Construction Of L-5-MTHF Producing Engineered E.coli And Preliminary Study Of Fermentation Conditions

Folic acid is a general term for pteroylglutamic acid and related compounds with the biological activity of folic acid.It belongs to vitamin B and is involved in the metabolism of one carbon unit in human body.However,human cannot synthesize folic acid and must take it in from food.Insufficient folic acid intake can lead to various diseases,including megaloblastic anemia,neural tube defects,and cardiovascular disease.In the body,folic acid is reduced to tetrahydrofolate,the coenzyme of one carbon unit transferase,by the catalysis of dihydrofolate reductase.Tetrahydrofolate is then ligated with one carbon to finally form the biologically active L-5-methyltetrahydrofolate(L-5-MTHF)through a series of enzymatic reactions,which further participates in the methylation process and DNA synthesis in vivo.L-5-MTHF is the only folic acid compound that can pass through the blood-brain barrier,and can be used to prevent Alzheimer’s disease.However,some mutations on the methylenetetrahydrofolate reductase(MTHFR)gene can affect the synthesis of L-5－MTHF in vivo,causing serious diseases in human.Therefore,it is necessary to take L-5-MTHF directly for the people with the gene mutation on MTHFR.Currently 5-MTHF is mainly synthesized by chemical methods.In the method,folic acid is used as the starting material,which is reduced to tetrahydrofolate acid by strong reducing agents.The product is further chemically methylated with formaldehyde to methylenetetrahydrofolate acid and then reduced to 5-MTHF.However,the chemically synthesized 5-MTHF is a racemic mixture,of which only(6S)-5-MTHF has physiological activity.Therefore,it needs to be purified by crystallization separation or chromatography separation,etc.The chemical synthesis of 5-MTHF has the disadvantages such as low production,low purity,environmental pollution,and harm to human health.In contrast,the biological synthesis of L-5-MTHF is promising because of its high specificity,high purity,environmentally friendly,and low cost.Therefore,modifying the metabolic pathway of Escherichia coli by genetic engineering to increase the production of L-5-MTHF will be very beneficial to the industrial production of L-5-MTHF in the future.The main experimental work in this study is as following.Firstly,the synthetic pathway of L-5-MTHF in E.coli was thoroughly analyzed and then a new L-5-MTHF-producing pathway was designed constructed in E.coli.At firstly,the intrinsic genes in the pathway of E.coli were overexpressed.The genes for dihydrofolate reductase(encoded by folA)and MTHFR(encoded by metF)from E.coli were selected,and their genes were inserted into two multiple cloning sites(MCSs)of pACYCDuet-1 vector,respectively,to construct the pACYCDuet-metF-folA,which was then transferred into the competent E.coli cells.Then,in order to construct a new pathway for L-5-MTHF production,the enzymes related to C1 transfer pathway from Methylobacterium extorquens AM1 were selected.The genes of formate-THF ligase,formyl-THF cyclohydrolase,and methylene-THF dehydrogenase(encoded by ftfL,fchA,and mtdA,respectively)were inserted into two MCSs of pETDuet-1 vector to construct pETDuet-C1T,which was co-transformed into the host E.coli with pACYCDuet-metF-folA.Additionally,the genes of formate-THF ligase,formyl-THF cyclohydrolase,and methylene-THF dehydrogenase(encoded by fhs,fchA,folD,respectively)in the Wood-Ljungdahl pathway of anaerobic Clostridium autoethanogenum DSM 10061 were selected to construct pETDuet-WL,which was co-transformed into the host E.coli with pACYCDuet-metF-folA.Further,the metH gene of host strain was also knocked out considering that the product of methionine synthase(encoded by metH gene)inhibits the activity of MTHFR.And the pACYCDuet-metF-folA was co-transformed into the mutant host strain with the pETDuet-C1T and pETDuet-WL,respectively.Finally,8 engineered strains were totally constructed for producing L-5-MTHF.Further,the growth curve of all the engineered strains was determined,and their growth was basically same as that of the original strain E.coli BL21(DE3)without adding IPTG,folic acid,sodium formate and glucose.The production of L-5-MTHF of all the engineered strains was then determined using HPLC under the optimized detection conditions,and their production were analyzed and compared.Two engineered strains[BL21-WL and BL21(△metH)-C1T]with higher production were screened out.The products of the two strains were detected by LC-MS,and the correctness of the target product was confirmed.The fermentation conditions of the two strains were further optimized,and it was found that the optimal induction time of the strain was 16 h(BL21-WL)and 12 h[BL21(△metH)-C1T],respectively,and the optimal induction temperature was 25℃.The optimum amount of IPTG was 0.6 mM,and the optimum time for adding IPTG was when the OD600nm of the culture reach 0.8.Based on the induction conditions,the influences of adding exogenic substances on the production of L-5-MTHF in the engineered strains were explored.It was found that in the medium supplemented with folic acid,sodium formate,and glucose,the strain BL21-WL had a highest production.The strain BL21(△metH)-C1T had the highest production in the medium supplemented with only folic acid.However,the optimal amounts of folic acid and sodium formate added to the medium still needed to be further optimized.Finally,in order to investigate the constructed pathway in the two engineered strains,the transcription and expression of exogenous genes,the activities of their encoding enzymes,and the accumulation of intermediate metabolites were analyzed and determined.It was found that methylenetetrahydrofolate dehydrogenase was the main rate-limiting step in this pathway.MTHFR also had a certain limiting effect on the production of target product,which points out the directions to further modify the strain to improve the production of L-5-MTHF.In summary,a series of engineered strains were successfully constructed by introducing exogenous genes,and a new metabolic pathway for L-5-MTHF production was constructed in E.coli and its mutant with the metH gene knockout.By developing a rapid HPLC detection method for detecting the product,the production of L-5-MTHF of all engineered strains were determined,and two engineered strains,BL21-WL and BL21(△dmetH)-C1T,were screened out.Preliminary optimization of the fermentation conditions showed that the production of BL21-WL was 445.94 μg·g-1,and the production of BL21(△metH)-C1T was 527.84 μg·g-1.Compared with the original E.coli BL21(DE3),the production has increased by 10.0 times and 11.8 times,respectively.Furthermore,the newly constructed pathway to synthesize L-5-MTHF was further studied,and the limitations of this metabolic pathway were discovered,which provides a way to further improve the production of L-5-MTHF.