Construction Of Escherichia Coli Engineering Strain For L-phenylalanine Production

As an intermediate of food chemicals and drugs,L-phenylalanine has been widely used in food,medicine,chemical industry and other fields.At present,due to the plasmid carrying of most strains used in production,antibiotics or inducers need to be added in the fermentation process to maintain cell production,which increase the cost of industrial production.Therefore,on the basis of the existing research,this study conducted genomic molecular modification of related genes in the L-phenylalanine synthesis pathway of wild E.coli by means of metabolic engineering,and constructed a plasmid free engineering strain of L-phenylalanine with high yield.The bifunctional enzymes PheA and DAHP synthase are the two key rate-limiting enzymes in the synthesis of L-phenylalanine.To remove feedback inhibition of key enzymes,improve the ability of L-phenylalanine accumulation,pheA gene in the absent R region and tyrB transaminase gene were combined into operons controlled by trc promoter.The operons were integrated into the PHE1 genome by Crispr/Cas9 technology in single,double and triple copies,respectively,to construct PHE2,PHE3 and PHE4 strains.The results showed that the phenylalanine yield of PHE2 increased from no to 6.1 g/L;The phenylalanine yield of PHE3 was 8.8 g/L,a 35.4%increase over PHE2,while the phenylalanine yield of PHE4 was 25%lower than that of PHE3.We then constructed PHE5 and PHE6 by integrating the aroG point mutation gene and two copies controlled by the trc promoter in the genome of PHE3.The results of flask fermentation showed that the phenylalanine yield of PHE5 was 13.1 g/L,an increase of 46.9%over that of PHE3;The PHE6 phenylalanine yield was 12.8 g/L,which was lower than PHE5.In conclusion,the release of feedback inhibition of key enzymes is beneficial to the accumulation of L-phenylalanine,and the appropriate enhancement of the key enzyme of feedback inhibition can significantly increase the yield of phenylalanine.The expression levels of six genes aroC,aroA,aroL,aroE,aroD and aroB in the shikimate pathway were strengthened to reduce the blockage of upstream metabolic flow and the accumulation of toxic intermediates.Shaking flask fermentation results showed that the overexpression of aroA gene and aroD gene could effectively increase the yield of phenylalanine.The production of phenylalanine for PHE8-2(gapC::ParoA-aroA)was 15.1 g/L,11.1%higher than that for PHE5;The production of phenylalanine for PHE11-1(aroD::PBBa_j23106-aroD)was 19.3 g/L,36.9%higher than that for PHE8-2.The modification of the other four genes did not show a positive effect on phenylalanine production and bacterial growth,indicating that AroA and AroD are two rate-limiting enzymes in the shikimate pathway,and the production capacity of L-phenylalanine could be improved by moderately strengthening the expression of aroA and aroD genes.PHE13 was constructed by knocking out the pykF gene in PHE11-1.The shaking flask results showed that the production of PHE13 was 23.7 g/L,24.1%higher than PHE11-1,and the bacterial growth was not affected,indicating that pykF gene knockout could increase the PEP supply and facilitate the synthesis of phenylalanine.Subsequently,pps gene and tktA gene controlled by promoters of different strengths were introduced into PHE13,and the results showed that phenylalanine production decreased to different degrees after the enhanced expression of the two genes compared with PHE13,indicating that the overexpression of pps gene and tktA gene after the knockout of pykF gene had little effect on the increase of phenylalanine production.PheP and AroP are two phenylalanine transporters in E.coli that transport extracellular phenylalanine to the cell for bacterial growth.In this study,PHE16 and PHE17 were constructed by knockout of pheP gene and aroP gene on PHE13,respectively.The results showed that the production of PHE16 was 18.1 g/L,19.9%lower than that of PHE13;The production of PHE17 was 19.8 g/L,which was 16.1%lower than that of PHE13,indicating that pheP gene and aroP gene knockout were not conducive to phenylalanine accumulation.PHE13 was used for batch feeding fermentation in a 5 L fermenter.The results showed that the production of phenylalanine reached 81.8 g/L after 48 hours of fermentation,and the average production intensity reached 1.7g/L/h,which was 12.2%and 21.4%higher than the highest yield(72.9 g/L,1.4 g/L/h)reported in the literature.The conversion rate is 24%,which has good industrialization potential.