| L-malic acid is an important four-carbon compound,which is widely used in food,medicine and chemical industry.At present,malic acid is synthesized by chemical synthesis,enzyme and fermentation methods.The synthesis of malic acid by microbial fermentation has the advantages of high stereoselectivity,low raw material cost and less by-products.C.Glutamicum is a classic food safety bacterium.Under anaerobic conditions,pyruvate carboxylase(PYC)catalyzes pyruvate into OAA,and then OAA is converted to L-malic acid.The conversion rate of sugar and acid in this pathway is 200%.In this study,the engineering strain to improve the yield of L-malic acid was obtained by strengthening the r TCA pathway and the trans-membrane transporter of malic acid.The details are as follows:(1)Enhancing Malate dehydrogenase(MDH)of r TCA pathway: Reverse screening was carried out by knockout system p K19 mobsac B,and a strong promoter gapA(Pgap)was knocked into the upstream of MDH gene to promote the conversion of OAA into L-malate by up-regulating MDH enzyme activity.The recombinant strain was named C10.The yield of malic acid in C10 strain was 23.2 g/L,34.5% higher than that of the control strain.(2)Up-regulating the activity of endogenous malic acid transporter: a strong promoter gapA was knocked into the upstream of Sdas gene of C.glutamicum,which promoted the trans-membrane transport of malic acid through up-regulated Sdas activity.The recombinant strain was named C11.The yield of malic acid in C11 strain was 25 g/L,7.2% higher than that of the control strain.(3)Overexpression of malate transporter genes from different sources: The malic acid transporter Dcu C from E.coli MG1655 and Sdas from C.glutamicum were overexpressed in C.glutamicum respectively.The corresponding recombinant strains were named C10-1 and C10-2.The results showed that the yield of malic acid of the recombinant strain C10-1 and C10-2 was 40.8 g/L and 31.6 g/L respectively,which was75.86% and 36.2% higher than that of the control strain respectively.(4)Improving cell growth and biomass: through the knockout adenosine triphosphate phosphatase gene amn of C.glutamicum,the content of intracellular ATP was increased which contributed to the growth of strains and higher biomass,thus improve the L-malic acid production,the recombinant strain named C12.And C12 malic acid production increased by 9.6% compared with the strain.(5)Comparative analysis of acid production of different recombinant strains: the metabolic modifications included: knocking out pyruvate bypass metabolic pathway,enhancing carboxylation pathway,modifying TCA cycling-related pathway and malic acid transport pathway.Pta-ack,ldh A,pqo and mal E genes were knocked out in pyruvate bypass metabolic pathway and the yield of malic acid in C04 was 11.8 g/L;The strong promoter Psod was knocked into the up-stream of ppc and pyc,and point mutation of ppc to eliminate product feedback inhibition.The yield of malic acid of recombinant strain C08 was 16.8 g/L.TCA cycle related pathway modification included the deletion of mqo and amn genes,the knockin of strong promoter Pgap in the up-stream of mdh and Sdas,The malic acid yield of recombinant strain C12 was 27.4 g/L.Malic acid transport pathway modification included overexpressing Sdas and Dcu C,and the yield of malic acid in recombinant strain C10-1 was 40.8 g/L.In this study,a series of genetic modifications were carried out on C.glutamicum through homologous recombination and double exchange.The wild-type C.glutamicum almost did not accumulate malic acid,and the malic acid yield of recombinant C10-1reached 40.8 g/L,while the content of by-products was significantly reduced,which provided a reference for obtaining high-yield L-malic acid engineering strain. |
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