A microbial factory for lactate-based polyesters using a lactate-polymerizing enzyme

Abstract

Polylactate (PLA) is synthesized as a representative bio-based polyester by the chemo-bio process on the basis of metal catalyst-mediated chemical polymerization of lactate (LA) supplied by microbial fermentation. To establish the one-step microbial process for synthesis of LA-based polyesters, we explored whether polyhydroxyalkanoate (PHA) synthase would exhibit polymerizing activity toward a LA-coenzyme A (CoA), based on the fact that PHA monomeric constituents, especially 3-hydroxybutyrate (3HB), are structurally analogous to LA. An engineered PHA synthase was discovered as a candidate by a two-phase in vitro polymerization system previously developed. An LA-CoA producing Escherichia coli strain with a CoA transferase gene was constructed, and the generation of LA-CoA was demonstrated by capillary electrophoresis/MS analysis. Next, when the engineered PHA synthase gene was introduced into the resultant recombinant strain, we confirmed the one-step biosynthesis of the LA-incorporated copolyester, P(6 mol% LA-co-94 mol% 3HB), with a number-average molecular weight of 1.9 x 10(5), as revealed by gel permeation chromatography, gas chromatography/MS, and NMR.

Fig. 1.

Microbial factory for LA-based polyester. PLA has been synthesized by the chemo-bio process. Copolymerization of LA with other monomer units has also been performed by basically the same chemo-synthetic procedures. In bio-process with microbes, LA monomer substrate is recognized as a CoA form (LA-CoA) by LA-polymerizing enzyme that is recruited from microbial PHA synthase. A candidate PHA synthase with acquired LA-polymerizing activity was selected based on the water-organic solvent two-phase in vitro polymerization system (5).

Fig. 2.

Construction of microbial production system for LA-based polyester. (A) CE/MS analysis of LA-CoA generated in recombinant Escherichia coli harboring the pct gene. The ions of m/z = 838 and 419 correspond to monovalent and bivalent ions of LA-CoA, respectively. pTV118N, control vector; pTV118Npct, expression vector for the pct gene. (B) LA-based polyester synthetic pathway in recombinant E. coli. The letters in boxes indicate the enzyme. LDH, lactate dehydrogenase; PCT, propionyl-CoA transferase; PhaA, β-ketothiolase; PhaB, NADPH-dependent acetoacetyl-CoA reductase; PhaC, PHA synthase. (C) Construction of the plasmids used in this study. P_Re and T_Re denote respectively the promoter and terminator regions of phbCAB operon from Ralstonia eutropha. P_lac denotes the lac promoter. The phaC1(STQK) gene encodes the Ser325Thr/Gln481Lys mutant of PHA synthase from Pseudomonas sp. 61–3. The phaA and phaB genes encode β-ketothiolase (PhaA) and NADPH-dependent acetoacetyl-CoA reductase (PhaB) derived from R. eutropha, respectively. The pct gene encodes a propionyl-CoA transferase from Megasphaera elsdenii.

Fig. 3.

Characterization of a P(LA-co-3HB) copolymer produced in the recombinant E. coli harboring the plasmid pTV118NpctC1AB(STQK). (A) GC/MS analysis of a P(LA-co-3HB) copolymer isolated from the recombinant E. coli harboring the plasmid pTV118NpctC1AB(STQK). Ethyl-LA, ethyl lactate; Ethyl-3HB, ethyl 3-hydroxybutyrate. (B) ¹³C-NMR spectrum of a P(LA-co-3HB) copolymer isolated from the recombinant E. coli harboring the plasmid pTV118NpctC1AB(STQK). (C) ¹H-NMR spectrum of a P(LA-co-3HB) copolymer isolated from the recombinant E. coli harboring the plasmid pTV118NpctC1AB(STQK). (D) 2d-NMR spectrum of a P(LA-co-3HB) copolymer isolated from the recombinant E. coli harboring the plasmid pTV118NpctC1AB (STQK).