Metabolic Engineering Of Corynebacterium Glutamicum For The Production Of L-valine

L-valine is an important branched-chain amino acid,which participates in protein synthesis and tissue repair in the body.It also plays an important role in mammalian growth and development.L-valine as a high value-added amino acid has broad production and application prospects is widely used in biomedicine,food additives,animal feed and other fields.At present,L-valine is mainly produced by biological fermentation,Corynebacterium glutamicum and Escherichia coli are the main production strains.In this study,a L-valine producing strain Corynebacterium glutamicum VHL-1 was used as the starting strain.Improve the synthesis of L-valine in the VHL-1 through metabolic modification and optimization of fermentation conditions.The main research conclusions are as follows:（1）Increased pyruvate precursor for L-valine synthesis by redistributing the carbon metabolic flow at the pyruvate node.Firstly,the competitive consumption of pyruvate was reduced by knocking out ldh,pox B and weakened ala T in VHL-1.Then,the pathway of pyruvate to oxaloacetic acid was blocked by knocking out the gene pyc coding pyruvate carboxylase.The recombination strain VHL-3 was built by knocking out pyc gene in strain VHL-2.Intracellular concentration of pyruvate was further increased in strain VHL-3.The flask fermentation results showed that the L-valine production of strain VHL-3 was 32.1±3.1 g·L^-1,which was 11.8%higher than that of the starting strain VHL-1.（2）Strengthen the synthesis pathway of L-valine in the strain.Acetohydroxylate synthase（AHAS）is the key rate-limiting enzyme in the process of L-valine synthesis.Compared the amino acid sequences of the acetylohydroxylate synthase from the starting strain VHL-1 and the wild-type strain ATCC13032.It was found that the acetylohydroxylate synthase from strain VHL-1 has K30Q,N156D and V233I three mutations in the catalytic subunit and A42V and H47L two mutations in the regulatory subunit.It was found that the combined mutation resulted in higher enzyme activity and anti-feedback inhibition effect of AHAS₁through in vitro enzyme activity test and anti-feedback inhibition analysis.Based on this result,in order to promote the synthesis of pyruvate to L-valine,the recombinant strain VHL-6 was constructed by replacing the natural promoter of ilv BNC₁ operon in the recombinant strain VHL-3 with the endogenous strong promoter P_tuf of Corynebacterium glutamicum and increasing the copy number of the ilv BN₁ gene.The L-valine production of strain VHL-6 in flask fermentation was 35.2±3.24g·L^-1,which was 21.8%higher than that of the original strain VHL-1.（3）Improve the extracellular transport efficiency of L-valine.Knocked out the transporter-coding gene brn Q in Corynebacterium glutamicum VHL-6.The flask fermentation results showed that it was not conducive to cell growth and L-valine synthesis.Subsequently,amino acid sequence analysis of Lrp₁ transcription factor and Brn FE transporter from strain VHL-1.The result showed that comparing with the Lrp from wild-type Corynebacterium glutamicum ATCC13032,there was S124N mutation in Lrp₁transcription factor.Overexpression of Lrp₁and Brn FE in strain VHL-6 built strain VHL-6/p EC-XK99E-lrp₁-brn FE.L-valine fermentation yield was 36.7±3.5 g·L^-1,which was 25.2%higher than that of the original strain VHL-1.（4）The fermentation medium components were optimized by single factor experiment,Plackett-Burman experiment and Box-Behnken experiment.The optimal composition of L-valine fermentation medium(g·L^-1):glucose 154.5,（NH₄）₂SO₄35,peptone 6,cornsyrup 4.5,K₂HPO₄ 3,Mn SO₄·H₂O 0.05,vitamin B₁ 2.5×10^-4,Ca CO₃ 40;The best fermentation conditions in shaking flask:p H 7.5,inoculum amount 5%,liquid content 35 m L/500 m L.The strain VHL-6/p EC-XK99E-lrp₁-brn FE was cultured in 5 L fermentation tank in fed-batch with optimized fermentation medium components.The final yield of L-valine was 86.2±2.2 g·L^-1,the production intensity was 1.20 g·L^-1·h-¹,and the conversion rate of sugar was 0.30 g·g^-1.