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Biosynthesis, Biodegradation And Molecular Evolution Of Polyhydroxyalkanoates In Escherichia Coli

Posted on:2010-03-11Degree:DoctorType:Dissertation
Country:ChinaCandidate:Q WangFull Text:PDF
GTID:1100360302483566Subject:Microbiology
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Polyhydroxyalkanoates(PHAs) are synthesized by micro-organism within the cell as a kind of bio-polyester.PHAs not only possess the properties as well as traditional petrochemical plastics but also are biodegradable,biocompatible,piezoelectric, optically active etc.these features makes them suitable for many applications in the packaging industry,medicine,pharmacy,agriculture,food industry,as raw materials for enantiomerically pure chemicals and in the production of paints.PHA can be obtained from renewable resources;hence,there has been a tremendous amount of commercial interest in these polymers.In contrast to bulk uses,PHAs have also been established as excellent materials in several niche applications,in particular for medical purposes,because PHAs are generally biocompatible with mammalian tissue and are resorbed at a slow rate;PHA can be used to develop tissue engineering scaffolds,which support cell growth and are degraded after implantation,leaving a viable tissue.Thus nowadays more and more efforts have been made for the applications in medical and tissue engineering.In conclusion,efforts in biopolymer research must be made to develop and enhance PHA production processes at low cost levels,and the development of PHA with better properties is necessary. PHA has been intensively studied for seventy years since the first PHA, poly(3-hydroxybutyrate)(PHB),was discovered in Bacillus megaterium by the French scientist Lemoigne in 1926.In the following years,research on purification of PHB granules from the cell and other forms of PHAs e.g.PHBV's production,and in succession investigations with other monomers,genes,microorganisms,metabolic pathways and the potential use of these biopolymers was realized.Recent research has focused on the use of alternative substrates,novel extraction methods,genetically enhanced species and mixed cultures with a view to make PHAs more commercially attractive.When we engineered Escerichia coli to produce different kind of PHAs,we found that Escerichia coli could not only accumulate approximate 90%PHB of the cell dry weight without any harmful effect on cell growth but also produce succinic acid and other organic acid.Thus we were devoted to investigate the mechanism that PHB can benefit the host Escerichia coli for survival under stress conditions such as cold shock, pH and cell density.A wide variety of microorganisms are able to accumulate polyhydroxyalkanoates (PHAs) as intracellular carbon/ energy storage compounds or reducing power for coping with changing,often oligotrophic environments.Various PHAs,as well as the best-known poly 3-β-hydroxybutyrate(PHB),were found to be accumulated and degraded as required under environmental conditions by most natural PHAs producing bacteria.When the environment is sufficient with carbon source or the C/ N ratio is quite high(>20),the PHAs accumulation is much faster than degradation. While facing different stresses,such as low nutrient availability and detrimental physical,chemical,or biological factors,these bacteria begin to mobilize PHAs to conquer those unfavorable environments.This paper we reported for the first time that a stress-induced system enabled E.coli,a non-PHB producer,to mobilize PHB in vivo by mimicking natural PHB accumulation bacteria.In this study,the successful expression of PHB biosynthesis and PHB depolymerase genes in E.coli was confirmed by PHB production and 3-hydroxybutyrate secretion.To confirm the capability of 3HB utilization by E.coli,an in vitro experiment of 3-hydroxybutyral-CoA synthetase activity measurement was performed.The consumption of 3HB as carbon and energy source when CoA and ATP existed in the reaction mixture indicated that the complete PHB mobilization in engineered E.coli was realized and 3HB can serve as an energy material.Starvation experiment demonstrated that the complete PHB mobilization system in E.coli served as an intracellular energy and carbon storage system,which increased the survival rate of the host when carbon resources were limited.Stress tolerance experiment indicated that E.coli strains with PHB production and mobilization system exhibited an enhanced stress resistance capability.This engineered E.coli with PHB mobilization has a potential biotechnological application as immobilized cell factories for biocatalysis and biotransformation. So far,PHAs with more than 150 types of monomers have been synthesized, ranging from stiff plastics to flexible elastomers,which properties are mainly depending on the monomer composition and molecular weight.PHB is a highly crystalline material which is stiffer and more brittle than synthetic plastics, Medium-chain-length(mcl)-PHAs are elastic polyesters but hard to manipulate, therefore,their industrial applications are limited.The copolymers consisting of both scl and mcl-PHAs greatly improve the flexibility and toughness owing to its diverse monomers.To a considerable extent,the substrate specificity of the PHA synthases determines the composition of the accumulated PHA.This section is aimed at obtain a broad substrate specificity hybrid PHA synthase which will posseses the substrate specificity of both parent PHA synthases and thus can incorporate both scl and mcl-hydroxyalkanyl-CoA precursors into copolymers. This paper introduced an incremental truncation hybrid enzyme library to create a broad substrate specificity hybrid enzyme.For hybrid or chimeric enzyme construction,it is difficult to predict exactly which fusion-points in domain swapping will produce an active hybrid enzyme.Thus,Incremental truncation was thought as a powerful strategy in the engineering of novel biocatalysts.In this study,we created a hybrid library with PHA synthase gene from Ralstonia eutropha as C-terminus and the gene from Pseudomonas aeruginosa as N-terminus using incremental truncation method.First,a recombinant strain harboring two PHA synthase genes was constructed,the polymer accumulated in this strain is a blend of PHB(about 90%) and PHA(about 10%).Then the incremental truncation was used to create a phaClpa-phbCRe hybrid protein library.As revealed by PHB production in recombinant E.coli,25 hybrids with different length were functional.The truncated mutants of PhbCRe showed a gradually decreased in vivo enzyme activity trends along with the increased truncation degree of PhbCRe.High degree truncation of PhbCRe at N-terminus can be complemented by N-terminus of typeⅡPHA synthase-PHA synthase from Pseudomonas aeruginosa.Importantly,three of the hybrids were found to have altered product specificity.They can produce P(3HB-co-3HA) with different monomer composition,which will broaden the variation of engineered PHA synthase. Accordingly,these results suggested that the N-terminal sequence of PHA synthase contributed to both enzyme activity and product specificity.The incremental truncation provides us a novel method to generate functional PHA synthase with desired properties and to study the reaction mechanism of the enzyme.PHB synthase from Ralstonia eutropha purified from recombinant E.coli cells exists in aqueous solution in both monomeric(single subunit) and homodimeric(two subunits) forms in equilibrium.Several lines of evidence suggest that the homodimer is the active form of the synthase.The initial mechanistic model for the polymerization reaction proposed that two different thiol groups form the catalytic site.The cysteine at 319 has been shown to provide one thiol group that is involved in the covalent catalysis,but a second thiol group on the same protein molecule has not yet been identified.In this paper,we confirmed the presence of synthase dimer in reaction by using complement GFP method based on reassembly of dissected fragments of green fluorescent protein fused to interacting proteins.The system used in this study consists of two plasmid vectors for indenendent expression of fusions with N-and C-terminal fragments of GFP,and allows for simple visual detection of protein-protein interactions.We demonstrate that a dimer synthase that has initiated the polymerization reaction(primed synthase) when the precursor is present,which means the dimer form of PHB synthase is significantly more stable against dissociation than the unprimed(unreacted) dimer synthase.Further study should be carried out to detect whether PHB granule formation begins at the inner site of the cytoplasmic membrane which is different from previous assumptions that PHB granule formation occurs randomly in the cytoplasm of PHB-accumulating bacteria.
Keywords/Search Tags:polyhydroxyalkanoate, PHB mobilization, Escerichia coli, PHA copolymer, enzyme molecule alteration
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