Metabolic engineering of a novel propionate-independent pathway for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in recombinant Salmonella enterica serovar typhimurium

Abstract

A pathway was metabolically engineered to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable thermoplastic with proven commercial applications, from a single, unrelated carbon source. An expression system was developed in which a prpC strain of Salmonella enterica serovar Typhimurium, with a mutation in the ability to metabolize propionyl coenzyme A (propionyl-CoA), served as the host for a plasmid harboring the Acinetobacter polyhydroxyalkanoate synthesis operon (phaBCA) and a second plasmid with the Escherichia coli sbm and ygfG genes under an independent promoter. The sbm and ygfG genes encode a novel (2R)-methylmalonyl-CoA mutase and a (2R)-methylmalonyl-CoA decarboxylase, respectively, which convert succinyl-CoA, derived from the tricarboxylic acid cycle, to propionyl-CoA, an essential precursor of 3-hydroxyvalerate (HV). The S. enterica system accumulated PHBV with significant HV incorporation when the organism was grown aerobically with glycerol as the sole carbon source. It was possible to vary the average HV fraction in the copolymer by adjusting the arabinose or cyanocobalamin (precursor of coenzyme B12) concentration in the medium.

FIG. 1.

Novel metabolic pathway for PHBV production from an unrelated carbon source. 1, Sbm; 2, methylmalonyl-CoA decarboxylase (YgfG); 3, 3-ketothiolase (PhaA); 4, acetoacetyl-CoA reductase (PhaB); 5, PHA synthase (PhaC); 6, 2-methylcitric acid synthase (PrpC). The citric acid cycle (left) and the 2-methylcitric acid cycle (right), which compete for acetyl-CoA and for propionyl-CoA, respectively, are shown. The 2-methylcitric acid cycle is blocked in the expression system described in this paper, as indicated. PHV, poly(3-hydroxyvalerate); PEP, phosphoenolpyruvate.

FIG. 2.

Variation of total polymer content (○) and polymer composition (•) in JE4199 harboring pMMB-pha and pBAD-Cro with arabinose induction. Cells were grown in MOPS-buffered minimal medium containing 274 mM glycerol with 0.1% succinate (A) and without succinate (B). Expression of the plasmid-encoded genes was induced with 500 μM IPTG and different arabinose concentrations when the cell density reached an OD₆₀₀ of 0.15. The points obtained when no arabinose was added are plotted at 0.00001% (wt/vol) arabinose because zero arabinose could not be plotted on the logarithmic scale.

FIG. 3.

Variation of total polymer content (○) and polymer composition (•) in JE4199 harboring pMMB-pha and pBAD-Cro with external CN-B₁₂ (precursor of coenzyme B₁₂). Cells were grown in MOPS-buffered minimal medium containing 274 mM glycerol. Expression of the plasmid-encoded genes was induced with 500 μM IPTG and 0.1% arabinose when the cell density reached an OD₆₀₀ of 0.15.