In Vitro Construction Of Reversible Glycine Cleavage System For Kinetic Study And Its Application In C1 Synthetic Biology

Most of the presently used fuels and chemicals are directly or indirectly derived from fossil carbons.With the imminent depletion of fossil carbons and the concomitant increase in atmospheric CO₂,the use of biorefineries for the production of fuels and value-added chemicals from bio-based carbon sources to replace fossil carbon sources has become an option.However,the use of simple sugars and starches directly competes with human consumption and hence can threaten food security and decrease biodiversity.There is an urgent need to search for sustainable and cheap resources as feedstocks for the production of high-value chemicals using microbial fermentation processes.One-carbon(C1)compounds,such as CO₂,formate and methanol have been proposed as ideal feedstocks to alleviate global energy shortage and environmental pollution problems.These compounds are either naturally abundant,or cheap to produce,or available as industrial by-products.However,in nature,only a small group of microbes can utilize C1 compounds to grow,and it is difficult for these microbes to meet the requirements of industrial production due to high cultivation costs and technical limitations.Reprogramming C1 metabolic pathways in industrial model microorganisms such as E.coli and yeast has become the key to solve this problem.The reductive glycine pathway(RGP)is considered to be the most potential artificial carbon fixation pathway with several advantages regarding the enzymes involved,such as oxygen tolerance,high energy efficiency,and little operation overlap with the central metabolism.Recently,this novel C1assimilation pathway has been successfully constructed in E.coli and yeast for the use of formate and CO₂.Theoretically,RGP can replace glycolytic pathway using only formate and CO₂ as substrates.However,the energy efficiency of RGP is so low that glucose is required to sustain the cell growth.Thus,to develop an engineering strain capable of growing solely on formate and CO₂ without glucose supplementation,further improvement of formate and CO₂ assimilation into pyruvate is needed.Related researches have shown that RGP can be improved mainly by enhancing the glycine synthase(also known as glycine cleavage system,GCS,when functioning in the reversed direction),which is the central part of this pathway.Currently,it is of great importance to study how to improve the efficiency of the glycine synthase for advancing C1 biorefinery toward the production of chemicals and materials from formate and CO₂.Therefore,the main research contents of this thesis were as follows:1.The reaction in the direction of glycine cleavage catalyzed by GCS:1.Glycine cleavage reaction in an in vitro glycine cleavage system(GCS)was successfully constructed,and kinetic parameters of the GCS were determined;the appropriate proportions of the four GCS protein components(P-,T-,L-and H-proteins)was obtained;the effect of serine hydroxymethyltransferase(SHMT)on the reaction rate of GCS was studied which showed that when the step catalyzed by T-protein was the rate-limiting step in the glycine cleavage reaction,the addition of SHMT increased the reaction rate by 40.4%;the effect of apo-H protein(H_apo)on the reaction rate of GCS in relation to the ratios of the other three GCS protein components was also examined,which showed that when the step catalyzed by P-protein was the rate-limiting step,the addition of H_apo would reduce the rate of the overall reaction.2.Formaldehyde formed as a by-product in the GCS reaction:formaldehyde as a by-product of the GCS reaction was proved by HPLC.Three causes are identified for its formation.First,the principal one is the decomposition of 5,10-CH₂-THF to form formaldehyde and THF under the given reaction conditions.Next,a certain amount of formaldehyde can be formed in the GCS due to oxidative degradation of THF.Finally,formaldehyde can be produced in the GCS even in the absence of THF.Based on this knowledge and the use of a formaldehyde-dependent aldolase,a glycine-based C1 metabolic pathway for the in vitro biosynthesis of1,3-propanediol was elaborated.3.The reaction in the direction of glycine synthesis catalyzed by GCS:Glycine synthesis reaction was successfully reconstructed in vitro catalyzed by reversed glycine cleavage system(also called glycine synthase,GS),and the rate-limiting step was determined to be the carboxylation reaction catalyzed by P-protein.By studying the effect of each protein component of GCS on glycine synthesis rate,it was found that increasing the ratio of H-protein in GCS protein can improve the affinity of GCS to NH₃ and CO₂,thus greatly improving the catalytic activity in glycine synthesis direction.In addition,adding dithiothreitol(DTT)to the reaction mixture can not only improve the resistance of GCS to formaldehyde,but also act as an activator to increase the rate of glycine synthesis.4.H-protein acts as an enzyme to catalyze glycine cleavage and synthesis:In this work,it was found that lipoylated H-protein(H_lip)alone shows surprisingly the catalytic function of GCS,and is able to catalyze the cleavage and synthesis of glycine with the help of the cofactor required by GCS proteins.The independent catalytic activity of H-protein is closely related to the cavity on the surface of H-protein.5.Construction and analysis of RGP dynamic model:By measuring the kinetic parameters of RGP enzymes under the near physiological conditions and mining the parameter information from database,the kinetic model of RGP was established.The model was successfully applied to simulate the main features of time courses of substrate,intermediate products and product concentration in RGP in vitro.Through parameter scanning,it was found that GCS catalyzed glycine synthesis had a great influence on the RGP flux,among which increasing the affinity of CO₂ and NH₃ to GCS was beneficial to improving the whole RGP flux.