Genetic Engineering Of Escherichia Coli To Produce L-5-methyltetrahydrofolate

Folic acid belongs to the B vitamin group and its metabolites play an important role in DNA synthesis and methylation,one-carbon unit metabolism,protein metabolism,etc.Among them,L-5-methyltetrahydrofolate(L-5-MTHF)is the only substance that can cross the blood-brain barrier after a multi-step enzymatic reaction.It has been widely used in the prevention and treatment of various diseases and has a broad market prospect.At present,research on the biosynthesis of L-5-MTHF has mainly focused on the metebolic engineering using Lactobacillus and Bacillus subtilis as the chassis cells,while less research has been conducted on the production of L-5MTHF using E.coli as the chassis cells.In our laboratory,three genes encoding enzymes of the Wood-Ljundah pathway(WLP pathway)were introduced,including formate-tetrahydrofolate ligase(fhs),formimido-tetrahydrofolate cyclodeaminase(fchA)and methylenetetrahydrofolate dehydrogenase(folD).The dihydrofolate reductase(folA)and 5,10-methylenetetrahydrofolate reductase(metF)of E.coli were also overexpressed.A new synthetic pathway for L-5-MTHF was successfully constructed in E.coli BL21(DE3),resulting in a significant increase in its yield.Based on this,the production of L-5-MTHF was improved by screening the promoter,optimizing the RBS sequence and regulating the expression of exogenous genes through the design of synthesis path and the screening of synthetic biology devices.At the same time,considering that the yield of L-5-MTHF using microorganisms includes multiple steps such as fermentation,purification and extraction,the preparation process is costly,so the chassis cell was replaced by probiotic bacteria E.coli Nissle 1917(EcN)that can colonize the intestine.Therefore,the target product L-5-MTHF can be used directly without purification.Firstly,the key enzymes were screened by sequence comparison and enzyme activity determination.The yield of L-5-MTHF in engineering bacteria BL21-D replacing FolD was higher than that of the original strain,which indicated that improving the FolD activity could relieve the rate-limiting step of the synthesis of the target product to some extent.The methylenetetrahydrofolate reductase(BpMTHFR)and methylenetetrahydrofolate dehydrogenase(BpFolD)from B.producta were heterologyously expressed and purified in E.coli BL21(DE3)using plasmid pET28b(+).The specific enzyme activities of BpMTHFR and BpFolD were detected(37.791 ± 3.95 U·mg-1 and 378.65±7.01 U·mg-1),indicating that these two enzymes have good activities and can be applied in the modification of the engineered strains.An engineered L-5-MTHFproducing bacterium constructed in the laboratory was optimized as a starting strain.Three engineered strains were further constructed by replacing the genes of the corresponding enzymes in the starting strains,and the yield of L-5-MTHF was detected by high performance liquid chromatography after fermentation.It was found that the yield of L-5-MTHF by engineering strain BL21-R with BpMRHFR was 315.94 μg·L-1,BL21-RD which replaced two enzyme was 363.26 μg·L-1.Compared with the original strain,BL21-D with BpFolD was 483.53 μg L-1,which increased by 37.34%.The synthesis of the target product L-5-MTHF was facilitated by removing the rate-limiting step of the synthesis of the target product.Secondly,the expression of foreign genes in E.coli BL21-WL were regulated by promoter screening and RBS sequence optimization.Compared with the original strain,the yield of BL21-RBS6 increased by 60.85%,and the refine regulation of genes related to L-5-MTHF synthesis pathway was realized.The pETDuet-1 plasmid was used as the backbone,and the lac and trc promoters were selected to replace the original T7 promoter on the plasmid to initiate the transcription of fhs,fchA and folD.Compared with the original strain,the yield of engineering bacteria BL21-99A was increased by 37.57%,which confirmed that trc promoter was more suitable for the expression of target genes.Further,three RBS sequences of different strength were selected from the MIT Registry of Biological Standard Parts database.Different RBS sequences were linked to the genes fhs,fchA and folD by fusion PCR technique to obtain six strains of the engineered bacteria with different gene expression strength.After fermentation,the protein expression in the extracts of the engineered bacteria was analyzed to determine the translation level of the exogenous genes,and the optimal combination of RBS sequences was selected by combining with the yield of L-5-MTHF.It was found that using strong,medium and weak RBS sequences to regulate the genes fhs.fchA and folD respectively was a more ideal combination.Thirdly,the L-5-MTHF synthesis pathway was introduced into EcN through genome modification and plasmid expression.The probiotic EcN-99A which can synthesize L-5-MTHF was constructed.Using the probiotic EcN as the chassis cell,the genes metF and folA,which were originally overexpressed on pACYCDuet-l,were integrated into the genome for expression.The promoters of both genes were replaced with the constitutive strong promoter Pup using Red recombination technology to reduce the stress caused to the cells by multiple plasmid overexpression.At the same time,the previously designed and optimized L-5-MTHF synthesis line was transferred into EcN.The yield of L-5MTHF was measured after induction fermentation.Finally,the probiotic EcN with a yield of 316.27 μg L-1 L-5-MTHF was obtained.In summary,in order to screen for optimal,the expression of exogenous genes such as fhs,fchA and folD were optimized from key enzymes,promoters,and RBS sequences.The synthetic pathway of L-5-MTHF was innovatively introduced into probiotic EcN with a view to achieving exogenous supplementation of L-5-MTHF in the form of oral probiotics.This method can avoid the extraction and preparation process of the target product and provides a new idea for the direct utilization of the product.The construction strategy and findings of this thesis provide a meaningful attempt for the efficient biosynthesis of L-5-MTHF.