Pathway Construction And Metabolic Engineering For Production Of Short-chain-length Polyhydroxyalkanoates-PHB And PHBV In Recombinant Escherichia Coli

Polyhydroxyalkanoates (PHAs), macromolecule-polyesters naturally produced by many species of microorganisms, are being considered as a replacement for conventional plastics. PHAs not only possess the properties as well as traditional petrochemical plastics but also are biodegradable, biocompatible, piezoelectric, optically active etc. Over the past years, PHAs, particularly poly-3-hydroxybutyrate (PHB) and copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV) have been used for packaging, coating, tissue engineering applications. In conclusion, efforts in biopolymer research must be made to develop and enhance PHAs production processes at low cost levels, and the development of PHA with better properties is necessary.PHAs can be divided into two broad classes based on the size of monomers incorporated into the polymer. Short-chain-length PHAs (scl-PHAs) consist of monomer units of C3 to C5; medium-chain-length PHAs (mcl-PHAs) consist of monomer units of C6 to C14. The large diversity of monomers found in PHAs provides a wide spectrum of polymers with varying physical properties. The homopolymer PHB is a relatively stiff and brittle bioplastic, which is of limited use. PHA copolymers composed of primarily 3HB with a fraction of longer chain monomers, such as 3HV,3HHx or 3HO, are more flexible and tougher plastics.PHB and PHBV have been produced on a commercial scale since 1970s. Promising strategies involve genetic engineering of microorganisms to introduce production pathways. This challenge requires the expression of several genes along with optimization of PHA synthesis in the host. Although excellent progress has been made in recombinant hosts, the barriers to obtaining high quantities of PHAs at low cost still remain to be solved. The aim of this paper is to reduce cost of PHB and PHBV production through pathway construction and metabolic engineering for scl-PHAs synthetic pathway in recombinant Escherichia coli.PHB is the best known PHA and has been studied intensively as a model product in the development of fermentation strategies. PHB production cost can be reduced by several means, including the use of cheap substrates or the enhancement of product yield, e.g., by using recombinant E. coli. E. coli has a number of advantages as a host for PHB production. These include a wide range of utilizable carbon sources, fast growth with a high level of productivity, the absence of an intracellular depolymerization system, easier purification due to fragile cells and large accumulated granules, as well as well-understood genetics and biochemistry. In this study, we compared PHB accumulation of different E. coli strain and chose recombinant E. coli DH5a as the PHB producer. We investigated the effect of oxygen supply on PHB synthesis in recombinant E.coli, and confirmed that PHB fermentation process in E. coli could be divided into two phage:(?) an active growth phase during which PHB content is kept relatively constant at a low level and (?) an active PHB synthesis phase during which PHB is actively accumulated with a concomitant increase of PHB content.The cost of the carbon source contributes significantly to the overall production cost of PHB. Then we investigated the PHB accumulation from glucose, xylose, arabinose, lactose and fructose in recombinant E. coli. We found that recombinant E. coli could produce PHB efficiently with each of these sugars as carbon source. While, Many cheap renewable carbon sources, such as corn cob hydrolysates, are mixture of sugars. Due to CCR, E. coli displays sequential sugar consumption when it is grown in media derived from mixture of sugars. Based on the fermentation analysis of E. coli strains and cheap renewable resources suitable for PHB production, we constructed aptsG mutant of E. coli DH5a AptsG. Application of E. coli DH5a AptsG/pBHR6 & mutant, we could product PHB efficiently from cheap renewable sugar mixture by the simultaneous consumption of different sugars. Batch fermentation at lab scale showed that E. coli DH5a AptsG/pBHR68 was able to produced PHB from corn cob molesses up to 84.6% of cell dry weight in 32 hours; meanwhile, the cell dry weight reached 8.24 g/L. Subsequently, we tried to produce PHB from Jerusalem artichoke sugar in recombinant E. coli, and yielded a high concentration of cell with 54.2% PHB content.PHBV is less brittle and less crystalline than PHB homopolymer, making it more suitable for commercial applications. The control of copolymer composition is important because the physical and mechanical material properties of the copolymer depend on the fraction of 3-hydroxyvalerate(3HV). To date, most attempts at producing PHBV copolymer with different compositions in recombinant E. coli have used the same strategy as that used with R. eutropha; that is, varying the propionate concentration to vary the 3HV fraction. However, many bacteria showed a low 3HV yield with propionate(Y3HV/Prop).The low yield may result from the activation of external propionate and the alternative pathways in propionate metabolism.In E. coli, prpBCDE operon encodes the enzymes for propionate activation and metabolism (also known as 2-methylcitrate pathway), allowing growth on propionate as a sole carbon and energy source. Transcription of the prpBCDE operon is down-regulated by glucose or glycerol due to the catabolite repression caused by phosphoenolpyruvate-dependent phosphotransferase system (PTS). To determine the influence of carbon sources on propionate metabolism and PHBV synthesis, we used glucose, glycerol and xylose as carbon source respectively to produce PHBV in recombinant E. coli DH5a/pBHR68. We found that E. coli showed higher ability to metabolite propionate and produce PHBV with xylose as carbon source than that with glucose or glycerol as carbon source. Furthermore, the experimental results of DH5??ptsG/pBHR68 also confirmed the catabolite repression in E. coli. To avoid the catabolite repression in PHBV production, a few researchers overexpressed prpE gene, which encodes propionyl-CoA synthetase. In this study, we found acetyl-CoA synthetase, encoded by acs, could also activate propionate and E. coli produced PHBV copolymer with higher 3HV fraction when it was overexpressed. Although E. coli produce PHBV copolymer with higher 3HV fraction, E. coli showed poor Y3HV/Prop about 0.15 g g-1, which is a small percentage of the maximum theoretical value of 1.35 g g-1. The low Y3HV/ProP resulted from the alternative pathways initiating complete oxidation of propionate in E. coli. To block the endogenous propionyl-CoA catabolism, we deleted the metabolic pathways diverted to MCC cycle and/or TCA cycle. To our surprise, deletion of prpC gene did not improve the 3HV fraction significantly, while deletion of scpC gene greatly improved the 3HV fraction in the copolymer. This result suggested that the metabolic flux from propionyl-CoA to methylmalonyl-CoA pathway was much more than that to MCC cycle in E. coli. Phosphate acetyltransferase (encoded by pta) catalyzes both acetyl-CoA and propionyl-CoA to acetate and propionate, respectively. To reduce the propionate formation, pta was deleted in mutant QW102. However, deletion of pta did not improve the 3HV fraction in the copolymer. It was notable that Y3HV/Prop of QW102/pBHR68 reached up to 0.64 g g-1, approximately five times higher than the Y3HV/Prop reached by the wild type DH5a/pBHR68.The prohibitively high price of PHBV hinders industrialization of the copolymer. A major factor of this condition was that propionate, which was activated to form the propionyl-CoA precursor of 3HV, is expensive to produce industrially and is considerably more costly than glucose. A more economical alternative is to produce propionyl-CoA from an inexpensive, unrelated carbon source.In previous experiments, we found that recombinant E. coli DH5a/pBHR68 produced PHBV with small amount of 3HV fraction even when it was not supplied with the precursor substrate, propionate. This suggested that propionyl-CoA and/or propionate can be generated in vivo through certain metabolic pathways from glucose. In this study, based on the analysis major propionyl-CoA origin in recombinant E. coli, we developed a PHBV biosynthesis pathway from single unrelated carbon source via threonine biosynthesis in E. coli. Firstly, we found that the limit step of propionyl-CoA formation was threonine deamination. Overexpression of ilvA effectively draws carbon flux towards the synthesis of 2-ketobutyrate under aerobic conditions. Therefore, we first overexpressed native ilvA from E. coli. Overexpression of ilvAEC achieved PHBV production with doubled 3HV fraction in the copolymer. To further improve the deamination efficiency, we tried the threonine deaminase from other bacteria and found overexpression of ilvACG could improve the 3HV fraction in the copolymer more than 10 times, from 0.43 mol% to 5.09 mol%.Afterwards, we removed the feedback inhibition of threonine by mutating and overexpressing the thrABC operon in E. coli. Finally, we constructed a series of strains and mutants, which were able to produce PHBV copolymer with varied monomer compositions. The highest 3-hydroxyvalerate fraction of 17.5 mol% in the copolymer was obtained by the mutant QW103/pHB-ilvA/pCL-thrABC. As a result, the PHBV production via this strategy not only provided PHBV copolymer with different properties at low cost, but also avoided complex control strategy when propionate was co-fed. Further improvement of the host strains should lead to a perspective and practical application.