Construction Of NADH Regeneration System And Ethanol Deficient Mutants And Preliminary Research Of 1, 3-propanediol Production

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.