Improvement of poly(3-hydroxybutyrate) [P(3HB)] production in Corynebacterium glutamicum by codon optimization, point mutation and gene dosage of P(3HB) biosynthetic genes

In our previous study, a system for producing poly(3-hydroxybutyrate) [P(3HB)] was established by introducing a polyhydroxyalkanoate (PHA) biosynthetic gene operon (phaCAB(Re)) derived from Ralstonia eutropha into Corynebacterium glutamicum. In this study, two experimental strategies have been applied to improve P(3HB) production in recombinant C glutamicum. One is a codon optimization of the N-terminal-coding region of the PHA synthase (PhaC(Re)) gene focusing on the codon usage preference for the translation system of C. glutamicum. The other is the replacement of wild-type phaC,, with a modified gene encoding a mutation of Gly4Asp (G4D), which enhanced the production of PhaCRe and P(3HB) in Escherichia coli. The introduction of these engineered PHA synthase genes into C glutamicum enhanced the production of PhaC(Re) and P(3HB). Interestingly, we found that these gene modifications also caused increases in the concentration of the translation products of the genes encoding monomer-supplying enzymes, beta-ketothiolase (PhaA(Re)) and acetoacetyl-CoA reductase (PhaB(Re)). This finding prompted us to carry out a gene dosage of phaAB(Re) for a double plasmid system, and the highest production (52.5 wt%) of P(3HB) was finally achieved by combining the gene dosage of phaAB(Re) with codon optimization. The molecular weight of P(3HB) was also increased by approximately 2-fold, as was P(3HB) content. Microscopic observation revealed that the volume of the cells accumulating P(31113) was increased by more than 4-fold compared with the non -P(3HB)-accumulating cells without filamentous morphologenesis observed in E. coli.