(The) enhanced biosynthesis and characterization of lactate containing PHAs by metabolically engineered recombinant Escherichia coli

Abstract

Our concerns are increasing about environmental pollution by solid wastes which are generally composed of non-biodegradable materials such as households and biomedical sources. Thus, there has recently been much interest in bio-based process for the production of industrial valuable materials from renewable resources. In this study, two independent researches have been carried out to produce biopolymers (PLA, PLGA) from genetically engineered microorganisms. Firstly, JLXF5 strain was engineered to express pPhaP437540 or pPhaP619C1437-PCT540 enzyme for enhanced production of PLA homopolymer. In fed-batch culture, the lactate fraction in the polymer was increased from 8.29 to 11.57 mol% and PHA content was increased from 10.52 to 18.81 wt% in the presence of the Phasin. Secondly, in order to produce PLGA, JLXF9 strain was constructed from JLXF7 by deleting the GlcDEFG genes. Poly(lactic acid-co-glycolic acid) [PLGA], the random copolymer of lactic acid (LA) and glycolic acid (GA), are biodegradable and biocompatible thermoplastic biopolymers with a broad range of industrial and biomedical applications such as drug delivery carriers. PLGA have received considerable interest because this is used in a host of Food and Drug Administration (FDA)-approved biopolymer. When JLXF9 strain harboring pTac15k-YcdW and pPs619C1437-CpPCT540 was cultivated by fed-batch fermentation, it was produced P(LA-co-GA) copolymers with 33 mol% of GA mol fraction and 41.56 wt% of PHA content. The PLGA copolymer of 24,000 Da was successfully produced from the final JLXF9 strain. In terms of industrial applications, the strain was further engineered to be capable of producing PLA from a cheaper carbon source than glucose, sucrose. Sucrose utilization pathway was constructed by introducing the Mannheimia succiniciproducens SacC gene encoding β-fructofuranosidase. The recombinant E. coli MKWL6 strain was able to produce PLA homopolymer with polymer content of 4.6wt% in the medium containing 20g/L glucose, and P(46.95mol% LA-co-53.05mol% 3HB) with polymer content of 41.84wt% in the medium supplemented with 20g/L glucose and 2g/L 3hydroxybutyreate (3HB). When overexpressing the M. succiniciproducens SacC gene and harboring $PhaC1437_{Ps6-19}$, $Pct540_{Cp}$, the recombinant E. coli MKWL6 strains could produce PLA homopolymer with polymer content 16.94 wt%, and that increased 2.86-fold compared with wild type W3110. These studies demonstrate that microorganisms can be engineered to produce industrially interesting polymers.