Study Of Synthesis Mevalonic Acid For Escherichia Coli Of Engineering

Mevalonic acid(MVA) as a key precursors of isopentenylpyrophosphate(IPP) synthesized, which can indirectly synthesis various isoprenoid compounds by MVA pathway, and widely use in food, medicine and chemical industry. Otherwise, it has an increasing market demand. However, the productions are subject to various problems by traditional methods, such as scarcity of raw materials, high cost, pollute environment and not suitable for large-scale industrial production. Biological synthesis of MVA use glucose and acetic acid as raw material, which were obtained from degradation of biomass and industrial effluents, respectively. Currently, with the advantages of resource sustainable and environment protecting, it has become the research focus. In this paper, the biosynthesis of MVA synthesis system was established by choosen industrial microbial strains Escherichia coli as a biocatalyst, glucose and acetate as the substrate, respectively.Fed-batch technique for high cell density fermentation of MVA production by recombinant Escherichia coli. YJM16 was studied. The result showed that the dry cell weight reached 54.48 g·L-1, the production of MVA was 40.43 g·L-1 and the yield was 20.2 %. The kinetics model was proposed by using the Logistic equation for cell growth, the Luedeking Piret equation for MVA production and the Luedeking-Piret-like equation for consumption of glucose as substrate. The result showed that high density fermentation kinetic of production of MVA by recombinant Escherichia coli. could be well expressed by these models. The goodness-of-fit scores R2 of models were 0.9319, 0.9578 and 0.9751, respectively.Overexpression of acs, maeB, sfcA, yfcC, pckA and ppsA gene, build the recombinant strains using acetate as carbon source, shake flask fermentation and select the optimal strain. We finded that overexpression ACS in favour of use acetic acid. Overexpression MAEB, SFCA, YFCC, PCKA and PPSA, however no enhancement of MVA production was observed. The result suggested that the level of NADPH and gluconeogenic pathway are not the main limiting factor. The optimal strain is FHR-2, the production of MVA was 368 mg·L-1 and the yield was 6.9 %. Compared with the original strain, 31.4% and 1.47% increased, respectively.5 L fermenter fermentation: using acetic acid as carbon source, optimization fermentation conditions. During cultivation, glucose as carbon source(20 g·L-1), pH was controlled at 7.0 with NH3·H2O and H2SO4, pO2 20 %, temperature 37 oC. When the initial glucose was depleted, reduced the temperature to 32 oC and induction by IPTG(final concentration was 0.5 mM), ammonium acetate solution(4 g·L-1·h-1) was added. After induction for 60 h, the concentration of bacteria OD600 nm reached about 20, and the concentration of MVA about 8.63 g·L-1, the yield was 29.5 %(theoretical yield 81.67%).