向的分析,本研究首先选择敲除大肠杆菌丙酮酸甲酸裂解酶基因(pflB),以期减少甲酸、乙酸的产生。采用λ噬菌体Red重组酶体系,构建了pflB缺失突变株,对突变株代谢产物测定结果显示乙酸的含量显著减少(仅为对照菌MG1655E的10.4%)。但是,乳酸含量却比对照提高了20.6%。当转入P4质粒后,获得产乙醇的pflB缺失突变株(MP-P4)乙醇产量其比MG1655E-P4提高了19.9%。
为进一步减少乳酸的积累,我们在pflB缺失突变株基础上,对编码乳酸脱氢酶的基因(ldhA)进行了敲除,构建得到ΔpflBΔldhA双突变菌株MPL。实验表明在厌氧条件下,当pflB基因被阻断后,细胞的生长速度下降;当pflB和ldhA基因同时被阻断后,细胞几乎不能在厌氧条件下生长。但是,pflB和ldhA基因的敲除却对菌体在好氧条件下的良好生长。转入P4质粒后,获得产乙醇的pflB和ldhA缺失突变株(MPL-P4)在好氧或厌氧条件下均能良好生长。
对所构建的乙醇工程菌(MPL-P4)进行发酵实验的结果表明,MPL-P4在含有10%葡萄糖的M9基本培养基中,发酵66小时的乙醇终产量达47.6g/L,发酵产物中乙酸、乳酸含量大大减少,检测不到乳酸的积累,乙醇产率达到了理论值的93.3%,比MP-P4产量提高了23%。
Lignocellulosic biomass is an attractive alternate to petroleum for production of biofuel. This conversion of biomass would require a new generation of microbial biocatalysts that can convert all the sugars present in the biomass to the desired compound. Eescherichia coli which has a extensive background of knowledge, is easy for genetic manipulation and able to utilize a wide range of carbon sources (cellulose and hemicellulose hydrolyzed), is selected for further research in this study. An ethanol-torerant E. coli MG1655 is isolated as a starting strain to knock out the pyruvate formate lyase gene (pflB) and alcohol dehydrogenase gene (ldhA), and to develop an ethanologenic strain using molecular technologies.
To enhance the ethanol tolerance of MG1655 strain, directed evolution was adopted by inoculating and transferring the strain serially into LB medium with increasing concentrations of ethanol. A final mutant that can tolerate up to 6.5% (w/v) of ethanol was obtained, which was selected to express the ethanol production genes from Zommonas mobilis.
Z. mobilis pdc and adhB genes were cloned in pZY507 to yield the tandem plasmid, P4, with both genes expressed under the lacIq-tac regulation system. P4 was introduced by transformation into both the selected ethanol-tolerant mutant, resulting recombinant was named MG1655E-P4, which can grow under 6.5% of ethanol, produced ethanol as main product, productivity of which researched 56.8% of the theory value.
To reduce the organic acids produced and improve ethanol productivity during fermentation, Gene pflB, which is highly expressed in E. coli MG1655E, was eliminated by using recombinase ofλphage system, the developed MP mutant showed a drastic decrease in acetate production (10.4% of parental strain), but an increase of 20.6% in lactate. To further reduce the lactate formation, the pflB and ldhA deficient double mutant (MPL) was constructed. In anaerobic condition, the growth of pflB- mutant was lower than the strain MG1655E, and the double mutant could not grow under the anaerobic condition, while both strains showed no siganificant difference with the strain MG1655E in aerobic growth. When the plasmid P4 was introduced into MP and MPL, respectively. The resulting strains MP-P4 and MPL-P4 could grows well in both aerobic and anaerobic circumstances.
Ethanol production by strain MPL-P4 was evaluated in M9 medium added with 10% of glucose, the amount of acetate was greatly reduced and no lactate was detected in the culture at 66 hours, the final concentration of ethanol reached 6.0%, 93% of the theory value, a 23% improvement over MP-P4.
本研究从10余种不同来源的大肠杆菌中筛选获得具有较高乙醇耐受性的MG1655菌株,通过连续在高乙醇浓度环境中定向筛选,获得了耐受乙醇的浓度达6.5%(w/v)的大肠杆菌菌株MG1655E。以该菌作为出发菌株,从运动运动发酵单胞菌(Zommonas Mobilis)中克隆高效乙醇代谢途径关键基因丙酮酸脱羧酶(PDC)和乙醇脱氢酶(ADHB)基因,构建含有强启动子Ptac及下游的pdc和adhB基因的表达质粒(P4),转化到耐乙醇大肠杆菌中,获得具有产乙醇能力的重组大肠杆菌(MG1655E-P4)。该重组菌在厌氧条件下,发酵葡萄糖生产的产物以乙醇为主,同时含有一定量的乙酸、乳酸等产物。其乙醇产率达到了理论值的56.8%,乙醇耐受浓度能保持在6.5%左右。
为了提高重组大肠杆菌的乙醇产率,减少副产物乙酸、乳酸产生。通过对大肠杆菌的糖代谢以及碳代谢流向的分析,本研究首先选择敲除大肠杆菌丙酮酸甲酸裂解酶基因(pflB),以期减少甲酸、乙酸的产生。采用λ噬菌体Red重组酶体系,构建了pflB缺失突变株,对突变株代谢产物测定结果显示乙酸的含量显著减少(仅为对照菌MG1655E的10.4%)。但是,乳酸含量却比对照提高了20.6%。当转入P4质粒后,获得产乙醇的pflB缺失突变株(MP-P4)乙醇产量其比MG1655E-P4提高了19.9%。
为进一步减少乳酸的积累,我们在pflB缺失突变株基础上,对编码乳酸脱氢酶的基因(ldhA)进行了敲除,构建得到ΔpflBΔldhA双突变菌株MPL。实验表明在厌氧条件下,当pflB基因被阻断后,细胞的生长速度下降;当pflB和ldhA基因同时被阻断后,细胞几乎不能在厌氧条件下生长。但是,pflB和ldhA基因的敲除却对菌体在好氧条件下的良好生长。转入P4质粒后,获得产乙醇的pflB和ldhA缺失突变株(MPL-P4)在好氧或厌氧条件下均能良好生长。
对所构建的乙醇工程菌(MPL-P4)进行发酵实验的结果表明,MPL-P4在含有10%葡萄糖的M9基本培养基中,发酵66小时的乙醇终产量达47.6g/L,发酵产物中乙酸、乳酸含量大大减少,检测不到乳酸的积累,乙醇产率达到了理论值的93.3%,比MP-P4产量提高了23%。
Lignocellulosic biomass is an attractive alternate to petroleum for production of biofuel. This conversion of biomass would require a new generation of microbial biocatalysts that can convert all the sugars present in the biomass to the desired compound. Eescherichia coli which has a extensive background of knowledge, is easy for genetic manipulation and able to utilize a wide range of carbon sources (cellulose and hemicellulose hydrolyzed), is selected for further research in this study. An ethanol-torerant E. coli MG1655 is isolated as a starting strain to knock out the pyruvate formate lyase gene (pflB) and alcohol dehydrogenase gene (ldhA), and to develop an ethanologenic strain using molecular technologies.
To enhance the ethanol tolerance of MG1655 strain, directed evolution was adopted by inoculating and transferring the strain serially into LB medium with increasing concentrations of ethanol. A final mutant that can tolerate up to 6.5% (w/v) of ethanol was obtained, which was selected to express the ethanol production genes from Zommonas mobilis.
Z. mobilis pdc and adhB genes were cloned in pZY507 to yield the tandem plasmid, P4, with both genes expressed under the lacIq-tac regulation system. P4 was introduced by transformation into both the selected ethanol-tolerant mutant, resulting recombinant was named MG1655E-P4, which can grow under 6.5% of ethanol, produced ethanol as main product, productivity of which researched 56.8% of the theory value.
To reduce the organic acids produced and improve ethanol productivity during fermentation, Gene pflB, which is highly expressed in E. coli MG1655E, was eliminated by using recombinase ofλphage system, the developed MP mutant showed a drastic decrease in acetate production (10.4% of parental strain), but an increase of 20.6% in lactate. To further reduce the lactate formation, the pflB and ldhA deficient double mutant (MPL) was constructed. In anaerobic condition, the growth of pflB- mutant was lower than the strain MG1655E, and the double mutant could not grow under the anaerobic condition, while both strains showed no siganificant difference with the strain MG1655E in aerobic growth. When the plasmid P4 was introduced into MP and MPL, respectively. The resulting strains MP-P4 and MPL-P4 could grows well in both aerobic and anaerobic circumstances.
Ethanol production by strain MPL-P4 was evaluated in M9 medium added with 10% of glucose, the amount of acetate was greatly reduced and no lactate was detected in the culture at 66 hours, the final concentration of ethanol reached 6.0%, 93% of the theory value, a 23% improvement over MP-P4.
引文
1.高卫华,张敏华,刘成,邹少兰,吴经才,能利用五碳糖和六碳糖生产乙醇的基因工程菌菌株的构建.工业微生物, 2004, 334:4～8
2.胡堃史兆兴赛道建, Red重组系统在在微生物基因敲除中的应用遗传.微生物学报, 2003, 25:628～632
3.辛芬,陈汉平,王贤华,杨海平,杨国来,张世红,高斌,刘德昌.木质纤维素生物质生产乙醇的预处理技术.新能源及工艺, 2006, 03:24～28
4.辛艳丽,贾玉艳,王恒梁,邓继先,利用Red系统快速改构含鼠β-酪蛋白基因的细菌人工染色体.中国生物工程杂志, 2005, 25:45-50
5. Alper H, Moxley J, Nevoigt E, Fink G R. and Stephanopoulos G. Engineering yeast transcription machinery for improved ethanol tolerance and production. Science, 2006, (New York, N.Y) 314:1565～1568.
6. Alterthum F, Ingram L O. Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli [J]. AEM, 1989, 55(8):1943～1948.
7. An H, Scopes R K, Rodriguez M, Keshav K F and Ingram L O. Gel electrophoretic analysis of Zymomonas mobilis glycolytic and fermentative enzymes: identification of alcohol dehydrogenase II as a stress protein. Journal of Bacteriology, 1991, 173: 5975～5982.
8. Anderlund M, Radstrom P, Hahn H et al. Expression of bifunctional enzymes with xylose reductase and xylitol dehydrogenase activity in Saccharomyces cerevisiae alters product formation during xylose fermentation [J].Metabolic Engineering, 2001, 3:226～235.
9. Aristos A, Penttila M. Metabolic engineering applications to renewable resource utilization. Curr Opin Biotechol, 2000, 1(2):187～198
10. Asghari A, Bothast R J, Doran J B, et al., Ethanol production from hemicellulose hydrolysates of agricultural residues using genetically engineered Escherichia coli strain KO11 [J]. J. ind. Microbiol., 1996, 16:42～47.
11. Bao XM, GAO D, Qu YB et al Effect on product formation in recombinant Saccharomyces cerevisiae strains expressing different levels of xylose metabolic genes. Chinese Journal of Biotechnology, 1997, 13(4):225～231
12. Bin Zhou, Gregory J.O. Martin, Neville B. Pamment. Increased Phenotypic Stability and Ethanol Tolerance of Recombinant Escherichia coli KO11 When Immobilized in Continuous fluidized Bed Culture. Biotechnology. and Bioengineering, 2008, Published online
13. BP.BP statistical review of world energy 2006. http: / /www. bp. com /Sectiongenericarticle. do? categoryId = 9017890&contentId = 7033493, 2007.
14. Bruinenber P M, de Bot P H M, Scheffers W A et al. The role of redox balances in the anaerobic fermentation of xylose by yeasts. J Microbiol Biotechnol, 1983, 18:287～292
15. Candida intermedia: first molecular characterization of ayeast xylose -H+ symporter. [J].Biochem J , 2006, 395:543～549.
16. Candy JM. and Duggleby RG. Investigation of the cofactor-binding site of Zymomonas mobilis pyruvate decarboxylase by site-directed mutagenesis. The Biochemical Journal, 1994, 300: 7～13.
17. Candy, J.M., and Duggleby, R.G. Structure and properties of pyruvate decarboxylase and site directed mutagenesis of the Zymomonas mobilis enzyme. Biochimica et biophysica acta, 1998, 1385:323～338.
18. Cassey B, Guest J R, Attwood M M. Environmental control of pyruvate dehydrogenase complex expression in Escherichia coli [J]. FEMS Microbiol. Lett., 1998, 159:325～329.
19. Clark DP. The fermentation pathways of Escherichia coli. FEMS Microbiol. Rev, 1989, 63:223～234.
20. Conway T, Osman YA, Konnan JI, Hoffmann EM. and Ingram LO. Promoter and nucleotide sequences of the Zymomonas mobilis pyruvate decarboxylase. Journal of Bacteriology, 1987, 169:949～954.
21. Conway T, Sewell GW, Osman YA. and Ingram L O. Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis. Journal of bacteriology, 1987, 169:2591～2597.
22. Deng MD and Coleman JR. Ethanol synthesis by genetic engineering in cyanobacteria. Applied and environmental microbiology, 1999, 65:523～528.
23. Diefenbach RJ and Duggleby RG. Pyruvate decarboxylase from Zymomonas mobilis. Struct- ure and re-activation of apoenzyme by the cofactors thiamin diphosphate and magnesiumion. the Biochemical Journal, 1991, 276:439～445.
24. Dien BS, Hespell R B, Ingram LO, et al., Conversion of corn milling fibrous co-products into ethanol by recombinant Escherichia coli strains K011 and SL40 [J]. J. Ind. Microbiol. Biotech., 1997, 13:19～625.
25. Dien BS, Nichols NN, O'Bryan PJ, et al., Development of new ethanologenic Escherichia coli strains for fermentation of lignocellulosic biomass [J]. Appl. Biochem. Biotechnol., 2000, 86:181～196.
26. Dobritzsch D, Konig S, Schneider G and Lu G. High resolution crystal structure of pyruvate decarboxylase from Zymomonas mobilis. the Journal of Biological Chemistry, 1998, 273:20196～20204.
27. Dombek KM, and Ingram LO. Effects of ethanol on the Escherichia coli plasma membrane. Journal of bacteriology , 1984, 157:233～239.
28. Dumsday GJ, Zhou B, Yaqin W, et al., Comparative stability of ethanol production by Escherichia coli [J]. J. ind. Microbiol. Biotech., 1999, 23:701～708.
29. Forster AC, Church GM. Towards synthesis of a minimal cell [J]. Molecular Systems Biology, 2006, 2:45.
30. Fraser CM, Gocayne JD, White O, et al., The minimal gene complement of Mycoplasma genitalium [J]., Science, 1995, 207:397～403.
31. Fried VA and A Novick. Organic solvents as probes for the structure and function of the bacterial membrane: effects of ethanol on the wild type and an ethanol-resistant mutant of Escherichia coli K-12, J Bacteriol, 1973, 114:239～248.
32. Gold RS, Meagher MM, Tong S, Hutkins RW and Conway T. Cloning and expression of the Zymomonas mobilis "production of ethanol" genes in Lactobacillus casei Current Microbiol- ogy, 1996, 33:256～260.
33. Gonzalez R, Tao H, Purvis JE, et al., Gene array-based identification of changes that contribute to ethanol tolerance in ethanologenic Escherichia coli: comparison of KO11 (parent) to LY01 (resistant mutant). Biotechnol. Prog., 2003, 19: 612～623.
34. Gregory Stephanopoulos. Challenges in Engineering Microbes for Biofuels Production Science, 2007, 315:801～804
35. Hahn-H?gerdal B, Jeppsson H, Olsson L et al, An interlaboratory comparison of the performance of ethanol-producing microorganisms in a xylose-rich acid hydrolysate [J]., Appl. Microbiol. Biotechnol., 1994, 41:62～72.
36. Hartner T, Straub KL, Kannenberg E. Occurrence of hopanoid lipids in anaerobic Geobacter species [J]. FEMS microbial. Lett., 2005, 243: 59～64.
37. Hespell RB, Wyckoff H, Dien BS, et al., Stabilization of PET operon plasmids and ethanol production in Escherichia coli strains lacking lactate dehydrogenase and pyruvate formate-lyase activitie [J]. Appl. Microbiol Biotechnol., 1996, 62:4594～4597.
38. Ingram LO, Aldrich HC, Borges AC, Causey TB, Martinez A, Morales F, Saleh A Underwo- od SA, Yomano LP, York SW, et al. Enteric bacterial catalysts for fuel ethanol production. Biote- chnology Progress, 1999, 15:855～866.
39. Ingram LO, Conway T, Clark DP, et al., Genetic engineering of ethanol production in Escherichia coli [J]. AEM., 1987, 53(10):2420–2425.
40. Ingram LO, Conway T, Clark DP, Sewell G.W and Preston JF. Genetic engineering of ethanol production in E.coli. AEM , 1987, 53:2420～2425
41. Ingram LO, Conway T. Expression of Different Levels of Ethanologenic Enzymes from Zymomonas mobilis in Recombinant Strains of Escherichia coli [J]. Appl. Environ. Microbiol, 1988, 54:397～404.
42. Jeppsson M, Bengtsson O, Franke K, Lee H, Hahn-Hagerdal B and Gorwa Grauslund M.F. The expression of a Pichia stipitis xylose reductase mutant with higher K(M) for NADPH increases ethanol production from xylose in recombinant Saccharomyces cere- visiae. Biotechnology and Bioengineering, 2006, 93:665～673.
43. Jeppsson M., Johansson B, Hahn-Hagerdal B and Gorwa-Grauslund M.F. Reduced oxi- dative pentose phosphate pathway flux in recombinant xylose-utilizing Saccharomyces cerevisiae strains improves the ethanol yield from xylose. Applied and environmental microbiology,2002, 68:1604～1609.
44. Kemppainen AJ,Shonnard D. Comparative life cycle assessments for biomass to ethanol production from different regional feedstocks [J]. Biotechno1 Prog, 2005, 2l:1075～1084.
45. Kenan C. Murphy. Use of Bacteriophage lambda Recombination Functions To Promote Gene Replacement in Escherichia coli. Journal of Bacteriology, 1998, 180:2063～2071
46. Keshav KF, Yomano LP, An HJ and Ingram LO. Cloning of the Zymomonas mobilis structural gene encoding alcohol dehydrogenase I (adhA): sequence comparison and expression in Escherichia coli. Journal of bacteriology, 1990, 172:2491～2497.
47. Kim Y, Ingram LO, Shanmugam KT. Construction of an Escherichia coli K-12 Mutant for Homo-ethanologenic Fermentation of Glucose or Xylose without Foreign Genes [J]. AEM., 2007, 73:1766～1771.
48. Kim Y, Ingram LO, Shanmugam KT. Dihydrolipoamide dehydrogenase mutation alters the NADH sensitivity of pyruvate dehydrogenase complex of Escherichia coli K-12 [J]. JB, 2008, 190:3851～3858.
49. Kirill AD and Barry LW. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A, 2000, 97:6640～6645.
50. Konig, S. Subunit structure, function and organisation of pyruvate decarboxylases from various organisms. Biochimica et biophysica acta, 1998, 1385, 271～286.
51. Kotter P, Ciriacy M. Xylose fermentation by Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol, 1993, 38:776～783
52. Kuyper M, Winkler AA, Van dijken JP et al. Minimal metabolic engineering of Saccha- romyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle [J]. FEMS Yeast Res, 2004, 4:655～664.
53. Lawford H G, Rousseau J D. Loss of ethanologenicity in Escherichia coli B recombinants pLOI297 and KO11 during growth in the absence of antibiotics [J]. Biotech. Lett., 1995, 17:751～756.
54. Leao C and van Uden N. Definition of ethanol tolerance[J]. Biochim. Biophys. Acta 1984, 774:43～48
55. Li Z. Karakousis G. Chiu S K, et a1. The beta protein of phage lambda promotes strand exchange Mol Biol, 1998, 276:733～744
56. Ligthelm M E, Prior B A, du Preez J C. the oxygen requirements of yeasts for the fermentation of D-xylose and D-glucose to ethanol. Appl Microbiol Biotechnol, 1988, 28:63
57. Lindsay SE, RJ Bothast and LO Ingram. Improved strains of recombinant Escherichia coli for ethanol production from sugar mixtures. Appl Microbiol Biotechnol, 1995, 43: 70–75.
58. Little JW. An exonuclease induced by bacteriophage. Nature of the enzymatic reaction.BiolChem, 1967, 242:679～686.
59. Ma YJ, Qi LL, Yang HB. The analysis and study of effect of atmospheric pollution on respiratory diseases. Liaoning Meteorological Quarterly, 2002, 2:233～234.
60. Martini S, Ricci M, Bonechi C, et al., In vivo 13C-NMR and modelling study of metabolic yield response to ethanol stress in a wild-type strain of Saccharomyces cerevisiae [J]. FEBS letters, 2004, 564: 63～68.
61. Mat-Jan F, Alam KY, and Clark DP. Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase. Journal of bacteriology, 1989, 171:342～348.
62. McKnight GL, Kato H, Upshall A., Parker MD, Saari, G and O'Hara PJ. Identification and molecular analysis of a third Aspergillus nidulans alcohol dehydrogenase gene. the EMBO Journal, 1985, 4:2093～2099.
63. Min Zhang, Christina Eddy, Kristine Deanda, Mark Finkelstein and Stephen Picataggio, Metabolic Engineering of a Pentose Metabolism Pathway in Ethanologenic Zymomonas mobilis. Science, 1995, 267: 240～243.
64. Neale AD, Scopes RK, Kelly JM. and Wettenhall RE. The two alcohol dehydrogenases of Zymomonas mobilis. Purification by differential dye ligand chromatography molec- ular character- risation and physiological roles. European Journal of Biochemistry. FEBS, 1986, 154:119～124.
65. Neale, A.D., Scopes, R.K., Wettenhall, R.E., and Hoogenraad, N.J.. Nucleotide seque- nce of the pyruvate decarboxylase gene from Zymomonas mobilis. Nucleic Acids Research, 1987, 15:1753～1761.
66. Neale, AD, Scopes RK, Wettenhall, RE and Hoogenraad NJ. Pyruvate decarboxylase of Zymomonas mobilis: isolation, properties, and genetic expression in Escherichia coli. Journal of Bacteriology, 1987, 169:1024～1028.
67. Nigam JM. Development of xylose- fermenting yeast pichia stipitis for ethanol production through adaptation on hardwood hemicellulose acid prehycrolysate[J]. Appl Microbiol, 2001, 90: 208～215
68. Ohta K, Beall D S, Mejia JP, et al., Genetic improvement of Escherichia coli for ethanol production chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II [J]. AEM, 1991, 57:903～900.
69. Ohta K, Beall DS, Mejia JP, Shanmugam KT, Ingram LO. Metabolic engineering of Klebsiella oxytoca M5A1 for ethanolproduction from xylose and glucose. Appl Environ Microbiol, 1991b, 57:2810–2815
70. Osman YA, Conway T, Bonetti SJ and Ingram LO. Glycolytic flux in Zymomonas mobi- lis enzyme and metabolite levels during batch fermentation. Journal of Bacteriology, 1987, 169:3726～3736.
71. Osvaldo D, Delgado M, Alejandra M, Carlos M, Abate and Faustino Si?eriz. Chromosomal integration and expression of green fluorescent protein in Zymomonas mobilis. Biotechnology Letters, 2002, 24:1285～1290.
72. Pearce F. Fuels gold: big risks of the biofuel revolution. New Scientist, 2006, 191 (2570) : 36241.
73. Peberdv J F. Use of the spheroplast fusion technique on ethanol tolerant yeasts. Enzyme Microb Technol, 1980, 2: 23～29.
74. Pohl M., Grotzinger J, Wollmer A., and Kula MR. Reversible dissociation and unfolding of pyruvate decarboxylase from Zymomonas mobilis. European Journal of Biochemistry, 1994, FEBS 224, 651～661.
75. Pohl M., Mesch K. and Rodenbrock A. Stability investigations on the pyruvate decarbo- xylase from Zymomonas mobilis. Biotechnol. Appl. Biochem, 1995, 22:95～105.
76. Preziosi-Belloy L, Nolleau V, Navarro JM. Fermentation of hemicellulosic sugars and sugar mixtures to xylitol by Candida parapsilosis [J]. Enzyme Micro. Technol., 1997, 21:124～129.
77. Purvis JE, Yomano LP, Ingram LO. Enhanced Trehalose Production Improves Growth of Escherichia coli under Osmotic Stress. Appli. Enviro. Microbio, 2005, 71(7)3761～3769
78. RFA,American Renewable fuels Association Statistics 2009 http://www.ethanolrfa.org /industry/ statistics/
79. Richard P, Verho R, Putkonen M, Londesborough J and Penttila M. Production of ethanol from L-arabinose by Saccharomyces cerevisiae containing a fungal L-arabinose pathway. FEMS Yeast Research, 2003, 3:185～189.
80. S BM, Schimz KL and Sahm H. Pyruvate decarboxylase from Zymomonas mobilis isolation and partial characterization. Arch. Microbiol. 1986, 146:105～110.
81. Sawers G. and A. Bock. Novel transcriptional control of the pyruvate formate-lyase gene: upstream regulatory sequences and multiple promoters regulate anaerobic expression. J. Bacteriol, 1989, 171:2485～2498.
82. Sawers G.and A. Bock. Anaerobic regulation of pyruvate formate-lyase from Escheri- chia coli K-12. J. Bacteriol, 1988, 170:5330～5336.
83. Shiloach J, Kaufman J, Guillard AS, Fass R. Effect of glucose supply strategy on acetate accumulation, growth, and recombinant protein production by Escherichia coli BL21 (λDE3) and Escherichia coli JM109. Biotechnol Bioeng, 1996, 49:421～428.
84. Sigrun D F, Hermann S and Georg A S. Pentose metabolism Z. mobilis wild type and recombi -nant strains. Appl microbial Biotechnol, 1992, 38:354～361.
85. Sreenath HK, Jeffries TW. Production of ethanol from wood hydrolyzate by yeasts . Bioresource Technology, 2000, 72: 253～260.
86. Stephanopoulos G. Challenges in engineering microbes for biofuels production [J]. Science, 2007, 315: 801～804.
87. Tamarit J, Cabiscol E, Aguilar J and Ros J. Differential inactivation of alcohol dehydr- ogenase isoenzymes in Zymomonas mobilis by oxygen. Journal of Bacteriology, 1997, 179:1102～1104.
88. Tarmy EM, Kaplan NO. Kinetics of Escherichia coli B D-Lactate Dehydrogenase and Evidence for Pyruvate-controlled Change in Conformation. J Biological Chemistry, 1968, 243:2587～2596.
89. Trinh C T, Unrean P, Srienc F. Minimal Escherichia coli cell for the most efficient production of ethanol from hexoses and pentoses [J]. AEM, 2008, 74:3634～3643
90. Underwood S A, Buszko A L, Shanmugam K T, et al., Lack of protective osmolytes limits final cell density and volumetric productivity of ethanologenic Escherichia coli KO11 during xylose fermentation [J]. AEM, 2004, 70: 2734.
91. Underwood SA, Zhou S, Causey TB, et al., Genetic changes to optimize carbon partitioning between ethanol and biosynthesis in ethanologenic Escherichia coli [J]. AEM, 2002, 68: 6263～6272.
92. Wang Z, Chen M, Xu Y, et al, An ethanol-tolerant recombinant Escherichia coli expressing Zymomonas mobilis pdc and adhB genes for enhanced ethanol production from xylose [J]. Biotech Lett 2008, 30:657～663.
93. Watanabe S, Kodaki T and Makino K. Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermo-stability by the introduction of structural zinc. Journal of Biological Chemistry, 2005, 280:10340～10349.
94. Williamson VM and Paquin CE. Homology of Saccharomyces cerevisiae ADH4 to an iron activated alcohol dehydrogenase from Zymomonas mobilis. Mol Gen Genet, 1987, 209: 374～381
95. Wills C, Kratofil P, Londo D and Martin T. Characterization of the two alcohol dehyd- rogenases of Zymomonas mobilis. Archives of Biochemistry and Biophysics, 1981, 210:775～785.
96. Wood BE, Ingram LO, Ethanol-production from cellobiose,amorphous cellulose, and crystalline cellulose by recombinant Klebsiella oxytoca containing chromosomally integrated Zymomonas mobilis genes for ethanol-production and plasmids expressing thermostable cellulase genes from Clostridium thermocellum. Appl Environ Microbiol, 1992, 58:2103～2110
97. Yomano L P, York S W, Zhou S, et al., Re-engineering Escherichia coli for ethanol production [J]. Biotechnol. Lett., 2008, 30:2097～2103.
98. Yomano L P, York S W, Ingram L O. Isolation and characterization of ethanol-tolerant mutan- ts of Escherichia coli KO11 for fuel ethanol production [J]. J. Ind. Microbiol. Biotech., 1998, 20:132～138
99. York S W and Ingrain L O. Ethanol production by recombinant Escherichia coli KO11 using crude yeast autolysate as a nutrient supplement [J]. Biotechnol Lett, 1996, 6:683～688.
100. Zhang L, Song GY. Child blood lead level if influence factor relevance. Medical Journal of Chinese People’s Health, 2006, 18 (10):832～834.
101. Zhang SM, Dai YH, Xie X H, et al. Study on blood lead level and related risk factors among children aged 0-26 years in 15 cities in China. Chin J Ep idemiol, 2005, 26 (9):2651～2654.
102. Zhou S, Davis FC, Ingram LO Gene integration and expression and extracellular secretion of Erwinia chrysanthemi endoglucanase CelY (celY) and CelZ (celZ) in ethanologenicKlebsiella oxytoca P2. Appl Environ Microbiol, 2001,67:6–14
103. 7Zhou S, Ingram LO, Synergistic hydrolysis of carboxymethyl cellulose and acid swollen cellulose by two endoglucanases (CelZ and CelY) from Erwinia chrysanthemi. J Bacteriol, 2000, 182:5676–5682
104. Zhou S, Yomano L P, Shanmugam K T, et al., Fermentation of 10% (w/v) sugar to D(-)-lactate by engineered Escherichia coli B [J]. Biotechnol. Lett., 2005, 1891～1896.
105. Zhu J, and Shimizu K. The effect of pfl gene knockout on the metabolism for optically pure D-lactate production by Escherichia coli. Applied microbiology and biote chnology, 2004, 64:367～375.
2.胡堃史兆兴赛道建, Red重组系统在在微生物基因敲除中的应用遗传.微生物学报, 2003, 25:628～632
3.辛芬,陈汉平,王贤华,杨海平,杨国来,张世红,高斌,刘德昌.木质纤维素生物质生产乙醇的预处理技术.新能源及工艺, 2006, 03:24～28
4.辛艳丽,贾玉艳,王恒梁,邓继先,利用Red系统快速改构含鼠β-酪蛋白基因的细菌人工染色体.中国生物工程杂志, 2005, 25:45-50
5. Alper H, Moxley J, Nevoigt E, Fink G R. and Stephanopoulos G. Engineering yeast transcription machinery for improved ethanol tolerance and production. Science, 2006, (New York, N.Y) 314:1565～1568.
6. Alterthum F, Ingram L O. Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli [J]. AEM, 1989, 55(8):1943～1948.
7. An H, Scopes R K, Rodriguez M, Keshav K F and Ingram L O. Gel electrophoretic analysis of Zymomonas mobilis glycolytic and fermentative enzymes: identification of alcohol dehydrogenase II as a stress protein. Journal of Bacteriology, 1991, 173: 5975～5982.
8. Anderlund M, Radstrom P, Hahn H et al. Expression of bifunctional enzymes with xylose reductase and xylitol dehydrogenase activity in Saccharomyces cerevisiae alters product formation during xylose fermentation [J].Metabolic Engineering, 2001, 3:226～235.
9. Aristos A, Penttila M. Metabolic engineering applications to renewable resource utilization. Curr Opin Biotechol, 2000, 1(2):187～198
10. Asghari A, Bothast R J, Doran J B, et al., Ethanol production from hemicellulose hydrolysates of agricultural residues using genetically engineered Escherichia coli strain KO11 [J]. J. ind. Microbiol., 1996, 16:42～47.
11. Bao XM, GAO D, Qu YB et al Effect on product formation in recombinant Saccharomyces cerevisiae strains expressing different levels of xylose metabolic genes. Chinese Journal of Biotechnology, 1997, 13(4):225～231
12. Bin Zhou, Gregory J.O. Martin, Neville B. Pamment. Increased Phenotypic Stability and Ethanol Tolerance of Recombinant Escherichia coli KO11 When Immobilized in Continuous fluidized Bed Culture. Biotechnology. and Bioengineering, 2008, Published online
13. BP.BP statistical review of world energy 2006. http: / /www. bp. com /Sectiongenericarticle. do? categoryId = 9017890&contentId = 7033493, 2007.
14. Bruinenber P M, de Bot P H M, Scheffers W A et al. The role of redox balances in the anaerobic fermentation of xylose by yeasts. J Microbiol Biotechnol, 1983, 18:287～292
15. Candida intermedia: first molecular characterization of ayeast xylose -H+ symporter. [J].Biochem J , 2006, 395:543～549.
16. Candy JM. and Duggleby RG. Investigation of the cofactor-binding site of Zymomonas mobilis pyruvate decarboxylase by site-directed mutagenesis. The Biochemical Journal, 1994, 300: 7～13.
17. Candy, J.M., and Duggleby, R.G. Structure and properties of pyruvate decarboxylase and site directed mutagenesis of the Zymomonas mobilis enzyme. Biochimica et biophysica acta, 1998, 1385:323～338.
18. Cassey B, Guest J R, Attwood M M. Environmental control of pyruvate dehydrogenase complex expression in Escherichia coli [J]. FEMS Microbiol. Lett., 1998, 159:325～329.
19. Clark DP. The fermentation pathways of Escherichia coli. FEMS Microbiol. Rev, 1989, 63:223～234.
20. Conway T, Osman YA, Konnan JI, Hoffmann EM. and Ingram LO. Promoter and nucleotide sequences of the Zymomonas mobilis pyruvate decarboxylase. Journal of Bacteriology, 1987, 169:949～954.
21. Conway T, Sewell GW, Osman YA. and Ingram L O. Cloning and sequencing of the alcohol dehydrogenase II gene from Zymomonas mobilis. Journal of bacteriology, 1987, 169:2591～2597.
22. Deng MD and Coleman JR. Ethanol synthesis by genetic engineering in cyanobacteria. Applied and environmental microbiology, 1999, 65:523～528.
23. Diefenbach RJ and Duggleby RG. Pyruvate decarboxylase from Zymomonas mobilis. Struct- ure and re-activation of apoenzyme by the cofactors thiamin diphosphate and magnesiumion. the Biochemical Journal, 1991, 276:439～445.
24. Dien BS, Hespell R B, Ingram LO, et al., Conversion of corn milling fibrous co-products into ethanol by recombinant Escherichia coli strains K011 and SL40 [J]. J. Ind. Microbiol. Biotech., 1997, 13:19～625.
25. Dien BS, Nichols NN, O'Bryan PJ, et al., Development of new ethanologenic Escherichia coli strains for fermentation of lignocellulosic biomass [J]. Appl. Biochem. Biotechnol., 2000, 86:181～196.
26. Dobritzsch D, Konig S, Schneider G and Lu G. High resolution crystal structure of pyruvate decarboxylase from Zymomonas mobilis. the Journal of Biological Chemistry, 1998, 273:20196～20204.
27. Dombek KM, and Ingram LO. Effects of ethanol on the Escherichia coli plasma membrane. Journal of bacteriology , 1984, 157:233～239.
28. Dumsday GJ, Zhou B, Yaqin W, et al., Comparative stability of ethanol production by Escherichia coli [J]. J. ind. Microbiol. Biotech., 1999, 23:701～708.
29. Forster AC, Church GM. Towards synthesis of a minimal cell [J]. Molecular Systems Biology, 2006, 2:45.
30. Fraser CM, Gocayne JD, White O, et al., The minimal gene complement of Mycoplasma genitalium [J]., Science, 1995, 207:397～403.
31. Fried VA and A Novick. Organic solvents as probes for the structure and function of the bacterial membrane: effects of ethanol on the wild type and an ethanol-resistant mutant of Escherichia coli K-12, J Bacteriol, 1973, 114:239～248.
32. Gold RS, Meagher MM, Tong S, Hutkins RW and Conway T. Cloning and expression of the Zymomonas mobilis "production of ethanol" genes in Lactobacillus casei Current Microbiol- ogy, 1996, 33:256～260.
33. Gonzalez R, Tao H, Purvis JE, et al., Gene array-based identification of changes that contribute to ethanol tolerance in ethanologenic Escherichia coli: comparison of KO11 (parent) to LY01 (resistant mutant). Biotechnol. Prog., 2003, 19: 612～623.
34. Gregory Stephanopoulos. Challenges in Engineering Microbes for Biofuels Production Science, 2007, 315:801～804
35. Hahn-H?gerdal B, Jeppsson H, Olsson L et al, An interlaboratory comparison of the performance of ethanol-producing microorganisms in a xylose-rich acid hydrolysate [J]., Appl. Microbiol. Biotechnol., 1994, 41:62～72.
36. Hartner T, Straub KL, Kannenberg E. Occurrence of hopanoid lipids in anaerobic Geobacter species [J]. FEMS microbial. Lett., 2005, 243: 59～64.
37. Hespell RB, Wyckoff H, Dien BS, et al., Stabilization of PET operon plasmids and ethanol production in Escherichia coli strains lacking lactate dehydrogenase and pyruvate formate-lyase activitie [J]. Appl. Microbiol Biotechnol., 1996, 62:4594～4597.
38. Ingram LO, Aldrich HC, Borges AC, Causey TB, Martinez A, Morales F, Saleh A Underwo- od SA, Yomano LP, York SW, et al. Enteric bacterial catalysts for fuel ethanol production. Biote- chnology Progress, 1999, 15:855～866.
39. Ingram LO, Conway T, Clark DP, et al., Genetic engineering of ethanol production in Escherichia coli [J]. AEM., 1987, 53(10):2420–2425.
40. Ingram LO, Conway T, Clark DP, Sewell G.W and Preston JF. Genetic engineering of ethanol production in E.coli. AEM , 1987, 53:2420～2425
41. Ingram LO, Conway T. Expression of Different Levels of Ethanologenic Enzymes from Zymomonas mobilis in Recombinant Strains of Escherichia coli [J]. Appl. Environ. Microbiol, 1988, 54:397～404.
42. Jeppsson M, Bengtsson O, Franke K, Lee H, Hahn-Hagerdal B and Gorwa Grauslund M.F. The expression of a Pichia stipitis xylose reductase mutant with higher K(M) for NADPH increases ethanol production from xylose in recombinant Saccharomyces cere- visiae. Biotechnology and Bioengineering, 2006, 93:665～673.
43. Jeppsson M., Johansson B, Hahn-Hagerdal B and Gorwa-Grauslund M.F. Reduced oxi- dative pentose phosphate pathway flux in recombinant xylose-utilizing Saccharomyces cerevisiae strains improves the ethanol yield from xylose. Applied and environmental microbiology,2002, 68:1604～1609.
44. Kemppainen AJ,Shonnard D. Comparative life cycle assessments for biomass to ethanol production from different regional feedstocks [J]. Biotechno1 Prog, 2005, 2l:1075～1084.
45. Kenan C. Murphy. Use of Bacteriophage lambda Recombination Functions To Promote Gene Replacement in Escherichia coli. Journal of Bacteriology, 1998, 180:2063～2071
46. Keshav KF, Yomano LP, An HJ and Ingram LO. Cloning of the Zymomonas mobilis structural gene encoding alcohol dehydrogenase I (adhA): sequence comparison and expression in Escherichia coli. Journal of bacteriology, 1990, 172:2491～2497.
47. Kim Y, Ingram LO, Shanmugam KT. Construction of an Escherichia coli K-12 Mutant for Homo-ethanologenic Fermentation of Glucose or Xylose without Foreign Genes [J]. AEM., 2007, 73:1766～1771.
48. Kim Y, Ingram LO, Shanmugam KT. Dihydrolipoamide dehydrogenase mutation alters the NADH sensitivity of pyruvate dehydrogenase complex of Escherichia coli K-12 [J]. JB, 2008, 190:3851～3858.
49. Kirill AD and Barry LW. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A, 2000, 97:6640～6645.
50. Konig, S. Subunit structure, function and organisation of pyruvate decarboxylases from various organisms. Biochimica et biophysica acta, 1998, 1385, 271～286.
51. Kotter P, Ciriacy M. Xylose fermentation by Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol, 1993, 38:776～783
52. Kuyper M, Winkler AA, Van dijken JP et al. Minimal metabolic engineering of Saccha- romyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle [J]. FEMS Yeast Res, 2004, 4:655～664.
53. Lawford H G, Rousseau J D. Loss of ethanologenicity in Escherichia coli B recombinants pLOI297 and KO11 during growth in the absence of antibiotics [J]. Biotech. Lett., 1995, 17:751～756.
54. Leao C and van Uden N. Definition of ethanol tolerance[J]. Biochim. Biophys. Acta 1984, 774:43～48
55. Li Z. Karakousis G. Chiu S K, et a1. The beta protein of phage lambda promotes strand exchange Mol Biol, 1998, 276:733～744
56. Ligthelm M E, Prior B A, du Preez J C. the oxygen requirements of yeasts for the fermentation of D-xylose and D-glucose to ethanol. Appl Microbiol Biotechnol, 1988, 28:63
57. Lindsay SE, RJ Bothast and LO Ingram. Improved strains of recombinant Escherichia coli for ethanol production from sugar mixtures. Appl Microbiol Biotechnol, 1995, 43: 70–75.
58. Little JW. An exonuclease induced by bacteriophage. Nature of the enzymatic reaction.BiolChem, 1967, 242:679～686.
59. Ma YJ, Qi LL, Yang HB. The analysis and study of effect of atmospheric pollution on respiratory diseases. Liaoning Meteorological Quarterly, 2002, 2:233～234.
60. Martini S, Ricci M, Bonechi C, et al., In vivo 13C-NMR and modelling study of metabolic yield response to ethanol stress in a wild-type strain of Saccharomyces cerevisiae [J]. FEBS letters, 2004, 564: 63～68.
61. Mat-Jan F, Alam KY, and Clark DP. Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase. Journal of bacteriology, 1989, 171:342～348.
62. McKnight GL, Kato H, Upshall A., Parker MD, Saari, G and O'Hara PJ. Identification and molecular analysis of a third Aspergillus nidulans alcohol dehydrogenase gene. the EMBO Journal, 1985, 4:2093～2099.
63. Min Zhang, Christina Eddy, Kristine Deanda, Mark Finkelstein and Stephen Picataggio, Metabolic Engineering of a Pentose Metabolism Pathway in Ethanologenic Zymomonas mobilis. Science, 1995, 267: 240～243.
64. Neale AD, Scopes RK, Kelly JM. and Wettenhall RE. The two alcohol dehydrogenases of Zymomonas mobilis. Purification by differential dye ligand chromatography molec- ular character- risation and physiological roles. European Journal of Biochemistry. FEBS, 1986, 154:119～124.
65. Neale, A.D., Scopes, R.K., Wettenhall, R.E., and Hoogenraad, N.J.. Nucleotide seque- nce of the pyruvate decarboxylase gene from Zymomonas mobilis. Nucleic Acids Research, 1987, 15:1753～1761.
66. Neale, AD, Scopes RK, Wettenhall, RE and Hoogenraad NJ. Pyruvate decarboxylase of Zymomonas mobilis: isolation, properties, and genetic expression in Escherichia coli. Journal of Bacteriology, 1987, 169:1024～1028.
67. Nigam JM. Development of xylose- fermenting yeast pichia stipitis for ethanol production through adaptation on hardwood hemicellulose acid prehycrolysate[J]. Appl Microbiol, 2001, 90: 208～215
68. Ohta K, Beall D S, Mejia JP, et al., Genetic improvement of Escherichia coli for ethanol production chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II [J]. AEM, 1991, 57:903～900.
69. Ohta K, Beall DS, Mejia JP, Shanmugam KT, Ingram LO. Metabolic engineering of Klebsiella oxytoca M5A1 for ethanolproduction from xylose and glucose. Appl Environ Microbiol, 1991b, 57:2810–2815
70. Osman YA, Conway T, Bonetti SJ and Ingram LO. Glycolytic flux in Zymomonas mobi- lis enzyme and metabolite levels during batch fermentation. Journal of Bacteriology, 1987, 169:3726～3736.
71. Osvaldo D, Delgado M, Alejandra M, Carlos M, Abate and Faustino Si?eriz. Chromosomal integration and expression of green fluorescent protein in Zymomonas mobilis. Biotechnology Letters, 2002, 24:1285～1290.
72. Pearce F. Fuels gold: big risks of the biofuel revolution. New Scientist, 2006, 191 (2570) : 36241.
73. Peberdv J F. Use of the spheroplast fusion technique on ethanol tolerant yeasts. Enzyme Microb Technol, 1980, 2: 23～29.
74. Pohl M., Grotzinger J, Wollmer A., and Kula MR. Reversible dissociation and unfolding of pyruvate decarboxylase from Zymomonas mobilis. European Journal of Biochemistry, 1994, FEBS 224, 651～661.
75. Pohl M., Mesch K. and Rodenbrock A. Stability investigations on the pyruvate decarbo- xylase from Zymomonas mobilis. Biotechnol. Appl. Biochem, 1995, 22:95～105.
76. Preziosi-Belloy L, Nolleau V, Navarro JM. Fermentation of hemicellulosic sugars and sugar mixtures to xylitol by Candida parapsilosis [J]. Enzyme Micro. Technol., 1997, 21:124～129.
77. Purvis JE, Yomano LP, Ingram LO. Enhanced Trehalose Production Improves Growth of Escherichia coli under Osmotic Stress. Appli. Enviro. Microbio, 2005, 71(7)3761～3769
78. RFA,American Renewable fuels Association Statistics 2009 http://www.ethanolrfa.org /industry/ statistics/
79. Richard P, Verho R, Putkonen M, Londesborough J and Penttila M. Production of ethanol from L-arabinose by Saccharomyces cerevisiae containing a fungal L-arabinose pathway. FEMS Yeast Research, 2003, 3:185～189.
80. S BM, Schimz KL and Sahm H. Pyruvate decarboxylase from Zymomonas mobilis isolation and partial characterization. Arch. Microbiol. 1986, 146:105～110.
81. Sawers G. and A. Bock. Novel transcriptional control of the pyruvate formate-lyase gene: upstream regulatory sequences and multiple promoters regulate anaerobic expression. J. Bacteriol, 1989, 171:2485～2498.
82. Sawers G.and A. Bock. Anaerobic regulation of pyruvate formate-lyase from Escheri- chia coli K-12. J. Bacteriol, 1988, 170:5330～5336.
83. Shiloach J, Kaufman J, Guillard AS, Fass R. Effect of glucose supply strategy on acetate accumulation, growth, and recombinant protein production by Escherichia coli BL21 (λDE3) and Escherichia coli JM109. Biotechnol Bioeng, 1996, 49:421～428.
84. Sigrun D F, Hermann S and Georg A S. Pentose metabolism Z. mobilis wild type and recombi -nant strains. Appl microbial Biotechnol, 1992, 38:354～361.
85. Sreenath HK, Jeffries TW. Production of ethanol from wood hydrolyzate by yeasts . Bioresource Technology, 2000, 72: 253～260.
86. Stephanopoulos G. Challenges in engineering microbes for biofuels production [J]. Science, 2007, 315: 801～804.
87. Tamarit J, Cabiscol E, Aguilar J and Ros J. Differential inactivation of alcohol dehydr- ogenase isoenzymes in Zymomonas mobilis by oxygen. Journal of Bacteriology, 1997, 179:1102～1104.
88. Tarmy EM, Kaplan NO. Kinetics of Escherichia coli B D-Lactate Dehydrogenase and Evidence for Pyruvate-controlled Change in Conformation. J Biological Chemistry, 1968, 243:2587～2596.
89. Trinh C T, Unrean P, Srienc F. Minimal Escherichia coli cell for the most efficient production of ethanol from hexoses and pentoses [J]. AEM, 2008, 74:3634～3643
90. Underwood S A, Buszko A L, Shanmugam K T, et al., Lack of protective osmolytes limits final cell density and volumetric productivity of ethanologenic Escherichia coli KO11 during xylose fermentation [J]. AEM, 2004, 70: 2734.
91. Underwood SA, Zhou S, Causey TB, et al., Genetic changes to optimize carbon partitioning between ethanol and biosynthesis in ethanologenic Escherichia coli [J]. AEM, 2002, 68: 6263～6272.
92. Wang Z, Chen M, Xu Y, et al, An ethanol-tolerant recombinant Escherichia coli expressing Zymomonas mobilis pdc and adhB genes for enhanced ethanol production from xylose [J]. Biotech Lett 2008, 30:657～663.
93. Watanabe S, Kodaki T and Makino K. Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermo-stability by the introduction of structural zinc. Journal of Biological Chemistry, 2005, 280:10340～10349.
94. Williamson VM and Paquin CE. Homology of Saccharomyces cerevisiae ADH4 to an iron activated alcohol dehydrogenase from Zymomonas mobilis. Mol Gen Genet, 1987, 209: 374～381
95. Wills C, Kratofil P, Londo D and Martin T. Characterization of the two alcohol dehyd- rogenases of Zymomonas mobilis. Archives of Biochemistry and Biophysics, 1981, 210:775～785.
96. Wood BE, Ingram LO, Ethanol-production from cellobiose,amorphous cellulose, and crystalline cellulose by recombinant Klebsiella oxytoca containing chromosomally integrated Zymomonas mobilis genes for ethanol-production and plasmids expressing thermostable cellulase genes from Clostridium thermocellum. Appl Environ Microbiol, 1992, 58:2103～2110
97. Yomano L P, York S W, Zhou S, et al., Re-engineering Escherichia coli for ethanol production [J]. Biotechnol. Lett., 2008, 30:2097～2103.
98. Yomano L P, York S W, Ingram L O. Isolation and characterization of ethanol-tolerant mutan- ts of Escherichia coli KO11 for fuel ethanol production [J]. J. Ind. Microbiol. Biotech., 1998, 20:132～138
99. York S W and Ingrain L O. Ethanol production by recombinant Escherichia coli KO11 using crude yeast autolysate as a nutrient supplement [J]. Biotechnol Lett, 1996, 6:683～688.
100. Zhang L, Song GY. Child blood lead level if influence factor relevance. Medical Journal of Chinese People’s Health, 2006, 18 (10):832～834.
101. Zhang SM, Dai YH, Xie X H, et al. Study on blood lead level and related risk factors among children aged 0-26 years in 15 cities in China. Chin J Ep idemiol, 2005, 26 (9):2651～2654.
102. Zhou S, Davis FC, Ingram LO Gene integration and expression and extracellular secretion of Erwinia chrysanthemi endoglucanase CelY (celY) and CelZ (celZ) in ethanologenicKlebsiella oxytoca P2. Appl Environ Microbiol, 2001,67:6–14
103. 7Zhou S, Ingram LO, Synergistic hydrolysis of carboxymethyl cellulose and acid swollen cellulose by two endoglucanases (CelZ and CelY) from Erwinia chrysanthemi. J Bacteriol, 2000, 182:5676–5682
104. Zhou S, Yomano L P, Shanmugam K T, et al., Fermentation of 10% (w/v) sugar to D(-)-lactate by engineered Escherichia coli B [J]. Biotechnol. Lett., 2005, 1891～1896.
105. Zhu J, and Shimizu K. The effect of pfl gene knockout on the metabolism for optically pure D-lactate production by Escherichia coli. Applied microbiology and biote chnology, 2004, 64:367～375.