Abstract: L-tyrosine serves as a common precursor for multiple valuable secondary metabolites,such as L-DOPA,melanin-like biomaterials,monolignols,betalain pigments,plant alkaloids and pharmaceuticals.At the same time,it can be used as a food additive in industry.The synthesis of this aromatic amino acid in Bacillus licheniformis occurs via the shikimate pathway with D-Erythrose 4-phosphate(E4P) and phosphoenolpyruvate(PEP) as precursors,but the underlying mechanisms involving metabolic regulation on L-tyrosine production remain unclear in this microorganism.In this work,we first optimized the carbon source,such as glucose,sucrose,lactose and xylose,in the fermentation of L-tyrosine.We found that lactose is more conducive to the synthesis of L-tyrosine by B.licheniformis,and the yield of L-tyrosine per OD600 reached 31.05 mg/L which is 54% higher than that with glucose as the carbon source.In addition,the culture conditions,such as temperature,dissolved oxygen(DO) and inoculum size,were optimized in the fermentation process of recombinant strain expressing HGPA.The optimization of cultural conditions obtained a result of inoculum size 3%(by volume),temperature 37 ℃,shaking speed 200 r/min by one-factor-at-a-time method.The production of L-tyrosine per OD600 by recombinant strain HGPA can reach 71 mg/L at the optimal fermentation condition.It has been verified that the overexpression of vgb,a gene encoding Vitreoscilla hemoglobin(VHb),can increase the yield of L-tyrosine per OD600 from 71 to 100 mg/L,increased by 40.8%.To evaluate the effect of gene vgb expression on the transcription of the shikimate pathway,we used quantitative RT-PCR for detecting the expression level of all genes in shikimate pathway of strain CICIM B6902::vgb with wild-type strain as control.The results indicated that the expression of aroC and aroD in strain overexpressing vgb gene was 2.57 and 2.91 times that of the original strain,respectively,which may result in the improvement of L-tyrosine production.Accordingly,we overexpressed genes aroC,aroD,and aroK,respectively,on the basis of HGPA and L-tyrosine production with expressing HGPAD and HGPAK can reach 1 200 mg/L.However,the production of L-tyrosine was constrained by a narrow range relative to recombinant strain expressing HGPA when aroC gene was co-expressed with 3-deoxy-7-phosphoheptulonate(DAHP) synthase(AroGfbr,D146 N) and dual-function chorismate mutase/prephenate dehydrogenase(TyrAfbr,M53I/A354V).It is worth noting that when 5 g/L shikimate was added during the fermentation process,the production of L-tyrosine with expressing HGPA and HGPAK could reach 1 311 and 1 490 mg/L,respectively.This indicated that the overexpression of genes aroD and aroK could not completely lift the restriction of nodal metabolic flux in shikimate pathway of B.licheniformis.This paper evaluated the effect of overexpression of bottleneck genes in shikimate pathway on aromatic amino acids production in B.licheniformis.Furthermore,this research provided the foundation for further realizing the application potential of B. licheniformis,a Generally Recognized As Safe(GRAS) workhorse,in the industrial production of L-tyrosine and its high-value derivatives.
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