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While poly(3-hydroxybutyrate- co -3-hydroxyvalerate) [P(3HB- co -3HV)] is a biodegradable commodity plastic with broad applications, its microbial synthesis is hindered by high production costs primarily associated with the supplementation of related carbon substrates (e.g. propionate or valerate). Here we report construction of engineered  Escherichia coli  strains for direct synthesis of P(3HB- co -3HV) from an unrelated carbon source (e.g. glucose or glycerol). First, an  E. coli  strain with an activated sleeping beauty mutase (Sbm) operon was used to generate propionyl-CoA as a precursor. Next, two acetyl-CoA moieties or acetyl-CoA and propionyl-CoA were condensed to form acetoacetyl-CoA and 3-ketovaleryl-CoA, respectively, by functional expression of β-ketothiolases from  Cupriavidus necator  (i.e. PhaA and BktB). The resulting thioester intermediates were channeled into the polyhydroxyalkanoate (PHA) biosynthetic pathway through functional expression of acetoacetyl-CoA reductase (PhaB) for thioester reduction and PHA synthase (PhaC) for subsequent polymerization. Metabolic engineering of  E. coli  host strains was further conducted to enhance total PHA content and the 3-hydroxyvaleryl (3HV) monomer fraction in the copolymer. Using a selection of engineered  E. coli  strains for batch cultivation with an unrelated carbon source, we achieved high-level P(3HB- co -3HV) production with the 3HV monomer fraction ranging from 3 to 19 mol%, demonstrating the potential industrial applicability of these whole-cell biocatalysts.

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Engineering of Escherichia coli for direct and modulated biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer using unrelated carbon sources