Construction Of L-valine Engineering Bacteria In Escherichia Coli

L-valine is an essential amino acid for human body,which is widely used in food additives,antibiotic precursors and feed industries,and has a wide application prospect and high commercial application value.However,L-valine has low production rate and high production cost in industrial production.Therefore,in this study,E.coli W3110 was used as the starting bacteria to redesign the metabolic network of valine production in Escherichia coli by using CRISPR/Cas9 gene editing technology.By removing the feedback inhibition of the key enzyme of L-valine,enhancing the metabolic flow of the pathway of L-valine synthesis,improving the extracellular transport efficiency of L-valine,and improving the supply of NADPH and precursors,we successfully constructed an E.coli engineering bacterium VHY20 that uses glucose as raw material and produces L-valine efficiently and stably.The main construction methods are as follows:1.The accumulation and yield of L-valine can be improved by strengthening L-valine synthesis pathway in E.coli.As a key enzyme in the L-valine synthesis pathway,AHAS is inhibited by L-valine feedback.In order to remove the feedback inhibition of L-valine on key enzyme,the gene Ptrc-alsS that encodes AHAS from Bacillus subtilis was introduced into E.coli.The flask shaking results showed that the production of L-valine in this strain was 4.7 g/L and the yield was 4.3%,indicating that the introduction of exogenous AHAS was beneficial to the accumulation of L-valine.In order to further improve the carbon flux in the L-valine synthesis pathway,double and triple copies of gene Ptrc-alsS were made on the E.coli genome.The production of L-valine and yield of strain Ptrc-alsS with double copies were 10.6 g/L and 9.7%,respectively,125.8%and 130.6%higher than the control strain.However,the production of L-valine and yield of the three-copy strain remained unchanged,indicating that the optimal expression of the exogenous key enzyme activity was double copies.Subsequently,the production of L-valine and yield reached 18.6 g/L and 17%,respectively,during the overexpression of other key enzymes in the synthesis of L-valine,namely AHAIR,DHAD and TA.It was proved that the strengthening of the branch synthesis pathway effectively increased the yield of L-valine.2.In order to increase intracellular L-valine transport capacity,this study tested the output efficiency of E.coli transporter and C.glutamicum transporter at different levels of promoter(native promote and Ptrc promoter).The results showed that the production of L-valine and yield of the strain incorporating the transporter gene Ptrc-brnFE encoding the C.glutamicum were the highest(32 g/L and 29.9%),respectively,which increased by 72%and 74.8%compared with the control strain.The results showed that the overexpression of the transporter protein of C.glutamicum could strengthen the transport system of L-valine and increase the extracellular accumulation of L-valine.3.The reaction in the L-valine synthesis pathway requires the supply of NADPH,that is,2 mol of NADPH is consumed for every 1 mol of L-valine synthesis.To improve the supply of NADPH,this study integrated the NAD kinase gene pntAB to convert more NADP into NADPH.The results of shaking fermentation showed that the production of L-valine and yield reached 34.1 g/L and 30.2%,and further double copies of the gene pntAB showed no change in the biomass and L-valine yield.It is proved that increasing the supply of NADPH is beneficial to increasing the yield of L-valine.4.As a key precursor of L-valine synthesis,pyruvate accumulation can promote the production of L-valine.In this study,ppGpp 3’pyrohydrolase gene spoT[R290E,K292D]was introduced to improve the reaction rate from glucose to pyruvate.Flask fermentation results showed that the production of L-valine and yield reached 41.2 g/L and 32%,increasing by 20.8%and 0.6%respectively.For inhibit the TCA cycle and allow the excess metabolic carbon flux to be used in the synthesis of L-valine,the thiE gene encoding thiamine phosphatase was knocked out and VB1 with a concentration of 5.3 mg/L was added.The shaking bottle results showed that the production of L-valine and yield reached 43 g/L and 35%,increasing by 3.6%and 9.3%respectively.In conclusion,increasing the reaction rate of glucose to pyruvate and inhibiting the TCA cycle are beneficial to the accumulation of L-valine.5.The strain that knocked out the gene thiE was fermented in a 5 L bioreactor-fermentation test to investigate the production of L-valine.The results showed that the batch fermentation for 48 h produced 85 g/L L-valine,the yield reached 30%,and the production intensity was 1.77 g/L/h.The strain has good industrial potential.