P[(R)-2-hydroxybutyrate] [P((R)-2HB)] is an aliphatic polyester analogous to poly(lactic acid) (PLA). However, little has been known for its properties because of a high cost of commercially available chiral 2HB as a starting substance for chemical polymer synthesis. In this study, P[(R)-2HB] and P[(R)-2HB-co-(R)-lactate] [P((R)-2HB-co-(R)-LA)] with a new monomer combination were successfully synthesized in recombinant Escherichia coli LS5218 from less-expensive racemic 2HB using an R-specific polyester synthase. The cells expressing an engineered polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3 and propionyl-CoA transferase from Megasphaera elsdenii were grown on LB medium containing 2HB and glucose in a shake flask and accumulated up to 17 wt % of P[(R)-2HB] with optical purity of >99.1%. In addition, the same cells cultured in a jar-fermentor produced P(86 mol % 2HB-co-LA) copolymer. Notably, the molecular weights (Mw) of P(2HB) (27000) and P(2HB-co-LA) (39000) were 2- and 3-fold higher than that of P(2HB) previously synthesized by chemical polycondensation. P(2HB) was processed into a transparent film by solvent-casting and it had flexible properties with elongation at break of 173%, which was contrast to the rigid PLA. Regarding mechanical properties, P(2HB-co-LA) was tougher but less stretchy than P(2HB). These results demonstrated that P(2HB) has useful properties and LA units in 2HB-based polymers can act as a controllable modulator of the material properties. In addition, P[(R)-2HB] was efficiently degraded by treatment of Novozym 42044 (lipase) but not Savinase 16L (protease), indicating that the degrading behavior of the polymer was similar to that of P[(R)-LA].
Glycolate(GL)-based polyesters were for the first time produced in the recombinant Escherichia coli fatty acid -oxidation pathway reinforcing mutant LS5218, using extracellularly added GL as a monomer precursor. Cells expressing a Ser325Thr/Gln481Lys mutant of polyhydroxyalkanoate synthase (PhaC1STQK) from Pseudomonas sp. 61-3, propionyl-CoA transferase from Megasphaera elsdenii and enoyl-CoA hydratase from Pseudomonas aeruginosa grown on GL and dodecanoate were found to produce novel copolymers of GL with 3-hydroxyalkanoates (3HAs) (C 4 -C 12 ), P(GL-co-3HA), with a weight-average molecular weight of 34 000. The 1 H NMR analysis of the copolymer revealed the incorporation of GL units into the polymer chain. This result demonstrates that PhaC1STQK polymerized glycolyl-CoA as a monomer substrate. Additionally, the novel lactate(LA)-based polyester P(LA-co-3HA) was produced by substituting GL with LA, indicating that the method is versatile and allows the production of a variety of biopolymers.
Keywords:poly (glycolide) poly (lactide) lactate-polymerizing enzyme biobased plastic polyhydroxyalkanoate
We constructed the broad-host-range human epidermal growth factor (EGF) secretion plasmid pKSEGF2 by inserting the Escherichia coli tac promoter, the signal sequence of Pseudomonas stutzeri amylase, and the synthesized EGF gene into the broad-host-range vector pKT230. E. coli JM109 carrying pKSEGF2 secreted EGF into the periplasm and the culture medium under the control of the tac promoter. Pseudomonas aeruginosa
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