| Isoprene is a kind of important C5 platform chemical, as well as the basic constitutional unit of terpenoid. As an important chemical material, it may be used to compound biological rubber which is extremely similar to natural rubber. In addition,isoprene may also be applied to compound all kinds of terpenoid which is widely applied to such fields as pesticide, medicine, perfume and adhesive. As the petroleum resource is becoming more and more exhausted, the bio-based C5 platform chemical,whose development does not relay on the petroleum resource, has become the strategic demand for realization of sustainable development of economic society.In the nature, the synthetic pathway of both isoprene and terpenoid are similar Namely, the –C-methyl-D-erythritol-4-phosphoric acid pathway and the mevalonate pathway.This research has successfully structured and produced isoprene engineering bacteria by bringing outsourced MVA pathway into colibacillus. It also has provided basis for isoprene’s large-scale biosynthesis by optimizing MVA pathway with synthetic biology and metabolic engineering. The main researches are as follows:1. Establishment of gas chromatographic method to measure isoprene productionThe standard of isoprene is regarded as the quantitative criteria. Agilent Techbologies 7890 A GC system type gas chromatograph(GC) is adopted by the system, with chromatographic column of HP-55% phenyl methyl siloxan(30 m×320μm×0.25 μm), as well as flame ionization detector. The temperature in the gasification room is 50℃ and that of the column box and detector is 240℃ and 280℃respectively. The standard curve of isoprene production and detection peak is obtained.2. Cloning and expression of isoprene synthase in different speciesClone the isoprene synthase genes from the genome of Mongolian oak, oriental white oak, sawtooth oak, Liaodong oak, English oak and polar. Structure the genes to the expression vector of p BAD-His B, and get the recombinant bacteria by co-transforming them, together with pYESs and pSPMIc, into Ecoli BW 25113. The objective protein is successfully expressed by auto-induction expression, and the isoprene production of BW-01 bacteria is 451.4 mg/L.3. Production improvement of isoprene resulting from optimization of expression vector Regulate the expression of key gene in the host bacteria by taking measures for 3expression vectors in the recombinant bacteria, such as adding rare codons AGA,adopting the promoters with fixed strength, as well as changing the copy number of expression vector, so as to change the isoprene production of recombinant bacterial strain. The isoprene production of optimum bacterial strain reaches 1341.6 mg/L,which has been improved for 197.2% than that before optimization.4. Production improvement of isoprene resulting from transformation of genetic background On the basis of λ-Red recombination technology, structure a part of expression genes into the genome of host bacteria, meanwhile kick off the unnecessary genes in the host bacteria genomes, and get new bacteria strain BW-13 to produce isoprene.The detected maximum isoprene production is 1267.3 mg/L, 60% of biomass more after induction than before.Colibacillus is regarded as the expression host in this research. The recombinant bacteria strain to produce isoprene by structuring outsourced MVA metabolic pathway into expression vector and transferring into the host. In addition, the whole recombinant system is set as the starting point. The protein expression quantity of the whole system is getting in a relatively balanced state by optimization, so as to maximize the system function of recombination and greatly improve the synthetic precursor of colibacillus terpenoid, as well as isoprene production of bacterial strain.The MVA pathway structured and optimized in this research may also be applied to synthesis of other terpenoid, and provide favorable reference for future research in biosynthesis. |
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