Directed Evolution Of Carbon Chain Elongating Enzyme GALS And Its Potential Application Value In Artificial Ecology

There are many types of one-carbon resources on the earth.Among them,methanol and formaldehyde are one of the basic intermediate products of biological metabolism.They are widely sourced and cheap,and are an important raw material for substrates.A highly active mutant Glycolaldehyde synthase(GALS)was finally obtained by modifying benzoyl formate decarboxylase from pseudomonas malodorous bacteria.It can catalyze the dimerization reaction of formaldehyde.The product is glycolaldehyde,and glycolaldehyde is an important intermediate product of life metabolic activities.It participates in various reactions in biological life activities and has many applications in daily life.Two metabolic pathways were designed with GALS,but the key rate-limiting enzymes in both reaction processes are GALS.Therefore,improving the enzyme activity of GALS can significantly improve the utilization efficiency of formaldehyde,which is not only particularly important for the two metabolic pathways designed,but also lays a foundation for subsequent studies.To this end,in the preliminary study,the laboratory obtained high-activity mutants by means of site-directed mutation and high-throughput screening.The first way of design is to start from methanol and use methanol dehydrogenase(BmMDH)GALS and alcohol dehydrogenase(aLd A)to generate glycolic acid through three-step catalytic reaction.Glycolic acid can participate in metabolic pathways such as tricarbonic acid cycle in organisms and be utilized by organisms.In order to achieve this pathway,the key enzymes in the process,BmMDH,GALS and aLdA,were first expressed in vitro and the activity of the enzyme was verified.The results showed that glycolic acid was detected by high-performance liquid chromatography(HPLC)and capillary electrophoresis,so This reaction can be achieved.After the in vitro reaction is realized,it is further achieved in vivo.The three-stage enzyme gene was introduced into E.coli with the formaldehyde dissimilation pathway,and methanol labeled with C13 was used as the only carbon source for further growth experiments.As a result,2-phosphoglycerate(2PG)and phosphoenolpyruvate(PEP),two C13-labeled metabolites introduced by the designedroute,were detected,which also indicates that the designed growth path of the glycolic acid strain has also been achieved.The second route takes formaldehyde as the substrate,and also goes through a three-step reaction to produce l-xylose.In this reaction process,two enzymes are used,one is GALS mentioned above,and the other is D-fructose-6 phosphate aldolase(FSA),which can form L-glyceraldehyde from formaldehyde and glycolaldehyde while catalyzing the formation of L-xylose from glycolaldehyde and L-glyceraldehyde.In order to achieve this process,as before,these two enzymes are first expressed and their activity verified.The results show that by derivatizing the reaction product first,L-xylose can also be detected by HPLC.After optimizing the pathway,the final in vitro conversion rate was as high as 64%.This reaction realizes the conversion from simple one-carbon substances to organic monosaccharides.The realization of this process provides a reference for the synthesis of other organic sugars.The design of catalytic pathways using methanol and formaldehyde as substrates provides a new possibility for the utilization of one-carbon resources and is of great significance for the development of biotechnology.