Synthesis Of L-cysteine And Screening And Characterization Of Cysteine Responsive Promoters From Engineered Escherichia Coli

L-cysteine is an important sulfur-containing amino acid.Biosynthesis of L-cysteine has recently attracted increasing attention owing to its wide application in cosmetics,medicine,food and other industries.In this study,the production of L-cysteine was enhanced through multi-gene combination regulation strategy in E.coli.Based on the results of comparative transcriptome analysis,we screened and characterized some promoters from Escherichia coli that show a significant response with changes in the L-cysteine concentration.Then,the responsiveness of promoters was optimized and verified,which lays a foundation for the construction of cysteine-specific biosensor.The main results are listed as follows:（1）Two rate-limiting enzymes 3-phosphoglycerate dehydrogenase and serine O-acetyltransferase derived from E.coli MG1655 were mutated to reduce the feedback inhibition,and then they were introduced into E.coli generated strain EC6 resulting in enhancement of the synthesis pathway of L-cysteine precursor and L-cysteine transport pathway.The 47.3 mg·L^-1of L-cysteine was produced by the shaking flask fermentation with strain EC6,which was165.7%higher than that of the original strain.（2）The gene tna A encoding L-cysteine desulfhydrase was knocked out to weak L-cysteine degradation pathway.The recombinant strain EC9 was constructed by overexpressing cys E^T167A,cys K,eam A and cys B genes.The 148.1 mg·L^-1 of L-cysteine was produced by the shaking flask fermentation with EC9,which was 213.1%higher than that of the recombinant strain EC6.Adding 2 g·L^-1 serine as precursor supplement and shaking flask fermentation for 48 h,the yield of L-cysteine with the recombinant strain EC9 reached 402.9 mg·L^-1.（3）E.coli W3110 was cultured in LB medium with different concentrations 0,3,6,10 g·L^-1 of L-cysteine,and genes which showed significant improvement at the transcription level were screened.Then,the promoter fragment was amplified and fused with the fluorescent reporter gene egfp to construct a library with 27 promoters.Furthermore,the sensitivity and specificity of the selected promoters in response to high and low concentration L-cysteine were analyzed,and the E2 and P1 promoters with a significantly higher response performance than other promoters were screened.（4）Softberry software was used to predict the core region of the promoter E2,and random mutation of AAAT region was designed in the-35 spacer region of promoter E2.A mutant library of the promoter E2 was constructed,and the mutant promoter E2-33 had a highest specific response to the L-cysteine concentration range of 1-7 g·L^-1 was screened.The overall relative fluorescence intensity increased by 15.6%than E2.（5）E2-33 and P1 were separately introduced into the recombinant strain EC9,and L-cysteine was added exogenously for shaking fermentation.The results showed that the relative fluorescence intensity of EC9/p E2-33 and EC9/p P1 was enhanced with the increase of L-cysteine concentration.Taking 0 g·L^-1 as control,when 0.5,1,3,5 g·L^-1 L-cysteine was added exogenously,the relative fluorescence intensities of EC9/p E2-33 were increased by 22.3%,43.5%,53.6%and 64.9%respectively.The relative fluorescence intensities of EC9/p P1 were increased by 28.9%,54.5%,69.6%and 63.3%respectively.These results indicate that both promoter E2-33 and P1 can respond to L-cysteine concentration changes during fermentation,and have the potential to construct cysteine-specific biosensors.