Study On The Catalytic Mechanism Of Key Enzymes In L-Serine Synthesis And L-Serine Production Through Multi-Enzyme Cascade Catalysis

L-Serine is an important bio-based chemical with widely applications in pharmaceutical,cosmetics and food industries.In recent years,the production of L-serine through microbial fermentation using renewable raw materials as substrates by engineering Escherichia coli and Corynebacterium glutamicum has been realized by many researchers.However,the key metrics of L-serine production such as concentration and yield are difficult to meet the requirements of industrial production.It is crucial to explore the mechanism of L-serine synthesis in microorganisms,examine the specific catalytic mechanisms of key enzymes and construct a multi-enzyme cascade system for effective biological production of L-serine.D-3-phosphoglycerate dehydrogenase SerA catalyzes the dehydrogenation of D-3phosphoglycerate(D-3-PG),which is the first step in L-serine synthesis.In the previous study,we found that SerA in Pseudomonas stutzeri A1501 cannot catalyze the dehydrogenation reaction of D-3-PG exclusively.Coupling with the reduction reaction of 2-ketoglutarate(2KG)is necessary to drive D-3-PG dehydrogenation.Through stoichiometric analysis,this study found that SerA catalyzed the transhydrogenation reaction between D-3-PG and 2-KG to generate 3-phosphohydroxypyruvate and D-2-hydroxyglutarate(D-2-HG).Then,the kinetics mechanism of transhydrogenation reaction catalyzed by SerA was analyzed from the perspective of kinetics,and it was confirmed that the dehydrogenation of one molecule of D3-PG catalyzed by SerA was accompanied by the generation of one molecule D-2-HG.In addition,metabolomic analysis showed that the mutation of D2HGDH which is responsible for the catabolism of D-2-HG in P.stutzeri A1501 would affect L-serine synthesis and a variety of core metabolic pathways.D2HGDH should also play a role in promoting the synthesis of L-serine.The distribution of SerA and D2HGDH in different bacteria was further analyzed.It was confirmed that SerA in E.coli can also catalyze the transhydrogenation reaction between D-3-PG and 2-KG.However,there is no homologous protein of D2HGDH in E.coli,which means there should be other unknown D-2-HG catabolic pathway in the strain.In this study,the catabolic mechanism of D-2-HG in E.coli was investigated,and it was found that the putative FAD-dependent dehydrogenase YdiJ had high activity towards D-2HG.Through the mutation of ydiJ,it was found that YdiJ plays crucial function in the catabolism of D-2-HG in E.coli MG 1655.The exogenous expression,purification and enzymatic property analysis confirmed that YdiJ could specifically catalyze the dehydrogenation of D-2-HG to produce 2-KG.The cellular localization and electron transport mechanism of YdiJ were further analyzed,and YdiJ was proved as a membrane-bound D-2HG quinone oxidoreductase.The electrons produced by D-2-HG dehydrogenation catalyzed by YdiJ can be transferred into the electron transport chain through menaquinone or coenzyme Q8.In addition,the double mutant strain obtained by knocking out serA in E.coli MG1655 ΔydiJ no longer accumulated D-2-HG extracellularly,indicating that SerA plays a key role in D-2-HG synthesis in E.coli MG 1655.Overexpression of SerA can cause the excessive accumulation of D-2-HG.We tried to overexpress YdiJ and D2HGDH in E.coli MG1655 and Vibrio natriegens,and found that the overexpression of YdiJ could more effectively eliminate the accumulation of D-2-HG produced by SerA than D2HGDH overexpression.Thus,YdiJ has some application potential in the construction of efficient Lserine-producing strains.Production of L-serine through E.coli fermentation is often accompanied by the generation of a large amount of D-2-HG,leading to the decrease in the concentration and yield of L-serine.This study designed a non-natural L-serine synthesis pathway that does not involve D-2-HG production by integration with thermodynamically favorable D-glycerate oxidation.It was found that the NAD+-independent D-lactate dehydrogenase EcDLD in E.coli has higher D-glycerate oxidase activity.Then,we constructed a multi-enzyme cascade system composed of five enzymes,which are alditol oxidase from Streptomyces coelicolor,L-alanine dehydrogenase from Archaeoglobus fulgidus,EcDLD,catalase from Aspergillus niger and formate dehydrogenase from Ogataea parapolymorpha DL-1.After optimizing the concentration of different enzymes,18.51 mM glycerol was converted into 17.58 mM Lserine within 12 h,and the molar yield of L-serine was 0.95 mol/mol.The concentration,yield and productivity of L-serine reported in this thesis are the highest level of L-serine production by in vitro multi-enzyme cascade system.In summary,this study researched the anabolism and catabolism of D-2-HG,a byproduct of L-serine fermentative production.We clarified the catalytic mechanism of SerA and YdiJ,which are the key enzymes in L-serine synthesis.It provided a certain theoretical basis for further efficient production of L-serine by fermentation.In addition,this study designed a non-natural L-serine synthesis pathway that does not involve the production of D2-HG.We performed the screening of key enzymes and the optimization of reaction conditions in the in vitro catalytic system.It provided a feasible technical·scheme to efficiently produce L-serine from glycerol through biocatalysis.