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Biosynthesis of a Novel Terpolymer P(LA-co-3HB-co-3HP) from Unrelated Carbon Source

Ren, Y., MOE Key Lab of Bioinformatics, Department of Biological Science and Biotechnology, School of Life Science, Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China

Polylactate (PLA) is currently commercially 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. PLA is turned into highly valuable materials for biomedical, food, and general purpose applications, but its poor ductility and heavy metal residue limited the application. Meanwhile, polyhydroxyalkanoates (PHA) are diverse polyesters synthesized by various bacteria as intracellular carbon and energy storage compounds. As bio-renewable and biodegradable materials, the diversity of PHA units confers to the different physical properties. So copolymerization of PLA with other hydroxyalkanoate (HA) units only using biological method is one of the effective methods to improve the physical properties of PLA and avoid the heavy metal contamination.

In this study, we established a mutant of PHA synthetase which could incorporate LA as well as other 3-HAs at the same time. We synthesized different PLA-PHA copolymers in recombinant Escherichia coliaiming at improving the properties of PLA and reducing the cost of copolymers by utilizing low cost carbon sources. The results obtained are summarized below:

The substrate specificity of the PHA synthases is the most important factor determining the monomer structures whether that could be incorporated into PHA or not. To introduce LA into the copolymer, we constructed mutated type II PHA synthetase derived from Pseudomonas stutzeri phaC1Ps, which has a versatile substrate specificity, referring to the former reports about PLA polymerization. The mutant phaC1Ps(E130D S325T S477G Q481K) could polymerize LA into PLA with precursor LA-CoA. Gene pct converting LA into LA-CoA and genes related to P3HB synthesis were co-expressed with the PHA synthetase mutant to produce P(3HB-co-LA). By optimizing the metabolite patheway and host strain, we finally produced P(3HB-co-23mol%LA) in recombinant E. coli. It is the most efficient PLA polymerizing enzyme in our system when we compared phaC1Ps(E130D S325T S477G Q481K) with those phaCs reported before.

P(3HB-co-LA), as a classic PLA-PHA copolymer, has been widely studied. The physical properties of the homopolymers PLA and P3HB are both high strength and low ductility. P3HP has an opposite character that it possesses exccellent mechanical strength, tensile stength and elongation break. To synthsize a PLA-PHA coplymer with an enhanced ductility, we constructed an P3HP synthetic pathway and combined with PLA and P3HB synthetic pathway. P(LA-co-3HB-co-3HP) terpolymer was produced from unrelated carbon sources firstly. The nuclear magnetic resonance (NMR) analysis revealed that the random terpolyester P(3HB-co-3HP-co-LA) contained 90.41 mol% 3HB, 7.78 mol% 3HP and 1.81 mol% LA respectively. In addition, the PLBP terpolymer was produced from two types of unrelated carbon sources, and the monomer ratio of PLBP terpolymer was variable, by regulating the proportion of glycerol and glucose and modifying the metabolism of the host strain. By analyzing these terpolymers, we characterized a new PLA-PHA copolymer with better mechanical porperties.

In conclusion, we synthesized new PLA-PHA terpolymers with variable components from unrelated carbon source which will promote the developemnt of LA based copolymer and its application.

Key Words: PHA; PLA; PHA synthetase; P(3HB-co-3HP-co-LA); unrelated carbon source; Escherichia coli