Cofactor Engineering In The Process Of 1,3-Propanediol Production By Klebsiella Pneumonaie

Biological oxidation-reduction is an important class of reactions that offers considerable potentials for the production of commodity and fine chemicals. The enzymes involved, oxidoreductases, are coenzyme-dependent. Availability and regeneration of coenzymes are often critical for the industrial applications of these enzymes. For example, biosynthesis of 1,3-propanediol (1,3-PD) from glycerol by Klebsiella pneumoniae is associated with the consumption of NADH, which in itself is generated/regenerated in the oxidative branch pathway of glycerol. These coupled oxidation and reduction pathways complicate the regulation of 1,3-PD synthesis and result in low yields of the target product.In this study, several new approaches were investigated to optimize in vivo NADH regeneration and to improve 1,3-PD biosynthesis. More specifically:(1) Along with 1,3-PD production, accumulation of the main by-product ethanol consumes a significant amount of NADH, which decreases the yield of 1,3-PD. The upstream enzyme for ethanol synthesis in K. pneumoniae YMU2, aldehyde dehydrogenase (ALDH), was inactivated, which should decrease the production of ethanol more effectively than inactivation of the downstream enzyme alcohol dehydrogenase in theory. An engineered strain DA-1HB was constructed by homologous recombination. In the fermentation of DA-1HB, the production of ethanol was decreased by more than 70%, the concentration of 1,3-PD reached 61 g.l-1, 10% higher than that of the parent strain, and the molar yield of 1,3-PD from glycerol reached 61.2% from 35.5%.(2) To improve the regeneration of NADH in vivo, an expression plasmid harboring fdh gene was constructed and transformed into K. pneumoniae YMU2, to yield a recombinant strain OF-1. In OF-1, formate/ formate dehydrogenase system was used for NADH regeneration. In the fermentation of OF-1, though the molar yield of 1,3-PD from glycerol (36%) was improved un-obviously, the final concentration of 1,3-PD was increased to 68 g.l-1.(3) The above two approches were combined: formate/ formate dehydrogenasewas established in DA-1HB and a new recombinant strain K. pneumoniae DAOF was obtained. By anaerobic culture of DAOF, the concentration and the molar yield of 1,3-PD reached 63 g.l-1 and 38.4% respectively.In addition, oxidoreduction potential of medium (ORP) was regulated by addition of electron receptors or controlling the ratio of air to nitrogen. By optimizing the fermentation conditions of DA-1HB, 70 g.l-1 1,3-PD was produced and the molar yield of 1,3-PD from glycerol reached 69.9%.