An Artificial Methanol Assimilation Pathway For Methanol Biological Conversion In Escherichia Coli

Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals.Although biological conversion of methanol can proceed at ambient conditions,there is a dearth of engineered microorganisms that use methanol to produce metabolites.Methylotrophic bacteria can utilize methanol and other reduced one-carbon compounds as their sole source of carbon and energy through a number of specialized enzymes and pathways.The natural metabolic pathway is complex,and that requires the participation of multiple enzymes,and the inability to transfer to a model strain to limited product diversity.Therefore,we have developed a linear artificial metabolism pathway for efficient methanol assimilation and dissimilation:the ability to use methanol is initiated by its oxidation to formaldehyde by methanol dehydrogenase(MDH),and formolase(FLS),which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule,and DHA can be used directly enter to the glycolysis pathway in E.coli.Compared with the natural methanol metabolic pathway,this pathway independent the the ribulose phosphate cycle as the formaldehyde receptor,and it only needs two steps to conversation of methanol in E.coli.We transferred the artificial pathway into E.coli,which proved the feasibility of this pathway in E.coli.In order to improve the utilization rate of methanol,we have directed evolution of key enzyme FLS in the artificial pathway and we have obtained five mutants with higher catalytic activity than original strains.At the same time,we have assembled the different MDH and FLS(and its variants)in vector pET28a and transferred in E.coli.We systematically analyzed their activities in vitro and vivo,detected the biomass(OD600)and the concentration of methanol.And then,we obtained the best engineering bacteria for methanol conversation in the artificial pathway.Finally,the metabolic pathways and metabolic profiles of methanol in vivo were further studied by 13C-methanol isotope tracing.1、The preliminary exploration of artificial metabolic pathway of methanol conversion in E.coli.Firstly,the strain E.coli DH5α were cultured in M9 medium with the sole carbon source of 10 mM 1,3-dihydroxyacetone.The results showed that DHA could as the sole carbon and energy source of E.coli DH5a.And then,we cloned and expressed different activity of methanol dehydrogenase and formolase,and analysis their enzymatic properties and kinetics,respectively.Next,we assembled the gene of mdh and fls on the vector pET28a and transferred to E.coli DH5α with a constitutive promoter for expression.The results showed that the engineering strain can growth in M9 medium with 100 mM methanol.After 48 h,the concentration of OD600 reached 0.68 and the methanol utilization reached 2.32 mM.In order to solve the problem of formaldehyde accumulation and low methanol utilization,we will improve the methanol utilization by molecular modification of the key enzymes in the pathway.2.Study on the directional modification of FLS.In order to improve the catalytic efficiency of FLS,we obtained six hot spots in FLS(PDB:NO:4PZQ)on the active center and substrate channel(T396,T446,M473,S477,L482 and L499)by the protein simulation software Hotspot Wizard 2.0.we carried out the six sites with random mutation and use the whole-cell catalytic screening with formaldehyde as substrate.The results showed that the amino acid sites of 473 and 482 have an obviously influence on the activity of FLS.Compared with FLS-wild,the variants activity of FLS:M473Y,FLS:L482D and FLS:L482R have increased.On the basis of two-point combination mutation,we obtained the beneficial mutations of FLS:M473Y/L482D and FLS:M473Y,Through the analysis of the enzyme kinetic parameters of five beneficial variants(M473Y,L482D,L482R,M473Y/L482D,and M473Y/L482R),the results showed that the catalytic efficiency of the five variants compared with FLS-wild increased by 52.74%,90.90%,129.95%,126.86%and 203.71%,respectively.Among them,the best highest catalytic activity of FLS:M473Y/L482R reach as 82.44 s-1 mM-1.3.Assembly screening of related genes in methanol metabolic pathway and optimization of culture conditions.To further improve the utilization of methanol in this pathway,we investigated two key enzymes:methanol dehydrogenase(MDH)and formolase(FLS).We assembly methanol dehydrogenase(CnMDH,BsMDH),promoter(J23100 and J23106)and fls(wild and the variants)in the vector pET28a and transferred to the Escherichia coli DH5α(ΔfrmA)and cultured in M9 and M9+1 g/L yeast liquid medium with 100 mM methanol,respectively.And compared the differently growth parameter with knock down of formaldehyde dehydrogenase(frmA).The results showed that the strain have higher activity grows better compared with the wild FLS,and the methanol effective utilization have improved.And the higher the methanol dehydrogenase activity with the higher the methanol utilization.By analysis and comparison,compared with inducible expression,the constitutive promoter performed better,the best combination was J23100,fls:M473Y/L482R and BsMDH,and the strain was DH5α(ΔfrmA)/pET28a-J23100-fls(M473Y/L482R)-BsMDH.After cultured for 72 h,the concentration of OD600 reached 1.32,and formaldehyde utilization reached 12.32 mM.Compared with the pre-optimization,the biomass with increased of 94.72%,and the methanol utilization with increased of 432.01%.TPP and Mn2+ can improve the substrate binding capacity(Km)and catalytic efficiency of FLS.Then,we studied the different concentrations of TPP and Mn2+ on the effect of strain growth and methanol utilization.At the same time,the culture temperature and pH were optimized.The experimental results show that the best cultured condition are temperature with 25℃,pH 7,0.6 mM TPP and 0.4 mM Mn2+.On the best culured condition,the optimized methanol utilization reached 16.11 mM with increase of 30.83%.4.Testing of C1-converting in vivo using dynamic 13C labeling and metabolic flow analysis.Under the methanol metabolic pathway,13C-methanol was used to analysis of the metabolic flow.We established the metabolism network in E.coli,and throuth the distribution of 13C-labeled substances in vivo and in vitro.And we calculate metabolic flux and metabolic distribution in E.coli.Th experimental results show that it have highly abundance of 13C with a highly in the glycolytic pathway and tricarboxylic acid cycle(TCA).And the metabolites such as 3-phosphoglycerate,pyruvate,and lysine and serine are highly labeled and more than 30%.By this means,the metabolic orientation and distribution of methanol under this linear pathway were further analyzed,which laid a foundation for subsequent research.