Cofactor Engineering For 3-hydroxypropionic Acid Production By Klebsiella Pneumonaie

3-hydroxypropionic acid (3-HP) is an important platform chemical, which will exert an direct influence on the development of many other high value chemicals, such as 1,3-propanediol and acrylic acid. klebsiella pneumoniae can naturally dissimilate glycerol and contain the completed enzymes of dha system, it was thereby employed in this study as host strain and was introduced a vector able to biosynthesize both glycerol and 3-HP. As a consequence, the NAD/NADH ratio in the engineered strain has been balanced and the yield of 3-HP was enhanced dramatically.To begin with, the metabolic pathway of glycerol synthesis was introduced into Escherichia coli BL-21 by construction of the co-expression vector containing genes encoding glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase that govern glycerol production in Saccharomyces cerevisiae. Consequently, a glycerol concentration of 1 g l-1 was achieved in flask culture and the recombinant could regulate intracellular osmotic pressure to resist environment stress by synthesis of glycerol in vivo.To improve the soluble protein expression and obviate the addition of IPTG, a vector pET-kp(derived from pET-28a) was constructed by replacement of T7 promoter with native constitutive promoter of K. pneumoniae. Then the glycerol biosynthesis cassette G12 was ligated to the plasmid pET-kp and the recombinant plasmid KG 12 was constructed and transformed. SDS-PAGE analysis showed the successful expression of key enzymes in tandem co-expression plasmid. Analysis of glycerol metabolic pathway in K. pneumoniae suggested that the 3-HP yield cannot be improved significantly without reasonable pathway design and oxidation-reduction balance in vivo. In order to improve the regeneration of NAD in vivo, an expression plasmid harboring GDP1, GPP1, ald4 and dhaB genes was constructed and transformed into K. pneumoniae to yield a recombinant strain KGAB. In KGAB, glycerol biosynthesis system was employed for NAD regeneration. SDS-PAGE analysis showed the expression of key enzymes in tandem co-expression plasmid. In the fermentation of KGAB, a 3-HP concentration of 0.52g/l was obtained from glucose in flask culture. These results indicated the successful construction of the pathway responsible for the biosynthesis of 3-HP from glucose.The flask culture of the recombinant K. pneumoniae under microaerobic condition was also performed. The cofactor regeneration system in K. pneumoniae was investigated by production of 3-HP using strains K1, KAB and KGAB at flask level. The results showed that the optimized metabolic pathway played crucial role in improving 3-HP concentration and productivity. Compared with KAB, the 3-HP concentration of KGAB is 6.6g/l, increased by 317%, glycerol conversion rate is 200% higher than KAB, and the productivity (0.82 g·L-1·OD-1 cell·h-1) was nearly improved to 600%. In addition, gradient concentrations of glucose of 2g/l,5g/l,10g/l,20g/l were added at the beginning of microaerobic fermentation. The glucose of 20g/l is most helpful to the biomass accumulation. In initial stage of fermentation, strains under 5g/l and 20g/l glucose have higher glycerol consumption rate. 3-HP concentration analysis showed that in the initial stage of fermentation, 3-HP concentration is in proportion to the glucose additive amount, which may attribute to the biomass. Considering unit cell concentration, three glucose amount (0g/l,10g/l,20g/l) will be benefical to 3-HP production.