Engineering The Shikimic Acid Pathway And Transport Pathway Of Bacillus Licheniformus For Synthesis Of L-phenylalanine

L-Phenylalanine（L-Phe）is a kind of essential amino acids,which widely applied in the fields of food,medicine and chemical.L-Phe is mainly produced by chemical synthesis,enzymatic method,and microbial fermentation.However,the chemical synthesis method causes environmental pollution,and the enzymatic method has some drawbacks,such as unstable enzyme activity.The microbial fermentation possesses excellent characteristics,such as low cost,fast growth and environmentally friendly.However,the production capacity of engineered strains is low,which restricts the application of L-Phe.In this study,2-phenylethanol-producing strain of Bacillus licheniformis DA1(DWc9nΔpykΔpheA-ldh::pheA^fbr-xkdE::aroG^fbr-xkd G::aroD-PbacA-aroK-PgroESL-aroCΔdhbCΔdhaSΔhisC-Pbay-glcU-Pbay-glcK-ParoK-ptsG)was used as the starting strain.To achieve the efficient production of L-Phe,the precursor synthesis pathway,the shikimic acid pathway,transport pathway,and L-Phe synthesis pathway were engineered.Finally,the fermentation conditions were optimized to enhance the L-Phe production.The transaminase HisC is a key enzyme that catalyzes phenylpyruvate to L-Phe.In this study,hisC was re-installed into DA1 to achieve de novo synthesis of L-Phe with a titer of 4.35g/L.Promoter engineering was used to optimize the hisC expression level and the recombinant strain DA3 was obtained.DA3 strain produced 5.9 g/L L-Phe.Secondly,the aromatic amino acid transport protein aroP was knocked out,and the aromatic amino acid export protein ydd G from E.coli was introduced into DA3 strain.The L-Phe titer of the recombinant strain DA7reached 6.43 g/L,increasing by 9%compared by DA3 strain.Then,glucose-6-phosphate dehydrogenase Zwf and transketase Tkt was overexpressed to enhance the supply of the precursor erythrosis-4-phosphate（E4P）,the L-Phe titer of the recombinant strain DA9 was increased to 6.6 g/L.Next,the expression of chorismate synthase AroF,shikimate dehydrogenase AroD and 5-enolpyruvyl-shikimate-3-phosphate synthase AroE in the shikimic acid synthesis pathway was enhanced.The results showed that the enhancement expression of the above gene had promoting effects on L-Phe production.The obtained recombinant strain DA13（DA1-Pbay-hisCΔaroP-yjiC::ydd G-Pbac AU12-zwf-PbacA-tkt-R36-aroF-R56-aroD-P43U12-aroE）produced 7.23 g/L L-Phe.Finally,the fermentation process of the best engineered strain DA13 was optimized,including carbon source concentrations,nitrogen source concentrations and ion concentrations.Under the optimized process,DA13 strain produced 12.5 g/L L-Phe and the glucose conversion rate was 23%,increasing by 1.87-fold compared to DA2 strain.