The Production Of PHB-co-PHBV Block Copolymer By 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. Therefore, PHAs have a bright future in many application fields.PHB and PHBV are the members of the short-chain-PHA family, and also the most wildly studied PHAs. PHB is the homopolymer of3-hydroxybutyrate (3HB), while PHBV is the random copolymer of3HB and3HV (3-hydroxyvalerate). Although the processing performance of PHBV is greatly improved than PHB, it still faces the isodimorphism problems, which making processing much more difficult. The block copolymer is not with this trouble as its special structure. But the research of block copolymer is one the most meanfui and challenge work. The work of this paper is to product PHB-co-PHBV block copolymer in recombinant E.coli.The first part of this paper is to construct the basic recombinant strain for block copolymer production. One of our earlier researches indicates that recombinant E.coli can put out PHBV with high3HV fraction from unrelated carbon sources. So we modification the pathway of threonine by mutating the aspartate kinase thrA and replaceing the promoter. Consequently, the thrA transcriptional level and the threonine production of the recombinant strain XL101improved11times and9.5times, respectively. After that, we intergrated as much as9copies of PHBV operons into the genome of the recombinant strain by using the Flp/FRT site specific recombination and named the strain with XL102. Thus, the basic strain for block copolymer production was ready. Then we checked the PHA production of strain XL102by expression the ilvACGcg or not. It turned out that,the recombinant strain XL102can product PHB without ilvACG, and can product PHBV with ilvACG-This is Consistent with our expect.The second part of this paper is to construct a genetic circuit to control the expression of ilvACG-We combinated the synthetic biology and metabolic engineering, introducing the principles of electrocircuit design to the gene expression controlling. The modules of the circuit include the quorum sensing system luxRI from Vibrio fischeri and the aiiA gene from Bacillus thuringiensis. Among the construction stage we used sfGFP as the report gene and modified the circuit with several factors, including the intensity of the promoter, the expression level of luxR, protein degradation efficiency, as well as transmission efficiency of AHL. Finally, we detected the flourescence oscillation when cultured the cells in shake flask, which improved the transmission of autoinducer AHL. All these work has laied a foundation for the production of PHB-co-PHBV block copolymer.