Research Of The Metabolic Regulation Mechanism Of NAD(H) Kinase On Poly-3-hydroxybutyrate Biosynthesis In Recombinant Escherichia Coli

Polyhydroxyalkanoates (PHA) is widely distributed in a variety of microorganisms as energy storage macromolecule compounds. PHA has attracted extensive attentions due to its diverse properties including biocompatibility, biodegradability and biorenewability. As one of the simplest representative of PHA, Poly-3-hydroxybutyrate (PHB) is the simplest, most well studied and firstly industrially produced member of PHA family. In this paper, we overexpressed NAD kinase and NADH kinase in recombinant Escherichia coli and studied its influence on PHB biosynthesis, the mail results are as follows.With the help of site-specific recombinantion technique of attB-attP, multiple copies of NAD kinase gene yfjB were integrated into the chromosome of E. coli. The yjjB integration mutant harbored similar metabolic profiles to that of the wild type control. PHB production titer was increased to 3.04 g.L-1 in 48 h shake flask cultivation, which was increased by 30%. In 72 h of shake flask cultivation,4.24 g.L-1 PHB production titer was obtained, reaching 1.84 folds of the control strain, and the substrate to PHB yield was increased by 43%. Transcriptional analysis showed that the transcription levels of genes within the PHB biosynthesis operon were increased by 6-8 folds with yfjB overexpression, which may be the primary reason for the improved PHB production.We next studied the effect of NADH kinase overexpression on PHB production with plasmid expression system. The co-expression vector of NADH kinase gene pos5p from Saccharomyces cerevisiae and PHB biosynthesis operon phaCAB was constructed. The recombinant strain harboring the co-expression vector showed improved PHB production, and PHB titer reached 3.82 g.L-1 in 72 h shake flask cultivation, which was increased by 66% compared to that of the control strain. In terms of substrate to PHB yield, a increasement of 28% was achieved. The analysis of intracellylar cofactor level showed that the ratio of (NADPH+NADP)/(NAD+NADH) was promoted to 2.2 folds, indicating that the NADH kinase gene pos5p from S.cerevisiae was successfully expressed in E. coli and functionaly played its role in PHB biosynthesis.