The Effect Of Over-expressing DhaT Gene On The Synthesis Of 1,3-Propanediol In Klebsiella Pneumoniae

1,3-Propanediol(1,3-PDO), an important three-carbon compound and industrial chemical used for fibres, medicines and cosmetics, attacts more and more attention because of its huge value even since the commercialization of a new polyester fiber polytrimethylene terephthalate(PTT). 1,3-Propanediol oxidoreductase encoded by dha T gene is a key enzyme in Klebsiella used glycerol as a substrate to product 1,3-PDO. In this study, we over-expressed dha T gene and strengthened the regeneration of the NADH to enhance the conversion of glycerol to 1,3-PDO and study the effects on synthesizing 1,3-PDO and the changes of metabolic flux in Klebsiella.In this study, engineered recombinant K. pneumoniae(p Etac-dha T) over-expressed dha T gene was constructed, in which the 1,3-propanediol oxidoreductase activity improved 94.3% to compare with K. pneumoniae(p Etac). The 1,3-PDO concentration of recombinant increased slightly and reached 19.5 g·L-1, while the 1,3-PDO conversion rate achieved 0.618. The results indicated that over-expressing dha T gene could not enhance the conversion of glycerol to 1,3-PDO, which is probably because the NADH cofactor limited the synthsis of 1,3-PDO catelyzed by DHAT.To improve the regeneration of the NADH, the formate dehydrogenase gene(fdh1) from S. cerevisiae and pyruvate formate-lyase gene(pfl B) from K. pneumoniae was over-expressed. The yield of 1,3-PDO of the K. pneumoniae(p Etac-pfl B), K. pneumoniae(p Etac-fdh1) and K. pneumoniae(p Etac-pfl B-tac-fdh1) increased by 8%, 12% and 18%, respectively and the conversion rate of 1,3-PDO increased by 7.3%, 13.2% and 19.5%, compared with K. pneumoniae(p Etac). The NADH and NAD+ concerntration of the three recombinants significantly increased compared with wild strain, while other by-products decreased except acetate.To further improve usage of NADH and the conversion rate of 1,3-PDO, we constructed three recombinants: K. pneumoniae(p Etac-pfl B/p RSF19-tac-dha T-tet), K. pneumoniae(p Etacfdh1/p RSF19-tac-dha T-tet) and K. pneumoniae(p Etac-pfl B-tac-fdh1/p RSF19-tac-dha T-tet). To compared with K. pneumoniae(p Etac/p RSF19-tac-dha T-tet), the yield of 1,3-PDO increased by 5.9%, 9.8%, 20.2%, reached 20.69 g·L-1, 21.45 g·L-1, 23.46 g·L-1, and the conversion rate of 1,3-PDO increased by 7.5%, 9.8%, 20.1%, respectively. The results showed that the restriction of NADH has a greater impact on synthesis of 1,3-PDO than DHAT. This study provided an advantage reference for improving the yield of 1,3-PDO fermented by Klebsiella.