Methane-based biosynthesis of 4-hydroxybutyrate and P(3-hydroxybutyrate-co-4-hydroxybutyrate) using engineered Methylosinus trichosporium OB3b.

4-Hydroxybutyric acid (4-HB) is a key platform chemical that serves as a precursor in a wide variety of industrial applications including 1,4-butanediol and bioplastics production. In this study, we reconstructed 4-HB biosynthetic pathway including CoA-dependent succinate semialdehyde dehydrogenase and NADPH-dependent succinate semialdehyde reductase in Type II methanotrophs, Methylosinus trichosporium OB3b, to synthesize 4-HB. These engineered strains were able to synthesize 4-HB from methane via tricarboxylic acid cycle. 4-HB synthesis was further improved to 10.5 mg/L by overexpressing phosphoenolpyruvate carboxylase, isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase genes in M. trichosporium OB3b. We combined the native poly(3-hydroxybutyrate) metabolic pathway and reconstructed 4-HB biosynthetic pathway to synthesize P(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer from structurally unrelated substrate methane as a single carbon source. These engineered strains could synthesize P(3HB-co-4HB) copolymer with 3.08 mol% 4-HB from methane. This study provides several engineering strategies to synthesize polyhydroxyalkanoates and their monomers from methane.