【作者】 张刚；

【导师】 李春； 曹竹安；

【作者基本信息】 石河子大学 ， 农产品加工及贮藏工程， 2008， 硕士

【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。更多还原

【Abstract】 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.更多还原