1，3-丙二醇产生菌肺炎克氏杆菌的分子育种
|
摘要
1，3-丙二醇(1，3-PD)是一种重要的化工原料，具有许多优良特性和很好的应用前景。微生物发酵法生产1，3-丙二醇与化学合成法相比具有显著的优点，因而越来越受到重视，成为当前的研究热点。底物和多产物抑制是造成微生物发酵法1，3-丙二醇产量和甘油转化率低，后期提取困难的主要原因，切断无益副产物的产生途径将提高1，3-丙二醇产量和甘油转化率，并简化后期提取工艺。
运用双底物两步法和微氧法对Klebsiella pneumoniae M5al菌株进行补料批式培养，发酵液中1，3-丙二醇浓度分别可达36.09gl~(-1)和45.22gl~(-1)，较国内所能达到的水平有很大提高。
以Klebsiella pneumoniae M5al为出发菌株，用mini-Tn5随机转座诱变结合自杀性底物筛选的方法得到了两株产乙酸途径pta基因缺失突变株XL-6和XL-11，应用到微氧法发酵中，突变株的乙酸产量为亲株的50％以下，甘油转化率有所提高，但1，3-丙二醇浓度和生产强度有所下降。
PCR扩增出产乙醇途径adhE基因的部分片段和产乳酸途径ldhA基因的部分片段，构建了分别带有部分adhE基因片段和部分ldhA基因片段的自杀质粒pWW和pVV，通过同源重组的手段分别得到了6个adhE~-突变株和4个ldlM~-突变株。6个adhE~-突变株的乙醛脱氢酶活性均降低为亲株的10％以下，乙醇脱氢酶活性降低为亲株的10％左右；4个ldhA~-突变株的乳酸脱氢酶活性均降低为亲株的7％以下。突变株将应用到发酵中以研究其能否提高1，3-丙二醇产量和甘油转化率。
As an important chemical material, 1,3-propanediol (1,3-PD) has numerous promising properties and applications. Compared with chemical synthesis, microbial fermentation processes to 1,3-propanedio! have many obvious advantages in that they typically use renewable feedstocks and reduce pollution to a great extent. And they have attracted particular attention and become the focuses of research. The inhibition potentials of substrate and products on the growth of microorganism cell will limit the product titer, the yield of 1,3-propanediol on glycerol and the productivity of 1,3-propanediol, and then make the product recovery and purification a troublesome task. This can be effectively solved by blocking the pathways leading to the byproducts that are of no benefit to 1,3-propanedil production.
The 1,3-propanediol titers of 36.09 g 1-1 and 45.22 g 1-1 have been obtained by using two-stage fermentation with two substrates and microaerobic fermentation in the fed-batch culture of Klebsiella pneumoniae M5al, respectively, which greatly exceeded the level that can be achieved in China.
Two pta- mutants have been selected by using suicide substrate after the mini-Tn5 transposon insertion mutagenesis of Klebsiella pneumoniae M5al. When used in microaerobic fermentation, the amount of acetate produced by the mutants reduced to less than 50% of the parent strain, and the yields improved whereas the 1,3-propanediol titers and productivities decreased.
Two suicide vectors of pWW and pVV carrying the 871bp segment ofadhE and 805bp segment of IdhA amplified by PCR respectively were constructed and six mutants ofadhE and four mutants of ldhA- were obtained by homogenous recombination between the partial adhE or IdhA in the vectors and the Klebsiella pneumoniae M5al chromosome. The acetaldehyde dehydrogenase (ACDH) and alcohol dehydrogenase (ADH) of the adhE mutants and parent strain were assayed with a result that the ACDH activities of mutants were less than 10% of the parent strain and the ADH activities of mutants are about 10% of the parent strain. The lactate dehydrogenase (LDH) were also assayed and the LDH activities of IdhA were less than 7% of the parent strain. Whether the mutants are able to improve the 1,3-propanediol titers, yields and productivities will be investigated in the fermentation later.
运用双底物两步法和微氧法对Klebsiella pneumoniae M5al菌株进行补料批式培养，发酵液中1，3-丙二醇浓度分别可达36.09gl~(-1)和45.22gl~(-1)，较国内所能达到的水平有很大提高。
以Klebsiella pneumoniae M5al为出发菌株，用mini-Tn5随机转座诱变结合自杀性底物筛选的方法得到了两株产乙酸途径pta基因缺失突变株XL-6和XL-11，应用到微氧法发酵中，突变株的乙酸产量为亲株的50％以下，甘油转化率有所提高，但1，3-丙二醇浓度和生产强度有所下降。
PCR扩增出产乙醇途径adhE基因的部分片段和产乳酸途径ldhA基因的部分片段，构建了分别带有部分adhE基因片段和部分ldhA基因片段的自杀质粒pWW和pVV，通过同源重组的手段分别得到了6个adhE~-突变株和4个ldlM~-突变株。6个adhE~-突变株的乙醛脱氢酶活性均降低为亲株的10％以下，乙醇脱氢酶活性降低为亲株的10％左右；4个ldhA~-突变株的乳酸脱氢酶活性均降低为亲株的7％以下。突变株将应用到发酵中以研究其能否提高1，3-丙二醇产量和甘油转化率。
As an important chemical material, 1,3-propanediol (1,3-PD) has numerous promising properties and applications. Compared with chemical synthesis, microbial fermentation processes to 1,3-propanedio! have many obvious advantages in that they typically use renewable feedstocks and reduce pollution to a great extent. And they have attracted particular attention and become the focuses of research. The inhibition potentials of substrate and products on the growth of microorganism cell will limit the product titer, the yield of 1,3-propanediol on glycerol and the productivity of 1,3-propanediol, and then make the product recovery and purification a troublesome task. This can be effectively solved by blocking the pathways leading to the byproducts that are of no benefit to 1,3-propanedil production.
The 1,3-propanediol titers of 36.09 g 1-1 and 45.22 g 1-1 have been obtained by using two-stage fermentation with two substrates and microaerobic fermentation in the fed-batch culture of Klebsiella pneumoniae M5al, respectively, which greatly exceeded the level that can be achieved in China.
Two pta- mutants have been selected by using suicide substrate after the mini-Tn5 transposon insertion mutagenesis of Klebsiella pneumoniae M5al. When used in microaerobic fermentation, the amount of acetate produced by the mutants reduced to less than 50% of the parent strain, and the yields improved whereas the 1,3-propanediol titers and productivities decreased.
Two suicide vectors of pWW and pVV carrying the 871bp segment ofadhE and 805bp segment of IdhA amplified by PCR respectively were constructed and six mutants ofadhE and four mutants of ldhA- were obtained by homogenous recombination between the partial adhE or IdhA in the vectors and the Klebsiella pneumoniae M5al chromosome. The acetaldehyde dehydrogenase (ACDH) and alcohol dehydrogenase (ADH) of the adhE mutants and parent strain were assayed with a result that the ACDH activities of mutants were less than 10% of the parent strain and the ADH activities of mutants are about 10% of the parent strain. The lactate dehydrogenase (LDH) were also assayed and the LDH activities of IdhA were less than 7% of the parent strain. Whether the mutants are able to improve the 1,3-propanediol titers, yields and productivities will be investigated in the fermentation later.
引文
1. Abbad-Andaloussi S, Manginot-Durr C, Amine J, et al. Isolation and characterization of Clostridium butyricum DSM 5431 mutants with increased resitance to 1,3-propanediol and altered production of acids. Appl Environ Microbiol 1995, 61:4413～4417.
2. Abbad-Andaloussi S, Guedon E, Spiesser E, et al. Glycerol dehydratase activity: the limiting step for 1,3-propanediol production by Clostridium butyricum. Lett Appl Microbiol 1996a, 22:311～314.
3. Abbad-Andaioussi S, Manginot-Durr C, Raval G, et al. Carbon and electron flow in Clostridium butyricum grown in chemostat culture on glycerol and on glucose. Microbiology 1996b, 142:1149～1158.
4. Abbad-Andaloussi S, Amine J, Gerard P, et al. Properties of allyl alcohol mutants of Clostridium butyricum grown on glycerol. Appl Environ Microbiol 1996c, 62:3499～3501.
5. Ahrens K, Menzel K, Zeng AP, et al. Kinetic, Dynamic, and Pathway Studies of Glycerol Metabolism by Klebsiella Pneumoniae in Anaerobic Continuous Culture:Ⅲ Enzymes and Fluxes of Glycerol Dissimilation and 1,3-Propanediol Formation. Biotechnol Bioeng 1998, 59:544～552.
6. Barbirato F, Camarasca-Claret C, Bories A, et al. Description of the glycerol fermentation by a new 1,3-propanediol producing microorganism: Enterobacter agglomerans. Appl Microbiol Biotechnol 1995, 43:786～793.
7. Barbirato F, Grivel JP, Soucaille P, et al. 3-Hydroxypropionaldehyde, an inhibitory metabolite of glycerol fermentation to 1,3-propanediol by enterobacterial species. Appl Environ Microbiol 1996, 62:1448～1451.
8. Barbirato F, Astruc S, Soucaille P, et al. Anaerobic pathways of glycerol dissimilation by Enterobacter agglomerans CNCM 1210: limitation an regulations. Microbiology 1997, 143: 2423～2432.
9. Biebl H. Glycerol fermentation to 1,3-propanediol by Clostridium butyricum. Measurement of product inhibition by use of a pH-auxostat. Appl Microbiol Biotechnol 1991, 35:701～705.
10. Biebl H, Marten S, Hippe H, et al. Glycerol conversion to 1,3-propanediol by newly isolated Clostridia. Appl Microbiol Biotechnol 1992, 36:592～597.
11. Biebl H, Marten S. Fermentation of glycerol to 1,3-propanediol: use of cosubstrates. Appl Microbiol Biotechnol 1995, 44:15～19.
12. Biebl H, Zeng AP, Menzel K, et al. Fermentation of glycerol to 1,3-propanediol and 2,3-butanediol by Klebsiella pneumoniae. Appl Microbiol Biotechnol 1998, 50:24～29.
13. Biebl H, Menzel K, Zeng AP, et al. Microbial production of 1,3-propanediol. Appl Microbiol Biotechnol 1999, 52:289～297.
14. Biebl H. Fermentation of glycerol by Clostridium pasteurianum-batch and continuous culture studies, J Indu Microbiol Biotechnol 2001,27:18～26.
15. 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.
16. Bulthuis, Ben A., Whited et al. Method for the production of 1,3-propanediol by recombinant organisms comprising genes for vitamin B_(12) transport. US 6,432,686, August 13, 2002.
17. Cameron DC, Altaras NE, Hoffman ML, et al. Metabolic engineering of propanediol pathways. Biotechnol Prog 1998, 14:116～125.
18. Chotani G, Dodge T, Hsu A, et al. The commercial production of chemicals using pathway engineering. Biochimica Biophysica Acta 2000, 1531:434～455.
19. Colin T, Bories A, Moulin G. Inhibition of Clostridium butyricum by 1,3-propanediol and diols during glycerol fermentation. Appl Microbiol Biotechno1 2000, 54:201～205.
20. Colin T, Bories A, Lavigne C, et al. Effects of acetate and butyrate during glycerol fermentation by Clostridium butyricum. Current Microbiology 2001, 43:238～243.
21. Cunningham PR and Clark PD. The use of suicide substrates to select mutants of Escherichia coli lacking enzymes of alcohol fermentation. Mol Gen Genet 1986, 205:487～493.
22. Dabrock B, Bahl H, Gottschalk G. Parameters affecting solvent production by Clostridium pasteurianum. Appl Environ Microbiol 1992, 58:1233～1239.
23. Daniel R and Gottschalk G. Growth temperature dependent activity of glycerol dehydratase in E. coli expressing the C. freundii dha regulon. FEMS Microbiology Letters 1992, 100:281～286.
24. Daniel R, Boenigk R and Gottschalk G. Purification of 1,3-propanediol dehydratase from Citrobacter freundii and cloning, sequencing and overexpression of the corresponding gene in E. coli. J Bacteriol 1995a, 177: 2151～2156.
25. Daniel R, Stuertz K and Gottschalk G. Biochemical and molecular characterization of the oxidative branch of glycerol utilization by Citrobacter freundii. d Bacteriol 1995b, 177:4392～4401.
26. Daniel R, Thomas A and Gottschalk G. Biochemistry of coenzyme B_(12)-denpendent glycerol and diol dehydratases and organization of the encoding genes. FEMS Microbiol Rev 1998, 22:553～566.
27. Deckwer WD. Microbial conversion of glycerol to 1,3-propanediol. FEMS Microbiol Rev 1995, 16:143～149.
28. Defretin, Sophie, Delelis et al. Process for the production of 1,3-propanediol by fermentation. US 6,406,895, June 18, 2002.
29. Diaz-Torres, Maria, Dunn-Coleman et al. Method for the recombinant production of 1,3-propanediol. US 6,136,576, October 24, 2000.
30. Emptage M., Haynie S. Laffend L. et al. Process for the biological production of 1,3-propanediol with high titer. WO 01/12833, February 22, 2001.
31. Fond O, Jansen NB, Tsao GT. A model of acetic acid and 2,3-butanediol inhibition of the growth and metabolism of Klebsiella oxytoca. Biotechnol Lett 1985, 7:727～732.
32. Forage RG, Foster MA. Glycerol fermentation in Klebsiella pneumoniae: functions of the coenzyme B_(12)-denpendent glycerol and diol dehydratases. J Bacteriol 1982, 149(2): 413～419.
33. Forage RG, Lin ECC. dha systems mediating aerobic and anaerobic dissimilation of glycerol in Klebsiella pneumoniae NCIB 418. J Bacteriol 1982, 151:591～599.
34. Forsberg C. Production of 1,3-propanediol from glycerol by Clostridium acetobutylicum and other Clotridium species. Appl Environ Microbiol 1987, 53:639～643
35. Gnzel B, Yonsel S, and Deckwer WD. Fermentative production of 1,3-propanediol from glycerol by Clostridiurn butyricum up to a scale of 2m~3. Appl Microbioi Biotechnol 1991, 36:289～295.
36. Hartlep M, Hussmann W, Prayitno N, et al. Study of two-stage processes for the microbial production of 1,3-propanediol from glucose. Appl Microbiol Biotechnol 2002, 60:60～66.
37. Haynie, Sharon L., Wagner et al. Process for making 1,3-propanediol from carbohydrates using mixed microbial cultures. US 5,599,689, February 4, 1997.
38. Herrero M, Lorenzo VD, Timmis KN. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol 1990,172(11): 6557～6567.
39. Homann T, Tag C, Biebl H, et al. Fermentation of glycerol to 1,3-propanediol by Klebsiella and Citrobacter strains. Appl Microbiol Biotechnol 1990, 33:121～126.
40. Huang H, Gong CS, Tsao GT. Production of 1,3-propanediol by Klebsiella pneumoniae. Appl Biochem Biotechnol 2002, 98-100:687～698.
41. Jin RZ, Forage RG, Lin ECC. Glycerol kinase as a substitute for dihydruxyacetone kinase in a mutant of Klebsiella pneumoniae. J Bacteriol 1982, 152(3): 1301～1307.
42. Kajiura H, Mori K, Tobimatsu T, et al. Characterization and mechanism of action of a reactivating factor for adenosylcobalamin-denpendent glycerol dehydratases. J Biol Chem 2001, 276(39): 36514～36519.
43. Laffend LA, Nagarajan, Vasantha et al. Bioconversion of a fermentable carbon source to 1,3-propanediol by a single microorganism. US 5,686,276, November 11, 1997.
44. 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.
45. Luers F, Laffend LA, Nagarajan, Vasantha. Bioconversion of a fermentable carbon source to 1,3-propanediol by a single microorganism. US 6,025,184, February 15, 2000.
46. Seyfried M, Daniel R and Gottschalk G. Glycerol conversion to 1,3-propanediol by Clostridium pasteurianum: cloning and expression of the gene encoding 1,3-propanediol dehydgenase. FEMS Microbiology Letters 1997, 154:337～345.
47. Macis L, Daniel R and Gottschaik G. Properties and sequence of the coenzyme B_(12)-denpendent glycerol dehydratases of Clostridium pasteurianum. FEMS Microbiol Lett 1998, 164:21～28.
48. Malaoui H and Marczak R. Purification and properties of the 1,3-propanediol dehydgenase of Clostridium butyricum E5. Enzyme and Microbial Technology 2000, 27:399～405.
49. Malaoui H and Marczak R. Separation and characterization of the 1,3-propanediol and glycerol dehydgenase activities from Clostridium butyricum E5 wide-type and mutant D. J Appl Microbiol 2001, 90:1006～1014.
50. Malaoui H and Marczak R. Influence of glycerol metabolism by wide-type and mutant strains of Clostridium butyricum E5 grown in chemostat culture. Appl Microbiol Biotechnol 2001, 55: 226～233.
51. Menzel K, Zeng AP, Biebl H, et al. Kinetic, Dynamic, and Pathway Studies of Glycerol Metabolism by Klebsiella Pneumoniae in Anaerobic Continuous Culture: ⅠThe phenomena and characterization of oscillation and hysteresis. Biotechnol Bioeng 1996, 52:549～560.
52. Menzel K, Ahrens K, Zeng AP, et al. Kinetic, Dynamic, and Pathway Studies of Glycerol Metabolism by Klebsiella Pneumoniae in Anaerobic Continuous Culture:Ⅳ Enzymes and fluxes of pyruvate metabolism. Biotechnol Bioeng 1996, 60(5):617～626.
53. Menzel K, Zeng AP, Deckwer WD. Enzymatic evidence for an involvement of pyruvate dehydrogenase in the anaerobic glycerol metabolism of Klebsiella pneumoniae. J Biotechnol 1997a, 56:135～142.
54. Menzel K, Zeng AP, Deckwer WD. High concentration and productivity of 1,3-propanediol from continuous fermentation of glycerol by Klebsiella pneumoniae. Enzyme Microbiol Technol 1997b, 20:82～86.
55. 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.
56. Mori K, Tobimatsu T, Hera T, et al. Characterization, sequencing, and expressing of the genes encoding a reactivating factor for glycerol-inactivated adenosylcobalamin-denpendent glycerol dehydratases. J Biol Chem 1997, 272:32034～32041.
57. Nagarajan, Vasantha, Nakamura et al. Production of 1,3-propanediol from glycerol by recombinant
bacteria expressing recombinant diol dehydratase. US 5,633,362, May 27, 1997.
58. Nagarajan, Vasantha, Nakamura et al. Production of 1,3-propanediol from glycerol by recombinant bacteria expressing recombinant diol dehydratase. US 5,821,092, October 13, 1998.
59. Nakamura, Charles E., Gatenby. Method for the production of 1,3-propanediol by recombinant microorganisms. US 6,103,494, January 11, 2000.
60. Nakas JP, Sehaedle M, Parkinson CM, et al. System development for linked-fermentation products of solvents from algal biomass. Appl Environ Microbiol 1983, 46:1017～1023
61. 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.
62. Papanikolaou S, Ruiz-Sanchez P, Pariset B, et al. High production of 1,3-propanediol from industrial glycerol by a newly isolated Clostridium butyricum strain. J Biotechnol 2000, 77:191～208.
63. Petitdemange E, Cherrier C, Raval G, et al. Regulation of the NADH-ferredoxin oxidoreductase in Clostridia of the butyric group. Biochim Biophys Acta 1976, 421:334～347.
64. Petitdemange E, Manginot-Durr C, Abbad-Andaloussi S, et al. Fermentation of raw glycerol to 1,3-propanediol by new strains of Clostridium butyricum. J Ind Microbiol 1995, 15:498～501.
65. Pflugmacher U, Gottschalk G. Development of an immobilized cell reactor for the production of 1,3-propanediol by Citrobacter freundii. Appl Microbiol Biotechnol 1994, 41:313～316.
66. Raynaud C, Sarcabal P, Meynial-Salles, et al. Molecular characterization of 1,3-propanediol (1,3-PD) operon of Clostridium butyricum. Proc Natl Acad Sci USA 2003, 100(9): 5010～5015.
67. Reimann A, Biebl H. Production of 1,3-propanediol by Clostridium butyricum DSM 5431 and product tolerant mutants in fedbatch culture. Feeding strategy for glycerol and ammonium. Biotechnol Lett 1996a, 18:827～832.
68. Reimann A, Biebl H, Decker WD. Influence of iron, phosphate and methyl vioiogen on glycerol fermentation of Clostridium butyricum. Appl Microbiol Biotechnol 1996b, 45:47～50.
69. Reimann A, Biebl H, Decker WD. Production of 1,3-propanediol by Clostridium butyricum in continuous culture with cell recycling. Appl Microbiol Biotechnol 1998a, 49:359～363.
70. Reimann A, Abbad-Andaloussi S, Biebl H. et al. 1,3-propanediol formation with product-tolerant mutants of Clostridium butyricum DSM 5431 in continuous culture: productivity, carbon and electron flow. J Appl Microbiol 1998b, 84:1125～1130.
71. Saint-Amans S, Perlot P, Goma G, et al. High production of 1,3-propanediol by Clostridium butyricum VPI 3266 in a simply controlled fed～batch system. Biotechnol Lett 1994, 16:832～836.
72. Saint-Amans S, Soucaille P. Carbon and electron flow in Clostridium butyricum grown in
chemostat culture on glucose～glycerol mixtures. Biotechnol Lett 1995, 17:211～216.
73. Saint-Amans S, Girbal L, Andrade J, et al. Regulation of carbon and flow in Clostridium butyricum VPI 3266 grown on glucose-glycerol mixtures. J Bacteriol 2001, 183(5): 1748～1754.
74. Sch#5utz H, Dadler F. Anaerobic reduction of glycerol to propanediol-1,3 by Lactobacillus brevis and Lactobacillus buchneri. Syst Appl Microbial 1984, 5:169～178.
75. Seifert C, Bowien S, Gottschalk G, et al. Identification and expression of the genes and purification of the gene products involved in reactivation of coenzyme B_(12)-denpendent glycerol dehydratases of Citrobacter freundii. Eur J Biochem 2001, 268:2369～2378.
76. Seyfried M, Daniel R and Gottschalk G. Cloning, sequencing and overexpression of the genes encoding coenzyme B_(12)-denpendent glycerol dehydratases of Citrobacter freundii. J Bacteriol 1996, 178:5793～5796.
77. Skraly FA, Lytle BL, Cameron DC. Construction and characterization of a 1,3-propanediol operon. Appl Environ Microbiol 1998, 64:98～105.
78. Skraly FA, Cameron DC. Purification and Characterization of a Bacillus licheniformis Phosphatase Specific for D-a-Glycerophosphate. Arch Biochem. Biophys 1998, 349(1): 27～35.
79. Solomon BO, Zeng A-P, Hibel H, et al. Comparison of the energetic efficiencies of hydrogen and oxychemicals formation in Klebsiella pneumoniae and Clostridium butyricum during anaerobic growth on glycerol. J Biotechnology 1995, 39(2): 107～117.
80. Sprenger GA, Hammer GA, Johnson EA, et al. Anaerobic growth of Escherichia coli on glycerol by importing genes of the dba regulon from Klebsiella pneumoniae. J Gen Microbiol 1989, 135:1255～1262.
81. Streekstra H, Teixeira de Mattos MJ, Neijssel OM, et al. Overflow metabolism during anaerobic growth of Klebsiella aerogenes NCTC 418 on glycerol and dihydroxyacetone in chemostst culture. Arch Microbiol 1987, 147:268～275.
82. Sun J, Van Den Heuvel J, Soucaille P, et al. Comparative Genomic Analysis of dha Regulon and Related Genes for Anaerobic Metabolism in Bacteria. Biotechnol prog 2003,19(2):263～272.
83. Tarmy EM and N.O. Kaplan. Chemical characterization of D-lactate dehydrogenase from Escherichia coil B. J Biol Chem. 1968, 271:2579～2586.
84. Tobimatsu T, Azuma M, Matsubara H, et al. Cloning, sequencing and high level expression of the genes encoding adenosylcobalamin-denpendent glycerol dehydratases of Klebsiella pneumoniae. J Biol Chem 1996, 271:22352～22357.
85. Tobimatsu T, Kajiura H, Yunoki M, et al. Identification and expression of the genes encoding a reactivating factor for adenosylcobalamin-denpendent glycerol dehydratases. J Bacteriol 1999, 181:4110～4113.
86. Tobimatsu T, Kajiura H, Toraya T. Specificities of reactivating factors for adenosylcobalamin-denpendent diol dehydratase and glycerol dehydratase. Arch Microbiol 2000, 174:81～88.
87. Tong IT, Liao HH, Cameron DC. 1,3-propanediol production by Escherichia coli expressinggenes from the Klebsiella pneumoniae dha regulon. Appl Environ Microbiol 1991, 57:3541～3546.
88. Tong IT, Cameron DC 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.
89. Toraya T and Mori K. A reactivating factor for coenzyme B_(12)-denpendent diol dehydratases. J Biol Chem 1999, 274:3372～3377.
90. Toraya T. Radical catalysis of B_(12) enzymes: structure, mechanism, inactivation, and reactivation of diol and glycerol dehydratases. Cell Mole Life Sci 2000, 57(1):106～127.
91. Xiu ZL, Zeng AP and Deckwer WD. Multiplicity and stability analysis of microorganisms in continuous culture: effects of metabolic overflow and growth inhibition. Biotechnol Bioeng 1998, 57:251～261.
92. Yanisch-Perron C, Vieera J and Messing J. Improved M13 phage cloning vecters and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 1985 33(1): 103～119.
93. Zeng AP. Pathway and kinetic analysis of 1,3-propanediol production from glycerol fermentation by Clostridium butyricum. Bioproc Eng 1996, 14:169～175.
94. Zeng AP, 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.
95. Zeng AP, Biebl H and Deckwer WD. Microbial conversion of glycerol to 1,3-propanediol [A]. In Saha BC and Woodward J, eds. Recent progress in Fuels and Chemicals from Biomass[C], American Chemical Society, 1997, 265～279.
96. Zeng AP, Biebl H, Schlieker H, et al. Pathway analysis of glycerol fermentation by Klebsiella pneumoniae: regulation of reducing equivalent balance and product formation. Enzyme and Microbial Technology 1993, 15:770～779.
97. Zeng AP, Menzel K, and Deckwer WD, et al. Kinetic, Dynamic, and Pathway Studies of Glycerol Metabolism by Klebsiella Pneumoniae in Anaerobic Continuous Culture: Ⅱ Analysis of metabolic rates and pathways under oscillation and steady-state conditions. Biotechnol Bioeng 1996, 52:561～571.
98. Zeng AP, Ross A, Hibel H, et al. Multiple product inhibition and growth modeling of Clostridium bulyricum and Klebsiella pneumoniae in glycerol fermentation. Biotechnol Bioeng 1994, 44:902～911.
99. 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.
100. Zhu MM, Lawman PD, Cameron DC. Improving 1,3-propanediol productions from glycerol in a metabolically engineered Escherichia coli by reducing accumulation of sn～glucerol～3～phosphate. Biotechnol Prog 2002, 18(4): 694～699.
101．北京化学试剂公司编．化学试剂目录手册．北京工业大学出版社，1993，586．
102．冯婧微，张洪林，蒋林时等．1，3-丙二醇合成方法研究．化工科技，2002，10(6)：43～47．
103．刘海军，王剑锋，张代佳等．用克雷伯氏菌批式流加发酵法生产 1，3-丙二醇．食品与发酵工业， 2001，27(7)4～7．
104．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)：45～52．
105．刘银乾，段启伟．1，3-丙二醇的制备方法．合成纤维工业，2002，25(2)：42～45．
106．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．
107．瞿国华．PTT的工业开发及1，3-丙二醇合成．合成纤维工业，2000，23(4)：31～34．
108．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．(第二版) 北京：科学出版社，1993．
109．邵敬伟，刘长江．1，3-丙二醇发酵生产的研究进展．沈阳农业大学学报，2001～2002，32(1)：57～60．
110．邵敬伟，刘长江，迟乃玉等．1，3-丙二醇发酵条件的正交设计实验．襄樊学院学报，2002，23(5)：45～49．
111．王剑锋，刘海军，修志龙等．生物转化生产 1，3-丙二醇的研究进展．化学通报，2001a，10：621～625。
112．王剑锋，刘海军，修志龙等．1，3-丙二醇间歇发酵培养基的优化．食品与发酵工业，2001b，27(8)：5～8。
113．王剑锋，修志龙，刘海军等．克雷伯氏菌微氧发酵生产 1，3-丙二醇的研究．现代化工，2001，21(5)：28～31．
114．王剑锋，修志龙，范圣第．甘油转化生产 1，3-丙二醇发酵液中甘油含量的测定．工业微生物，2001，31(2)：33～35．
115．王熙庭，陈曼华．1，3-丙二醇生产方法及用途．煤化工，2000，4：38～40．
116．修志龙．1，3-丙二醇的微生物法生产和市场分析．现代化工，1999，19(3)：33～35．
117．修志龙．甘油连续生物歧化过程培养基和pH调控策略研究．高校化学工程学报，2001，15(4)：397～402。
118．修志龙，曾安平．微生物细胞连续培养过程中振荡和混沌行为的研究进展．生物工程进展，1999，19(6)：58～63．
119．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，2000，27(4)：300～302．
120．修志龙，曾安平，安利佳．甘油生物歧化过程动力学数学模拟和多稳态研究．大连理工大学学报，2000，40(4)：428～433．
121．薛丽梅，王琲，韩大伟．1，3-丙二醇的化学合成法．化学与粘合，2001，1：38～40．
122．杨菊群，王幸宜，戚蕴石．1，3-丙二醇的合成新进展．石油化工，2002，31(11)：943～947．
123．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(2)：11～15．
124．张健，赵红英，刘宏娟等．以葡萄糖为辅助底物发酵生产 1，3-丙二醇的研究．现代化工，2002，22(6)：32～35．
125．赵红英，张健，刘宏娟等．1，3-丙二醇发酵过程中底物抑制及其对策的研究．现代化工，2002，22(7)：34～38．
126．赵育．新型热塑性聚酯—聚对苯二甲酸丙二醇酯．塑料科技，2000，136：33～36．
127．中华人民共和国国家技术监督局．GB7479—87．中华人民共和国国家标准—水质 铵的测定．北京：中国标准出版社，1987～08～01．
128．朱丙田，刘德华，任海玉等．1，3-丙二醇发酵条件的探索．化工冶金，2000，21(4)：420～422．
2. Abbad-Andaloussi S, Guedon E, Spiesser E, et al. Glycerol dehydratase activity: the limiting step for 1,3-propanediol production by Clostridium butyricum. Lett Appl Microbiol 1996a, 22:311～314.
3. Abbad-Andaioussi S, Manginot-Durr C, Raval G, et al. Carbon and electron flow in Clostridium butyricum grown in chemostat culture on glycerol and on glucose. Microbiology 1996b, 142:1149～1158.
4. Abbad-Andaloussi S, Amine J, Gerard P, et al. Properties of allyl alcohol mutants of Clostridium butyricum grown on glycerol. Appl Environ Microbiol 1996c, 62:3499～3501.
5. Ahrens K, Menzel K, Zeng AP, et al. Kinetic, Dynamic, and Pathway Studies of Glycerol Metabolism by Klebsiella Pneumoniae in Anaerobic Continuous Culture:Ⅲ Enzymes and Fluxes of Glycerol Dissimilation and 1,3-Propanediol Formation. Biotechnol Bioeng 1998, 59:544～552.
6. Barbirato F, Camarasca-Claret C, Bories A, et al. Description of the glycerol fermentation by a new 1,3-propanediol producing microorganism: Enterobacter agglomerans. Appl Microbiol Biotechnol 1995, 43:786～793.
7. Barbirato F, Grivel JP, Soucaille P, et al. 3-Hydroxypropionaldehyde, an inhibitory metabolite of glycerol fermentation to 1,3-propanediol by enterobacterial species. Appl Environ Microbiol 1996, 62:1448～1451.
8. Barbirato F, Astruc S, Soucaille P, et al. Anaerobic pathways of glycerol dissimilation by Enterobacter agglomerans CNCM 1210: limitation an regulations. Microbiology 1997, 143: 2423～2432.
9. Biebl H. Glycerol fermentation to 1,3-propanediol by Clostridium butyricum. Measurement of product inhibition by use of a pH-auxostat. Appl Microbiol Biotechnol 1991, 35:701～705.
10. Biebl H, Marten S, Hippe H, et al. Glycerol conversion to 1,3-propanediol by newly isolated Clostridia. Appl Microbiol Biotechnol 1992, 36:592～597.
11. Biebl H, Marten S. Fermentation of glycerol to 1,3-propanediol: use of cosubstrates. Appl Microbiol Biotechnol 1995, 44:15～19.
12. Biebl H, Zeng AP, Menzel K, et al. Fermentation of glycerol to 1,3-propanediol and 2,3-butanediol by Klebsiella pneumoniae. Appl Microbiol Biotechnol 1998, 50:24～29.
13. Biebl H, Menzel K, Zeng AP, et al. Microbial production of 1,3-propanediol. Appl Microbiol Biotechnol 1999, 52:289～297.
14. Biebl H. Fermentation of glycerol by Clostridium pasteurianum-batch and continuous culture studies, J Indu Microbiol Biotechnol 2001,27:18～26.
15. 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.
16. Bulthuis, Ben A., Whited et al. Method for the production of 1,3-propanediol by recombinant organisms comprising genes for vitamin B_(12) transport. US 6,432,686, August 13, 2002.
17. Cameron DC, Altaras NE, Hoffman ML, et al. Metabolic engineering of propanediol pathways. Biotechnol Prog 1998, 14:116～125.
18. Chotani G, Dodge T, Hsu A, et al. The commercial production of chemicals using pathway engineering. Biochimica Biophysica Acta 2000, 1531:434～455.
19. Colin T, Bories A, Moulin G. Inhibition of Clostridium butyricum by 1,3-propanediol and diols during glycerol fermentation. Appl Microbiol Biotechno1 2000, 54:201～205.
20. Colin T, Bories A, Lavigne C, et al. Effects of acetate and butyrate during glycerol fermentation by Clostridium butyricum. Current Microbiology 2001, 43:238～243.
21. Cunningham PR and Clark PD. The use of suicide substrates to select mutants of Escherichia coli lacking enzymes of alcohol fermentation. Mol Gen Genet 1986, 205:487～493.
22. Dabrock B, Bahl H, Gottschalk G. Parameters affecting solvent production by Clostridium pasteurianum. Appl Environ Microbiol 1992, 58:1233～1239.
23. Daniel R and Gottschalk G. Growth temperature dependent activity of glycerol dehydratase in E. coli expressing the C. freundii dha regulon. FEMS Microbiology Letters 1992, 100:281～286.
24. Daniel R, Boenigk R and Gottschalk G. Purification of 1,3-propanediol dehydratase from Citrobacter freundii and cloning, sequencing and overexpression of the corresponding gene in E. coli. J Bacteriol 1995a, 177: 2151～2156.
25. Daniel R, Stuertz K and Gottschalk G. Biochemical and molecular characterization of the oxidative branch of glycerol utilization by Citrobacter freundii. d Bacteriol 1995b, 177:4392～4401.
26. Daniel R, Thomas A and Gottschalk G. Biochemistry of coenzyme B_(12)-denpendent glycerol and diol dehydratases and organization of the encoding genes. FEMS Microbiol Rev 1998, 22:553～566.
27. Deckwer WD. Microbial conversion of glycerol to 1,3-propanediol. FEMS Microbiol Rev 1995, 16:143～149.
28. Defretin, Sophie, Delelis et al. Process for the production of 1,3-propanediol by fermentation. US 6,406,895, June 18, 2002.
29. Diaz-Torres, Maria, Dunn-Coleman et al. Method for the recombinant production of 1,3-propanediol. US 6,136,576, October 24, 2000.
30. Emptage M., Haynie S. Laffend L. et al. Process for the biological production of 1,3-propanediol with high titer. WO 01/12833, February 22, 2001.
31. Fond O, Jansen NB, Tsao GT. A model of acetic acid and 2,3-butanediol inhibition of the growth and metabolism of Klebsiella oxytoca. Biotechnol Lett 1985, 7:727～732.
32. Forage RG, Foster MA. Glycerol fermentation in Klebsiella pneumoniae: functions of the coenzyme B_(12)-denpendent glycerol and diol dehydratases. J Bacteriol 1982, 149(2): 413～419.
33. Forage RG, Lin ECC. dha systems mediating aerobic and anaerobic dissimilation of glycerol in Klebsiella pneumoniae NCIB 418. J Bacteriol 1982, 151:591～599.
34. Forsberg C. Production of 1,3-propanediol from glycerol by Clostridium acetobutylicum and other Clotridium species. Appl Environ Microbiol 1987, 53:639～643
35. Gnzel B, Yonsel S, and Deckwer WD. Fermentative production of 1,3-propanediol from glycerol by Clostridiurn butyricum up to a scale of 2m~3. Appl Microbioi Biotechnol 1991, 36:289～295.
36. Hartlep M, Hussmann W, Prayitno N, et al. Study of two-stage processes for the microbial production of 1,3-propanediol from glucose. Appl Microbiol Biotechnol 2002, 60:60～66.
37. Haynie, Sharon L., Wagner et al. Process for making 1,3-propanediol from carbohydrates using mixed microbial cultures. US 5,599,689, February 4, 1997.
38. Herrero M, Lorenzo VD, Timmis KN. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol 1990,172(11): 6557～6567.
39. Homann T, Tag C, Biebl H, et al. Fermentation of glycerol to 1,3-propanediol by Klebsiella and Citrobacter strains. Appl Microbiol Biotechnol 1990, 33:121～126.
40. Huang H, Gong CS, Tsao GT. Production of 1,3-propanediol by Klebsiella pneumoniae. Appl Biochem Biotechnol 2002, 98-100:687～698.
41. Jin RZ, Forage RG, Lin ECC. Glycerol kinase as a substitute for dihydruxyacetone kinase in a mutant of Klebsiella pneumoniae. J Bacteriol 1982, 152(3): 1301～1307.
42. Kajiura H, Mori K, Tobimatsu T, et al. Characterization and mechanism of action of a reactivating factor for adenosylcobalamin-denpendent glycerol dehydratases. J Biol Chem 2001, 276(39): 36514～36519.
43. Laffend LA, Nagarajan, Vasantha et al. Bioconversion of a fermentable carbon source to 1,3-propanediol by a single microorganism. US 5,686,276, November 11, 1997.
44. 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.
45. Luers F, Laffend LA, Nagarajan, Vasantha. Bioconversion of a fermentable carbon source to 1,3-propanediol by a single microorganism. US 6,025,184, February 15, 2000.
46. Seyfried M, Daniel R and Gottschalk G. Glycerol conversion to 1,3-propanediol by Clostridium pasteurianum: cloning and expression of the gene encoding 1,3-propanediol dehydgenase. FEMS Microbiology Letters 1997, 154:337～345.
47. Macis L, Daniel R and Gottschaik G. Properties and sequence of the coenzyme B_(12)-denpendent glycerol dehydratases of Clostridium pasteurianum. FEMS Microbiol Lett 1998, 164:21～28.
48. Malaoui H and Marczak R. Purification and properties of the 1,3-propanediol dehydgenase of Clostridium butyricum E5. Enzyme and Microbial Technology 2000, 27:399～405.
49. Malaoui H and Marczak R. Separation and characterization of the 1,3-propanediol and glycerol dehydgenase activities from Clostridium butyricum E5 wide-type and mutant D. J Appl Microbiol 2001, 90:1006～1014.
50. Malaoui H and Marczak R. Influence of glycerol metabolism by wide-type and mutant strains of Clostridium butyricum E5 grown in chemostat culture. Appl Microbiol Biotechnol 2001, 55: 226～233.
51. Menzel K, Zeng AP, Biebl H, et al. Kinetic, Dynamic, and Pathway Studies of Glycerol Metabolism by Klebsiella Pneumoniae in Anaerobic Continuous Culture: ⅠThe phenomena and characterization of oscillation and hysteresis. Biotechnol Bioeng 1996, 52:549～560.
52. Menzel K, Ahrens K, Zeng AP, et al. Kinetic, Dynamic, and Pathway Studies of Glycerol Metabolism by Klebsiella Pneumoniae in Anaerobic Continuous Culture:Ⅳ Enzymes and fluxes of pyruvate metabolism. Biotechnol Bioeng 1996, 60(5):617～626.
53. Menzel K, Zeng AP, Deckwer WD. Enzymatic evidence for an involvement of pyruvate dehydrogenase in the anaerobic glycerol metabolism of Klebsiella pneumoniae. J Biotechnol 1997a, 56:135～142.
54. Menzel K, Zeng AP, Deckwer WD. High concentration and productivity of 1,3-propanediol from continuous fermentation of glycerol by Klebsiella pneumoniae. Enzyme Microbiol Technol 1997b, 20:82～86.
55. 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.
56. Mori K, Tobimatsu T, Hera T, et al. Characterization, sequencing, and expressing of the genes encoding a reactivating factor for glycerol-inactivated adenosylcobalamin-denpendent glycerol dehydratases. J Biol Chem 1997, 272:32034～32041.
57. Nagarajan, Vasantha, Nakamura et al. Production of 1,3-propanediol from glycerol by recombinant
bacteria expressing recombinant diol dehydratase. US 5,633,362, May 27, 1997.
58. Nagarajan, Vasantha, Nakamura et al. Production of 1,3-propanediol from glycerol by recombinant bacteria expressing recombinant diol dehydratase. US 5,821,092, October 13, 1998.
59. Nakamura, Charles E., Gatenby. Method for the production of 1,3-propanediol by recombinant microorganisms. US 6,103,494, January 11, 2000.
60. Nakas JP, Sehaedle M, Parkinson CM, et al. System development for linked-fermentation products of solvents from algal biomass. Appl Environ Microbiol 1983, 46:1017～1023
61. 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.
62. Papanikolaou S, Ruiz-Sanchez P, Pariset B, et al. High production of 1,3-propanediol from industrial glycerol by a newly isolated Clostridium butyricum strain. J Biotechnol 2000, 77:191～208.
63. Petitdemange E, Cherrier C, Raval G, et al. Regulation of the NADH-ferredoxin oxidoreductase in Clostridia of the butyric group. Biochim Biophys Acta 1976, 421:334～347.
64. Petitdemange E, Manginot-Durr C, Abbad-Andaloussi S, et al. Fermentation of raw glycerol to 1,3-propanediol by new strains of Clostridium butyricum. J Ind Microbiol 1995, 15:498～501.
65. Pflugmacher U, Gottschalk G. Development of an immobilized cell reactor for the production of 1,3-propanediol by Citrobacter freundii. Appl Microbiol Biotechnol 1994, 41:313～316.
66. Raynaud C, Sarcabal P, Meynial-Salles, et al. Molecular characterization of 1,3-propanediol (1,3-PD) operon of Clostridium butyricum. Proc Natl Acad Sci USA 2003, 100(9): 5010～5015.
67. Reimann A, Biebl H. Production of 1,3-propanediol by Clostridium butyricum DSM 5431 and product tolerant mutants in fedbatch culture. Feeding strategy for glycerol and ammonium. Biotechnol Lett 1996a, 18:827～832.
68. Reimann A, Biebl H, Decker WD. Influence of iron, phosphate and methyl vioiogen on glycerol fermentation of Clostridium butyricum. Appl Microbiol Biotechnol 1996b, 45:47～50.
69. Reimann A, Biebl H, Decker WD. Production of 1,3-propanediol by Clostridium butyricum in continuous culture with cell recycling. Appl Microbiol Biotechnol 1998a, 49:359～363.
70. Reimann A, Abbad-Andaloussi S, Biebl H. et al. 1,3-propanediol formation with product-tolerant mutants of Clostridium butyricum DSM 5431 in continuous culture: productivity, carbon and electron flow. J Appl Microbiol 1998b, 84:1125～1130.
71. Saint-Amans S, Perlot P, Goma G, et al. High production of 1,3-propanediol by Clostridium butyricum VPI 3266 in a simply controlled fed～batch system. Biotechnol Lett 1994, 16:832～836.
72. Saint-Amans S, Soucaille P. Carbon and electron flow in Clostridium butyricum grown in
chemostat culture on glucose～glycerol mixtures. Biotechnol Lett 1995, 17:211～216.
73. Saint-Amans S, Girbal L, Andrade J, et al. Regulation of carbon and flow in Clostridium butyricum VPI 3266 grown on glucose-glycerol mixtures. J Bacteriol 2001, 183(5): 1748～1754.
74. Sch#5utz H, Dadler F. Anaerobic reduction of glycerol to propanediol-1,3 by Lactobacillus brevis and Lactobacillus buchneri. Syst Appl Microbial 1984, 5:169～178.
75. Seifert C, Bowien S, Gottschalk G, et al. Identification and expression of the genes and purification of the gene products involved in reactivation of coenzyme B_(12)-denpendent glycerol dehydratases of Citrobacter freundii. Eur J Biochem 2001, 268:2369～2378.
76. Seyfried M, Daniel R and Gottschalk G. Cloning, sequencing and overexpression of the genes encoding coenzyme B_(12)-denpendent glycerol dehydratases of Citrobacter freundii. J Bacteriol 1996, 178:5793～5796.
77. Skraly FA, Lytle BL, Cameron DC. Construction and characterization of a 1,3-propanediol operon. Appl Environ Microbiol 1998, 64:98～105.
78. Skraly FA, Cameron DC. Purification and Characterization of a Bacillus licheniformis Phosphatase Specific for D-a-Glycerophosphate. Arch Biochem. Biophys 1998, 349(1): 27～35.
79. Solomon BO, Zeng A-P, Hibel H, et al. Comparison of the energetic efficiencies of hydrogen and oxychemicals formation in Klebsiella pneumoniae and Clostridium butyricum during anaerobic growth on glycerol. J Biotechnology 1995, 39(2): 107～117.
80. Sprenger GA, Hammer GA, Johnson EA, et al. Anaerobic growth of Escherichia coli on glycerol by importing genes of the dba regulon from Klebsiella pneumoniae. J Gen Microbiol 1989, 135:1255～1262.
81. Streekstra H, Teixeira de Mattos MJ, Neijssel OM, et al. Overflow metabolism during anaerobic growth of Klebsiella aerogenes NCTC 418 on glycerol and dihydroxyacetone in chemostst culture. Arch Microbiol 1987, 147:268～275.
82. Sun J, Van Den Heuvel J, Soucaille P, et al. Comparative Genomic Analysis of dha Regulon and Related Genes for Anaerobic Metabolism in Bacteria. Biotechnol prog 2003,19(2):263～272.
83. Tarmy EM and N.O. Kaplan. Chemical characterization of D-lactate dehydrogenase from Escherichia coil B. J Biol Chem. 1968, 271:2579～2586.
84. Tobimatsu T, Azuma M, Matsubara H, et al. Cloning, sequencing and high level expression of the genes encoding adenosylcobalamin-denpendent glycerol dehydratases of Klebsiella pneumoniae. J Biol Chem 1996, 271:22352～22357.
85. Tobimatsu T, Kajiura H, Yunoki M, et al. Identification and expression of the genes encoding a reactivating factor for adenosylcobalamin-denpendent glycerol dehydratases. J Bacteriol 1999, 181:4110～4113.
86. Tobimatsu T, Kajiura H, Toraya T. Specificities of reactivating factors for adenosylcobalamin-denpendent diol dehydratase and glycerol dehydratase. Arch Microbiol 2000, 174:81～88.
87. Tong IT, Liao HH, Cameron DC. 1,3-propanediol production by Escherichia coli expressinggenes from the Klebsiella pneumoniae dha regulon. Appl Environ Microbiol 1991, 57:3541～3546.
88. Tong IT, Cameron DC 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.
89. Toraya T and Mori K. A reactivating factor for coenzyme B_(12)-denpendent diol dehydratases. J Biol Chem 1999, 274:3372～3377.
90. Toraya T. Radical catalysis of B_(12) enzymes: structure, mechanism, inactivation, and reactivation of diol and glycerol dehydratases. Cell Mole Life Sci 2000, 57(1):106～127.
91. Xiu ZL, Zeng AP and Deckwer WD. Multiplicity and stability analysis of microorganisms in continuous culture: effects of metabolic overflow and growth inhibition. Biotechnol Bioeng 1998, 57:251～261.
92. Yanisch-Perron C, Vieera J and Messing J. Improved M13 phage cloning vecters and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 1985 33(1): 103～119.
93. Zeng AP. Pathway and kinetic analysis of 1,3-propanediol production from glycerol fermentation by Clostridium butyricum. Bioproc Eng 1996, 14:169～175.
94. Zeng AP, 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.
95. Zeng AP, Biebl H and Deckwer WD. Microbial conversion of glycerol to 1,3-propanediol [A]. In Saha BC and Woodward J, eds. Recent progress in Fuels and Chemicals from Biomass[C], American Chemical Society, 1997, 265～279.
96. Zeng AP, Biebl H, Schlieker H, et al. Pathway analysis of glycerol fermentation by Klebsiella pneumoniae: regulation of reducing equivalent balance and product formation. Enzyme and Microbial Technology 1993, 15:770～779.
97. Zeng AP, Menzel K, and Deckwer WD, et al. Kinetic, Dynamic, and Pathway Studies of Glycerol Metabolism by Klebsiella Pneumoniae in Anaerobic Continuous Culture: Ⅱ Analysis of metabolic rates and pathways under oscillation and steady-state conditions. Biotechnol Bioeng 1996, 52:561～571.
98. Zeng AP, Ross A, Hibel H, et al. Multiple product inhibition and growth modeling of Clostridium bulyricum and Klebsiella pneumoniae in glycerol fermentation. Biotechnol Bioeng 1994, 44:902～911.
99. 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.
100. Zhu MM, Lawman PD, Cameron DC. Improving 1,3-propanediol productions from glycerol in a metabolically engineered Escherichia coli by reducing accumulation of sn～glucerol～3～phosphate. Biotechnol Prog 2002, 18(4): 694～699.
101．北京化学试剂公司编．化学试剂目录手册．北京工业大学出版社，1993，586．
102．冯婧微，张洪林，蒋林时等．1，3-丙二醇合成方法研究．化工科技，2002，10(6)：43～47．
103．刘海军，王剑锋，张代佳等．用克雷伯氏菌批式流加发酵法生产 1，3-丙二醇．食品与发酵工业， 2001，27(7)4～7．
104．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)：45～52．
105．刘银乾，段启伟．1，3-丙二醇的制备方法．合成纤维工业，2002，25(2)：42～45．
106．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．
107．瞿国华．PTT的工业开发及1，3-丙二醇合成．合成纤维工业，2000，23(4)：31～34．
108．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．(第二版) 北京：科学出版社，1993．
109．邵敬伟，刘长江．1，3-丙二醇发酵生产的研究进展．沈阳农业大学学报，2001～2002，32(1)：57～60．
110．邵敬伟，刘长江，迟乃玉等．1，3-丙二醇发酵条件的正交设计实验．襄樊学院学报，2002，23(5)：45～49．
111．王剑锋，刘海军，修志龙等．生物转化生产 1，3-丙二醇的研究进展．化学通报，2001a，10：621～625。
112．王剑锋，刘海军，修志龙等．1，3-丙二醇间歇发酵培养基的优化．食品与发酵工业，2001b，27(8)：5～8。
113．王剑锋，修志龙，刘海军等．克雷伯氏菌微氧发酵生产 1，3-丙二醇的研究．现代化工，2001，21(5)：28～31．
114．王剑锋，修志龙，范圣第．甘油转化生产 1，3-丙二醇发酵液中甘油含量的测定．工业微生物，2001，31(2)：33～35．
115．王熙庭，陈曼华．1，3-丙二醇生产方法及用途．煤化工，2000，4：38～40．
116．修志龙．1，3-丙二醇的微生物法生产和市场分析．现代化工，1999，19(3)：33～35．
117．修志龙．甘油连续生物歧化过程培养基和pH调控策略研究．高校化学工程学报，2001，15(4)：397～402。
118．修志龙，曾安平．微生物细胞连续培养过程中振荡和混沌行为的研究进展．生物工程进展，1999，19(6)：58～63．
119．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，2000，27(4)：300～302．
120．修志龙，曾安平，安利佳．甘油生物歧化过程动力学数学模拟和多稳态研究．大连理工大学学报，2000，40(4)：428～433．
121．薛丽梅，王琲，韩大伟．1，3-丙二醇的化学合成法．化学与粘合，2001，1：38～40．
122．杨菊群，王幸宜，戚蕴石．1，3-丙二醇的合成新进展．石油化工，2002，31(11)：943～947．
123．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(2)：11～15．
124．张健，赵红英，刘宏娟等．以葡萄糖为辅助底物发酵生产 1，3-丙二醇的研究．现代化工，2002，22(6)：32～35．
125．赵红英，张健，刘宏娟等．1，3-丙二醇发酵过程中底物抑制及其对策的研究．现代化工，2002，22(7)：34～38．
126．赵育．新型热塑性聚酯—聚对苯二甲酸丙二醇酯．塑料科技，2000，136：33～36．
127．中华人民共和国国家技术监督局．GB7479—87．中华人民共和国国家标准—水质 铵的测定．北京：中国标准出版社，1987～08～01．
128．朱丙田，刘德华，任海玉等．1，3-丙二醇发酵条件的探索．化工冶金，2000，21(4)：420～422．