Microbial production of various lactate containing polyhydroxyalkanoates by systems metabolically engineered Escherichia coli

Abstract

Increasing concerns about limited fossil fuels and environmental pollution have great attention to develop sustainable production of various chemicals from renewable resources. The petroleum-based plastics are world widely used in enormous applications such as packaging, automobiles because of their low cost, ease of manufacture, versatility and imperviousness to water. Polyhydroxyalkanoates (PHAs), bacterial polyesters, are one of promising alternatives owing to its potential to be used as performance plastics and superior to other plastics in many aspects, including its biodegradability, biocompatibility for potential biomedical applications. In this study, the important synthetic biopolymers used in biomedical field, poly(lactate-co-glycolate) [PLGA] and its various copolymers were produced by systems metabolically engineered E. coli by adapting bacterial PHA synthesis system. Poly(lactate-co-glycolate) (PLGA) is a random copolymer of lactic and glycolic acids and is widely used biodegradable and biocompatible polymer. It is also FDA-approved for various medical applications such as medical sutures, scaffolds and drug delivery carrier. To develop one-step fermentative production of PLGA by engineered E. coli, first the novel metabolic pathway was constructed: It consists of evolved propionyl-CoA transferase and evolved polyhydroxyalkanoate (PHA) synthase in which lactate and glycolate are converted into lactyl-CoA and glycolyl-CoA, respectively, and then these hydroxyacyl-CoAs are polymerized into PLGA. Next, to produce PLGA from unrelated carbon sources, we employed two glycolate biosynthesis pathway, glyoxylate shunt and Dahms pathway while E. coli can produce lactate natively. The metabolic engineering strategies employing glyoxylate shunt and Dahms pathway were described in chapter 2 and 3, respectively. The resulting PLGA producing E. coli strains were applied for generating diverse forms of PLGA is shown by the production of copolymers containing 3-hydroxybutyrate, 4-hydroxybutyrate or 2-hydroxyisovalerate (Chapter 4 and 5). Finally, to enhance the PLGA production, the metabolic flux between Dahms pathway and E. coli native xylose metabolic pathway (pentose phosphate pathway) was optimized by using synthetic promoters with different strengths (Chapter 6). In conclusion, through this thesis, the biosynthesis of non-natural polymer, PLGA was developed for the first time based on microbial PHA biosynthesis. This bacterial PLGA producing system also can be adapted to other various copolymers production. The synthetic biology strategies could further optimize the metabolic flux resulting in enhanced production of PLGA and its copolymers. The systems metabolic engineering strategies used here is meaningful in that it proposes a platform strategy, which can be further utilized in the development of numerous useful polymers and also other chemicals.