Application of a propionyl coenzyme A synthetase for poly(3-hydroxypropionate-co-3-hydroxybutyrate) accumulation in recombinant Escherichia coli

The genetic operon for propionic acid degradation in Salmonella enterica se rovar Typhimurium contains an open reading frame designated prpE which enco des a propionyl coenzyme A (propionyl-CoA) synthetase (A. R. Horswill and J . C. Escalante-Semerena, Microbiology 145:1381-1388, 1999), In this paper w e report the cloning of prpE by PCR its overexpression in Escherichia coli, and the substrate specificity of the enzyme. When propionate was utilized as the substrate for PrpE, a K-m of 50 muM and a specific activity of 120 m u mol.min(-1).mg(-1) were found at the saturating substrate concentration. PrpE also activated acetate, 3-hydroxypropionate (3HP), and butyrate to the ir corresponding coenzyme A esters but did so much less efficiently than pr opionate, When prpE was coexpressed with the polyhydroxyalkanoate (PHA) bio synthetic genes from Ralstonia eutropha in recombinant E, coli, a PHA copol ymer containing 3HP units accumulated when 3HP was supplied with the growth medium. To compare the utility of acyl-CoA synthetases to that of an acyl- CoA transferase for PHA production, PHA-producing recombinant strains were constructed to coexpress the PHA biosynthetic genes with prpE, with acoE (a n acetyl-CoA synthetase gene from R, eutropha [H, Priefert and A. Steinbuch el, J, Bacteriol. 174:6590-6599, 1992]), or with orfZ (an acetyl-CoA:4-hydr oxybutyrate-CoA transferase gene from Clostridium propionicum [H. E. Valent in, S. Reiser, and K. J. Gruys, Biotechnol, Bioeng, 67:291-299, 2000]). Of the three enzymes, PrpE and OrfZ enabled similar levels of 3HP incorporatio n into PHA, whereas AcoE was significantly less effective in this capacity.