Metabolic Engineering Of Escherichia Coli For The Production Of Fumaric Acid

In this paper, we first knockout frdB genes in SPUEC101 produced succinic acid which catalyze the succinic acid to fumaric acid to get strain SPUEC103, then in this paper, its growth characteristics was studied and its metabolic pathway was modified, this paper try to amplify pyruvate carboxylase pyc and knockout ptsG gene in order to improve the production of fumaric acid, the study found that expressing pyruvate carboxylase can improve the production of fumaric acid, knockout ptsG gene of fumaric acid production was slightly increased, but lower strain rate of the growth rate and utilization of glucose. Experiments prove that knockout frdB and ptsG, at the same time expressing the bacillus subtilis pyruvate carboxylase combination is can produce fumaric.1.Get a strain SPUEC101 of producting higher succinic acid.In this article, through early bromocresol green flat screen, succinic acid qualitative semi-quantitative screening and HPLC final selection, set up a simple and rapid, Economical and practical scientific selection method of succinic acid producing strains, and determined by this method the strains of succinic acid production up to 5.16 g/L strains, named SPUEC101.2.Successfully construct SPUEC103 (ΔfrdB)The mutants of Escherichia coli deficient in frdB showed slower growth,together with exploiting less glucose.At the same time,the defection of frdB could change the yields of succinate and fumarate,the yield of succinate and fumarate was the highest in LB media supplemented with 30 g/L glucosethe,the yield of succinate of dual-phase fermentation decreased from 24.6% to 15.4%.The portion of fumarate and malate in SPUEC 103 increased,the final concentration of the fumarate and malate was 0.182±0.002 g/L and 0.023±0.002 g/L.And the concentration of pyruvate and acetate decreased from 1.87±0.02 g/L and 0.012±0.002 g/L to 2.36±0.03 g/L and 0.862±0.012 g/L,respectively.3.The pyc of Bacillus subtilis expression plasmid pET-28a(+)-pyc was constructed.This article will pyruvate carboxylase expression vector pET-28 (+)-pyc import frdB knockout bacteria SPUEC 103 delta frdB successfully constructed SPUEC107/pyc delta frdB.Contrast SPUEC103 delta frdB and SPUEC107/pyc delta frdB, it can be seen after the heterologous gene amplification pyc, e. coli can improve gene knockout frdB growth ability weakened condition, but also can improve the utilization of glucose.Break the aseptic SPUEC103 frdB delta frdB expressing pyruvate carboxylase, succinic acid content increased by 65.3%, yanhusuo acid content increased by 39.6%.4.Successfully construct SPUEC108 (ΔfrdBΔptsG/pyc)Contrast SPUEC107 ptsG knockout strains SPUEC108 grow under anaerobic conditions and the larger changes in the production of fermentation product.Specific changes as shown in figure 2 to 15, figure 2-16 and shown in table 2 to 5.First of all, SPUEC108 slow growth rate than wild fungus anaerobic fermentation, needs a much longer to achieve stable growth period, and the final concentration of bacteria than wild fungus has greatly reduced.In terms of glucose consumption has similar phenomenon.After the glucose consumption rate lower, fermented product has bigger change happened, the manifestation of succinic acid from 4.33 g/L rose to 4.83 g/L, yanhusuo acid content by 0.254 g/L rose to 0.286 g/L, increased by 12.5%, while other fermented product has dropped.