Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxy-4-methylvalerate) by recombinant Escherichia coli expressing leucine metabolism-related enzymes derived from Clostridium difficile

Abstract

An obligate anaerobic bacterium Clostridium difficile has a unique metabolic pathway to convert leucine to 4-methylvalerate, in which 4-methyl-2-pentenoyl-CoA (4M2PE-CoA) is an intermediate of this pathway. 4M2PE-CoA is also able to be converted to 3-hydroxy-4-methylvalerate (3H4MV), a branched side chain monomer unit, for synthesis of polyhydroxyalkanoate (PHA) copolymer. In this study, to synthesize 3H4MV-containing PHA copolymer from leucine, the leucine metabolism-related enzymes (LdhA and HadAIBC) derived from C. difficile and PHA biosynthesis enzymes (PhaPCJAc and PhaABRe) derived from Aeromonas caviae and Ralstonia eutropha were co-expressed in the codon usage-improved Escherichia coli. Under microaerobic culture conditions, this E. coli was able to synthesize P(3HB-co-12.2 mol% 3H4MV) from glucose with the supplementation of 1 g/L leucine. This strain also produced P(3HB-co-12.6 mol% 3H4MV) using the culture supernatant of leucine overproducer E. coli strain NS1391 as the medium for PHA production, achieving 3H4MV copolymer synthesis only from glucose. Furthermore, we tested the feasibility of the 3H4MV copolymer synthesis in E. coli strain NS1391 from glucose. The recombinant E. coli NS1391 was able to synthesize P(3HB-co-3.0 mol% 3H4MV) from glucose without any leucine supplementation. This study demonstrates the potential of the new metabolic pathway for 3H4MV synthesis using leucine metabolism-related enzymes from C. difficile.

Keywords: 3-Hydroxy-4-methylvalerate; Aeromonas caviae; Clostridium difficile; Leucine metabolism; Polyhydroxyalkanoate; Ralstonia eutropha.