Metabolic Engineering Of The Synthesis Of 4C-Dicarboxylic Acids By Corynebacterium Glutamicum

4-carbon-dicarboxylic acids(fumaric acid,succinic acid,L-malic acid,oxaloacetic acid)are important intermediates in the acetaldehyde acid cycle and TCA cycle,which have important metabolic significance in organisms.As a classical safe model strain,C.glutamicum ATCC 13032 has clear genetic background and relatively simple gene operation,which makes C.glutamicum ATCC 13032 to be widely used as a cell factory to produce various chemicals in a biological manner.In this study,C.glutamicum C07 was used as the starting strain and its metabolism pathway was modified by gene editing to explore the effect on 4C dicarboxylic acid synthesis.The research results were as shown below:(1)The recombination vector p K19 mobsac B-?ppc was successfully constructed with one base mutation site in the ppc gene fragment of C.glutamicum by SOE PCR.And then the 299 th site of ppc gene was successfully mutated with G replaced by A.The point mutation strain was named CY02.The site binding with Asp was mutated to bind with the amino acid Asn.Because of removing the substrate inhibition,the enzyme activity of PPC was increased from 50 U/mg protein to 92.22 U/mg protein.Though fermentation cultivation,the production of L-malate reached the amount of 14.16 g/L,and fumaric acid and succinate production were 19.82 mg/L and 2.698 g/L respectively.The yield of pyruvic acid was 5.95 g / L.(2)The upstream and downstream arms of C.glutamicum gene mqo were amplified and the recombinant vector p K19 mobsac B-?mqo was constructed which contained the upstream and downstream homology arms.The gene mqo was knockouted by using the reverse screening of p K19 mobsac B vector.Deletion mqo could prevent the conversion of malic acid to oxaloacetate.This deletion mqo strain was named CY09.By fermentation cultivation,CY09 could produced L-malic acid 16.12 g/L,fumaric acid 21.32 mg/L and succinic acid 2.78 g/L.The production of the pyruvic acid was 3.512 g/L.(3)The fermentation medium and fermentation conditions for the production of organic acid by recombinant strains were optimized through the two-stage fermentation with early aerobic and limited oxygen in late stage.Using optimized fermentation parameters,the L-malic acid yield of CY09 was 24.768 g/L,fumaric acid 25.95 mg/L,succinic acid 3.087 g/L and the producion of pyruvic acid was 2.547g/L.