The Study Of NAD(P)H-NAD(P) For Biosynthesis Of 1,3-Propanediol By Xylose Pathway In Klebsiella Pneumoniae

A valuable three carbon platform compound, 1,3-propandediol(1,3-PDO) has great commercial applications as a chief monomer of polytrimethylene-tereph-thalate(PTT). The biosynthesis of 1,3-propandediol has always been focused on around the world. Reducing equivalent NAD(P)H plays a vital role in the bioproduction of 1,3-propanediol. In order to enhance the production of 1,3-propandiol, the xylose isomerase gene xyl A and xylulokinase gene xyl B from Escherichia coli were cloned and heterologous expressed in Klebsiella pneumoniae, resulted in the recombinant K. pneumoniae(p Etac-xyl A), K. pneumoniae(p Etacxyl B) and K. pneumoniae(p Etac-xyl A-tac-xyl B), strengthening intracellular reducing equivalent NAD(P)H and xylose metabolism in the present study.The recombinant K. pneumoniae(p Etac-xyl A) was cultured with different concentrations of xylose and glycerol as co-substrates in flask fermentation. Compared with the parent K. pneumoniae(p Etac), the NADPH and NADH of K. pneumoniae(p Etac-xyl A) were increased approximately by 0.39 and 0.34 folds adding 8 g·L-1 xylose when induced with 1.0 mmol·L-1 IPTG. The titer of 1,3-propanediol of the recombinant K. pneumoniae(p Etac-xyl A) reached 23.31 g·L-1, which was 20% higher than that of the parent strain. The conversion rate of 1,3-propanediol was 0.73 mol·mol-1 in the genetic engineered strain.Via optimizating expression-level in the recombinant K. pneumoniae(p Etac-xyl B), the NADPH and NADH were improved approximately by 0.37 and 0.32 folds when added with 8 g·L-1 xylose. The yield of 1,3-propanediol reached 22.97 g·L-1, which was 19.9% higher than that of the parent strain. The conversion rate of 1,3-propanediol was 0.72 mol·mol-1 in the K. pneumoniae(p Etac-xyl B), generating a similar fermentation result of the K. pneumoniae(p Etac-xyl A).In contrast to K. pneumoniae(p Etac-xyl A) and K. pneumoniae(p Etac-xyl B), the fermentability was further improved through expression-level optimization by the recombinant K. pneumoniae(p Etac-xyl A-tac-xyl B). Compared with the parent K. pneumoniae(p Etac), the NADPH and NADH of K. pneumoniae(p Etac-xyl A-tac-xyl B) were improved approximately by 0.43 and 0.37 folds when adding 8 g·L-1 xylose. The final titer of 1,3-propanediol were 24.46 g·L-1, which was 23.6% increased. The conversion rate of 1,3-propanediol of the K. pneumoniae(p Etac-xyl A-tac-xyl B) reached 0.76 mol·mol-1, which was a higher level in the previous reports. In conclusion, the introduced xylose metabolism caused a effective regeneration of the NAD(P)H, and meanwhile made part of the metabolic carbon flux flowing to 1,3-propanediol synthesis route. This study provides a new method and idea in the fermentative production of 1,3-propanediol.