Microbial synthesis of a novel terpolyester P(LA-co-3HB-co-3HP) from low-cost substrates

Abstract

Polylactide (PLA) is a bio-based plastic commonly synthesized by chemical catalytic reaction using lactic acid (LA) as a substrate. Here, novel LA-containing terpolyesters, namely, P[LA-co-3-hydroxybutyrate (3HB)-co-3-hydroxypropionate (3HP)], short as PLBP, were successfully synthesized for the first time by a recombinant Escherichia coli harbouring polyhydroxyalkanoate (PHA) synthase from Pseudomonas stutzeri (PhaC1_Ps ) with 4-point mutations at E130D, S325T, S477G and Q481K, and 3-hydroxypropionyl-CoA (3HP-CoA) synthesis pathway from glycerol, 3-hydroxybutyryl-CoA (3HB-CoA) as well as lactyl-CoA (LA-CoA) pathways from glucose. Combining these pathways with the PHA synthase mutant phaC1_Ps (E130D S325T S477G Q481K), the random terpolyester P(LA-co-3HB-co-3HP), or PLBP, was structurally confirmed by nuclear magnetic resonance to consist of 2 mol% LA, 90 mol% 3HB, and 8 mol% 3HP respectively. Remarkably, the PLBP terpolyester was produced from low-cost sustainable glycerol and glucose. Monomer ratios of PLBP could be regulated by ratios of glycerol to glucose. Other terpolyester thermal and mechanical properties can be manipulated by adjusting the monomer ratios. More PLBP applications are to be expected.

Figure 1

Metabolically engineered pathways for production of terpolyester P(LA‐co‐3HB‐co‐3HP) or PLBP from unrelated carbon source. Enzymes encoded by each gene are described below: phaA, β‐ketothiolase; phaB, NADPH‐dependent acetoacetyl‐CoA reductase; ldhA, lactate dehydrogenase; pct, propionyl‐CoA transferase; dhaB, glycerol dehydratase; dhaT, 1,3‐propanediol dehydrogenase; aldD, aldehyde dehydrogenase; pcs', propanoyl‐CoA synthetase; phaC, PHA synthase from Pseudomonas stutzeri strain 1317 (PhaC1_Ps) with 4‐point mutations at E130D, S325T, S477G and Q481K.

Figure 2

Alignment of phaC1 _Ps and phaC2 _Ps with previous LA polymerizing enzymes reported (Taguchi et al., 2008; Yamada et al., 2010; Yang et al., 2011). phaC1 _Ps and phaC2 _Ps from Pseudomonas stutzeri were aligned with reported LA polymerizing enzymes which were phaC1 _Ps61‐3 from Pseudomonas sp. 61‐3 and phaC1 _Ps6‐19 from Pseudomonas sp. MEBL6‐19. Several constitutive sites were found dominating the LA polymerizing capacity (in the red frames). Abbreviations: phaC1_Ps, Pseudomonas stutzeri phaC1; phaC2_Ps, Pseudomonas stutzeri phaC2; phaC1_Ps61‐3, Pseudomonas sp. 61‐3 phaC1; phaC1_Ps6‐19, Pseudomonas sp. MEBL6‐19 phaC1; E, glutamic acid; D, aspartic acid; S, serine; F, phenylalanine; Q, glutamine. Five mutation sites on phaC1 _ps (phaC2 _ps), including E130D, S325(326)T, F392(393)S, S477(478)G and Q481(482)K, were selected based on the alignment result with the LA polymerizing enzymes.

Figure 3

NMR analysis of PLBP terpolyester. ¹H NMR spectra (A) and ¹³C NMR spectra (B) of random copolyester P(3HB‐co‐3HP‐co‐LA) containing 90.41 mol% 3HB, 7.78 mol% 3HP and 1.81 mol% LA, respectively, and its expanded ¹³C NMR spectra of carboxyl carbon [B(1), P(1), A(1)] area (C) and methylene regions (D) in the terpolyester. B, P and A refer to 3HB, 3HP and LA; numbering schemes were the same as molecular formulations of polyester indicated in the inset in (A). N*M represents the interaction of monomer N and M. “i” indicates “isotactic.” Chemical shifts are in ppm and tetramethylsilane (TMS) was employed as an internal chemical shift standard.

Figure 4

The concentration of glucose, glycerol and lactate in shake‐flask studies. E. coli S17‐1 ΔpflA harbouring two plasmids of pLA' and p3HP2p was cultured in LB medium supplemented with 20 g L⁻¹ glucose, 10 g L⁻¹ glycerol and 2 g L⁻¹ lactate. Blue line, glucose; red line, glycerol; green line, lactate. Error bars represent the standard deviation of experiments conducted in triplicates.