Metabolic Engineering Of Corynebacterium Glutamicum For The Production Of β-alanine

In this study,based on the mutant Corynebacterium glutamicum with high L-lysine production,a recombinant Corynebacterium glutamicum strain which can use glucose as carbon source and produceβ-alanine efficiently by microbial fermentation was constructed.It has great production potential for microbial fermentation to produceβ-alanine.Therefore,in this study,the key enzymes ofβ-alanine biosynthesis pathway were screened and optimized,and the carbon flux in biosynthesis pathway was improved and the carbon flux in competitive pathway was limited in L-lysine producing strain XQ-5.This is the first report through genetic modificationβ-alanine biosynthesis pathway to improve the production ofβ-alanine in L-lysine producing bacteria.These results provide new insights for the construction of other valuable biochemical reactors.The main results are as follows:（1）Firstly,by replacing aspartate kinase（AK,coded by gene lysC）in L-lysine producing strain XQ-5,which was relieved of feedback inhibition after mutation with wild-type AK,L-lysine synthesis was inhibited by the feedback of AK₁₃₀₃₂ without large accumulation.L-lysine is no longer accumulated in large quantities,indicating that the metabolic flux of L-aspartic acid will flow as far as possible toβ-alanine synthesis pathway.The recombinant strain XQ-5.1,produced 5.6±0.11 g·L^-1β-alanine by fermentation,which 11 times higher than the original strain XQ-5(0.5±0.04 g·L^-1).（2）aspartate-α-decarboxylase（ADC,coded by gene pan D）is a key enzyme of the decarboxylation of L-aspartic acid to formβ-alanine.A phylogenetic tree of ADCs was constructed about 15 ADCs from different species and common industrial bacteria were screened.By comparing the enzyme activity and fermentation performance,the ADC from Bacillus subtilis was finally selected as the optimal enzyme.Through the combination of three effective single mutations of BsADC,the mutants that further improve the catalytic ability and catalytic stability were screened.It was found that its variant(BsADC^E56S/I88M)has the highest catalytic activity and an effect on the production ofβ-alanine,which improving 60%.In order to further improve the production ofβ-alanine.The heterologous expression level of BsADC was controlled by optimizing the promoter and RBS.By comparing the fermentation performance,P_gro and thir RBS were finally selected to control and improve the expression of BsADC.Replaced gene Cgpan D expression frame with P_gro-thir RBS-BsADC^E56S/I88M-terminator expression frame to obtain recombinant strain XQ-5.2,which produced 25.8±0.73g·L^-1β-alanine by fermentation and 4.6 times higher than the recomniantion strain XQ-5.1.（3）In order to further increase the yield ofβ-alanine,through overexpressing aspartase from Thermophilic bacterium YM55-1 to increase flux of the precursor L-aspartate.Then the recombinant strain XQ-5.3 was obtained,which produced 26.9±0.90 g·L^-1β-alanine by fermentation and 4%higher than the recomniantion strain XQ-5.2.（4）Through deleting gene ldh to block the accumulation of the main by-products lactic acid.Through deleting avtA and weakening gene ala T by inserting terminator in front of gene to reduce the accumulation of the main by-products L-alanine.The production ofβ-alanine was 30.7±2.3 g·L^-1,which 61 times higher than the original strain XQ-5 and the accumulation of by-product lactic acid(from 5.2±0.14 g·L^-1 to 0.2±0.09 g·L^-1)and L-alanine(from 7.6±0.22 g·L^-1 to 3.8±0.32 g·L^-1)decreased in recombinant strain XQ-5.5 by shake flask fermentation.Finally,the recombinant strain XQ-5.5 was tested in 5 L fermentor to determineβ-alanine yield(56.5±3.2 g·L^-1),the productivity(0.79 g·L^-1·h^-1)and the glucose conversion efficiency（39.5%）.In addition,the production of byproducts decreased.