Metabolic Engineering Of Bacillus Subtilis For Efficient Biosynthesis Of 5-methyltetrahydrofolate

Folate(vitamin B9)is a water-soluble vitamin that refers to folic acid(pteroyl monoglutamic acid)and related compounds with folate biological activity.L-5methyltetrahydrofolate(5-MTHF)is the only active form of folate that is important for human body,which can prevent neural tube defects in newborns and treat Alzheimer’s disease.However,the human body cannot synthesize 5-MTHF naturally and must obtain it from food.At present,the chemical synthesis of 5-MTHF and its precursor folic acid has the problem of environmental pollution.Compared with chemical synthesis,microbial synthesis is an important way to realize the green and sustainable supply of 5-MTHF.At present,there are three main problems in the production of 5-MTHF by microbial synthesis:(1)lack of methods for regulating the ratio of different forms of folate;(2)insufficient supply of key precursors guanosine 5’-triphosphate(GTP)and para-aminobenzoic acid(pABA);(3)the difficulty in identifying key genes that affect 5-MTHF synthesis through global metabolic regulation.In order to solve the above problems,in this study,Bacillus subtilis 168 was used as the host strain to achieve the accumulation of 5-MTHF by strengthening the synthesis pathway of 5-MTHF and dihydrofolate(DHF).By introducing the Clustered Regularly Interspaced Short Palindromic Repeats interference(CRISPRi)system,5-MTHF catabolism pathways and competitive pathways were down-regulated,which improved 5MTHF synthesis efficiency.Based on transcriptome analysis of global metabolic network,key genes affecting 5-MTHF synthesis were identified.On this basis,inhibiting expression of the key gene comGC and combining with the modular precursor supply engineering,the production of 5-MTHF was significantly increased.The main research results of this study are summarized as follows:(1)5,10-methylenetetrahydrofolate reductase(MetF)from Escherichia coli MG1655 was expressed in B.subtilis,which replaced the endogenous bifunctional YitJ enzyme of B.subtilis with 5,10-methylenetetrahydrofolate reductase and homocysteine S-methyltransferase activities.This strategy reduced the degradation of 5-MTHF caused by the activity of homocysteine S-methyltransferase,realizing 229.59 μg/L 5-MTHF production in strain A2;through enhancing the expression of dihydrofolate reductase(DHFR),dihydrofolate synthase/folate polyglutamate synthase(DHFS/FPGS),and GTP cyclohydrolase Ⅰ(GCHⅠ),the first rate-limiting enzyme in folate biosynthesis,DHF and tetrahydrofolate(THF)supply increased,resulting 960.27 μg/L 5-MTHF production in strain A8,which was 4.2-fold that of A2.(2)Inhibition of branched pathways involving 5-MTHF intermediates was realized by the CRISPRi system for key genes,including the pyk and tkt genes in the central metabolic pathway,the purN,purH and thyA genes in the purine pathway,the panB and ykpB genes in the coenzyme A synthesis pathway and the ribA,pheA,tyrA and trpE genes in competing metabolic pathways.When panB,thyA,trpE and pheA were separately inhibited,the titer of 5-MTHF of the recombinant strain increased significantly,which was 1.3-fold that of the starting strain A8,reaching 1.58 mg/L.The concentration of 5-MTHF and its different forms of folate were detected by ultra-performance liquid chromatography-tandem mass spectrometry(UHPLCMS/MS).The results showed that the total folate titer was 3.31 mg/L,which is 8.5-fold that of the control strain A0.Compared with A0,the proportion of precursor folic acid in strain A8 decreased from 41.9%to 15.7%,and the proportion of target product 5-MTHF increased to 61.8%.(3)5-MTHF precursor GTP and pABA supply was modularly strengthened.Module M1 is the synthesis pathway of precursor GTP,and module M2 is the downstream synthesis pathway of GTP.In module M1,the genes ndk,gmk,guaA and deoD were overexpressed by promoters P1,P6 and P10 with different strengths,and guaA and deoD genes were identified as key genes.In module M2,the genes folE,yciA,phoA,folB and folK were overexpressed,and phoA and folBK genes were identified as key genes.Key genes guaA and deoD screened in M1 and phoA and folBK in M2 were modularly assembled.When guaA and folBK were under the control of promoters P6 and P10,respectively,the strain B89 had the highest 5-MTHF titer,reaching 2.77 mg/L,which was 60.9%higher than that of control strain A9(1.72 mg/L).The supply of precursors GTP and pABA was strengthened through modular engineering,which balanced the supply of multiple modular precursors and effectively promoted the synthesis of 5-MTHF.(4)Transcriptome analysis of three strains of B.subtilis with low,medium and high levels of 5-MTHF titer was performed,resulting identification of significantly up-and downregulated genes.The expression of 50 significantly down-regulated genes was inhibited by CRISPRi.When the genes comGB,comGF,thiO,thiD,coiA,thiF,ygaK,comGC,cypX and yhcV were inhibited,respectively,the titer of 5-MTHF increased by more than 10%compared with A9.Inhibiting the expression of comGB led to the highest titer of 5-MTHF,reaching 2.89 mg/L.The expression of 25 genes with significantly up-regulated transcription levels was overexpressed by the strong promoter P566.When the genes gatC,yopO,yoqC,yrvD and yciB were overexpressed,respectively,the titer of 5-MTHF increased by more than 17%compared with A9.Overexpressing yciB led to the highest titer of 5-MTHF,reaching 2.34 mg/L.The 15 key genes(comGB,comGF,thiO,thiD,coiA,thiF,ygaK,comGC,cypX,yhcV,gatC,yopO,yoqC,yrvD and yciB)screened for improving 5-MTHF biosynthesis were combined with the modularly strengthened precursor supplying strain B89.When the gene comGC was inhibited in B89,the titer of 5-MTHF increased by 23.1%compared with that of B89,reaching 3.41 mg/L,which is the highest titer achieved by microbial synthesis of 5-MTHF.