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 (phaCABRe) 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 (PhaCRe) gene focusing on the codon usage preference for the translation system of C. glutamicum. The other is the replacement of wild-type phaCRe 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 PhaCRe 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, β-ketothiolase (PhaARe) and acetoacetyl-CoA reductase (PhaBRe). This finding prompted us to carry out a gene dosage of phaABRe for a double plasmid system, and the highest production (52.5 wt%) of P(3HB) was finally achieved by combining the gene dosage of phaABRe 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(3HB) was increased by more than 4-fold compared with the non-P(3HB)-accumulating cells without filamentous morphologenesis observed in E. coli.