Rational Metabolic Engineering Of Corynebacterium Glutamicum For Efficient Synthesis Of L-glutamate

L-glutamate（L-Glu）is the first commercialized amino acid that is widely used in food,pharmaceutical,daily chemical and agricultural industries because of its high nutritional value.At present,the fermentation efficiency of glutamic acid production bacteria used in industrial fermentation in China is relatively low and the sugar utilization rate is not high,and there is an urgent need for industrial production strains with high acid production rate and sugar-acid conversion rate.In this study,a strain of Corynebacterium glutamicum G01 with L-Glu titer of96.53±2.32 g·L^-1 in the laboratory collection was used as the starting strain（C.glutamicum G01was obtained from C.glutamicum E01 by stepwise mutagenesis）,and a series of metabolic engineering modifications were implemented to improve L-Glu yield,and the main findings were as follows:（1）Reducing L-alanine by-product content.L-alanine was main byproduct by measuring the content of various amino acids of C.glutamicum G01 fermentation broth.The ala T gene encoding alanine aminotransferase was knocked out to reduce the production of the by-product alanine.Compared with G01,the alanine content of the obtained strain decreased by 72.57%to2.23±0.09 g·L^-1,the L-Glu production increased to 101.33±1.67 g·L^-1,the growth of the knockout strain was not affected.（2）Enhancing the carbon metabolic flux.α-Ketoglutarate nodal carbon flux plays an important role in glutamate synthesis,therefore,the RBS sequence of the E1 subunit ofα-ketoglutarate dehydrogenase is replaced to reduce its activity.The strain G01-RBS4,which underwent RBS sequence optimization,showed a significant reduction in enzyme activity,with a specific enzyme activity of 0.469±0.13 U·mg^-1,decreased by 73.87%compared to G01,and the L-Glu production increased by 23.97%to 119.67±1.98 g·L^-1.（3）Transcriptional analysis in the key pathway of glutamate synthesis was performed on C.glutamicum E01 and C.glutamicum G01,and genes with large differences were overexpressed in G01.The yield of L-Glu in the strain overexpressing ppc was 108.33±2.08g·L^-1,and the yield of L-Glu in the strain overexpressing pfk was 107.66±1.89 g·L^-1,the yield of L-Glu in the strain overexpressing pyk was 106.33±2.45 g·L^-1,which increased by 12.22%,10.15%,and 11.53%respectively,compared with G01 strain.（4）Enhancing the catalytic activity of the key enzyme glutamate dehydrogenase（GDH）.Comparison of the catalytic activity of GDH from different sources revealed that GDH derived Aspergillus niger with higher enzyme activity.The strain G01/p XMJ19-An-gdh,which overexpressed GDH derived A.niger was fermented.The fermentation results showed that the L-Glu production of the GDH derived A.niger overexpression strain increased by 17.89%to113.80 g·L^-1.（5）Rational design was performed for glutamate transporter protein（Msc CG）,the L-Glu production of the A100V mutant G01/p XMJ19-Msc CG^A100V increased by 23.58%to 119.30g·L^-1 compared to G01,indicating that the mutant promoted glutamate efflux.（6）By combining the above strategies,the original RBS sequence of odh A sequence was replaced by the RBS4 sequence in the ala T knockout strain,while a rational genomic modification of Msc CG and genomic integration of GDH derived A.niger were achieved,and finally a recombinant strain was constructed by overexpression of ppc,pfk,and pyk.L-Glu yield was 136.33±4.68 g·L^-1,the glycolic acid conversion rate was 55.80%,which was 11.60%higher compared with G01,and this study with good industrial prospects.