Metabolic Engineering And Fermentation Characteristics Of Corynebacterium Glutamicum For L-Leucine Production

A high-quality bacterial strain is the decisive factor in the production of L-leucine by fermentation,and it is also the key to determining whether it has industrial value and if fermentation will succeed or fail.Based on the Corynebacterium glutamicum ML04strain,a high-yield L-leucine strain was engineered using a combination of protoplast ARTP mutagenesis and systemic metabolic engineering.The media composition and the fermentation process were optimized.Finally,a 60T-scale fermentation production was successfully achieved.The main findings are summarized as follows:(1)The protoplast preparation and regeneration conditions of the C.glutamicum ML04 strain were optimized.Cells were cultured to an OD_562nm of 0.5-0.7 in media containing 3.0 g/L glycine and 5?g/m L ampicillin.Lysozyme was added to a final concentration of 5.0 mg/m L,and the cells were incubated at 30?for 60 min until the protoplast formation rate reached 95.2%and the regeneration rate reached 22.6%.Next,the protoplasts were subjected to ARTP mutagenesis and structural analog screening.After 96 well plate-PC–HPLC(96-PH)high-throughput screening and shake flask rescreening,the genetically stable L-leucine high-yield strain ML1-9 was obtained,and fermented in shake flasks.After 48 hours of fermentation in shake flasks,the yield of L-leucine was approximately 15.04 g/L,which was 70.8%higher than that of the original C.glutamicum ML04 strain.(2)Considering the characteristics of C.glutamicum ML1-9 and a published protocol,a high-efficiency electroporation scheme suitable for a variety of strains was established.First,the cells were cultured in media containing 3.0 g/L glycine,5?g/m L ampicillin and 1.0 g/L Tween 80 to an OD_562nm of 0.5-0.7,which weakened the synthesis of cell walls and enhanced the performance of the competent cells.Then,2.5-3.0?g of a xenogeneic plasmid was added,and the electric field intensity was controlled at 0.9-1.0k V/mm.Immediately after the shock,the resuscitation media containing 0.75 mol/L sorbitol was added,and heat shock was performed at 46?for 8 min to passivate the cell modification system.After the heat shock,pre-incubation was performed at 37?for 1 h,followed by resuscitation at 30?.Coating a low-concentration antibiotic selection plate increased the efficiency of exogenous genes entering C.glutamicum ML1-9.(3)The systemic metabolic modification strain C.glutamicum MDLeu-19 was successfully constructed by homologous recombination.The shake flask fermentation showed a significant increase in L-leucine production that reached 21.8 g/L,the glucose conversion rate reached 27.5%,which were 45.3%and 47.3%higher than the C.glutamicum ML1-9,respectively.Moreover,the proportion of miscellaneous acid also decreased significantly,which was 56.1%lower than that of C.glutamicum ML1-9.(4)The differences between the IPMS and Lrp protein coding genes of C.glutamicum MDLeu-19 and C.glutamicum ML1-9 were cloned and analyzed.The specific amino acid mutation sites were obtained for the first time,and the effects of mutation on the synthesis and secretion of L-leucine were verified.The results showed that the mutation of IPMS(R529H,G532D and L535V)facilitated its binding to the substrate and promoted the synthesis of L-leucine.The mutation of Lrp(A78G and L80R)effectively enhanced the positive regulation of Brn FE and IPMS and was beneficial to the enhancement of the synthesis and secretion of L-leucine.(5)Using the P_tac promoter as a regulatory element,we co-expressed the mutated IPMS and Lrp proteins and constructed the C.glutamicum MDLeu-19/p Z-leu A^r-lrp^*strain.The results of the shake flask fermentation showed that the L-leucine yield reached approximately 26.7 g/L,which was 22.2%higher than the yield of the C.glutamicum MDLeu-19 strain,77.7%higher than the yield of the C.glutamicum ML1-9 strain,and204.6%higher than the yield of the original C.glutamicum ML04 strain.(6)The combination of the innovative development of the seed strengthening process of C.glutamicum MDLeu-19/p Z-leu A^r-lrp*,the improvement in the organic nitrogen source and glucose control technology in the fermentation media,and the optimization of the organic nitrogen source and improvement in the glucose control process greatly improved the L-leucine fermentation yields.The yield of L-leucine in a30-L fermenter reached 42.8g/L,and the highest glucose conversion rate reached 33.2%,which was 20.1%and 10.3%higher than the original formulation process,respectively.In addition,the impurities were reduced and the utilization rate of the media was enhanced,which improved the characteristics of the fermentation broth and improved post-processing efficiency.(7)Production of the C.glutamicum MDLeu-19/p Z-leu A^r-lrp^* strain was successful in a 60-T fermenter.The L-leucine yield reached 40.5 g/L,and the glucose conversion rate reached 30.5%,which reflected an industrial scale fermentation capacity.