Determination Of Key Enzymes In Lacate Synthesis Pathway Through In Vitro Multi-Enzyme Catalysis

The study of the key enzymes plays an important role for the optimization of metabolic pathways because the key enzymes of metabolic pathways control the flux of the whole pathway.The objective of this study is to find the key enzymes in the lactate synthesis pathway which includes glycolytic pathway,that converts glucose to pyruvate and the lactate dehydrogenase reaction.Based on the metabolic control analysis theory,an in vitro metabolic pathway analysis method was presented for key enzyme determination in a lactate producing E.coli strain.First crude enzyme extracts were obtained from this strain and the absolute protein concentrations for all enzymes in the pathway were measured.Then individual pure enzymes were added to the crude extract and the pathway flux changes were measured to calculate the flux control coefficients(FCCs)of the enzymes.The enzymes with the largest FCCs are the key enzymes in the pathway.All enzymes in the pathway were purified by gene cloning and expression in E.coli BL21.The purified enzymes were then mixed in different ratios to build the in vitro pathway.A surprising finding from the in vitro analysis is that fructose bisphosphate aldolase class II(FbaA)also have phosphoglycerate kinase(Pgk)activity catalyzing 1,3-bisphospho-D-glycerate to 3-phosphate-D-glycerate.The FbaA and Pgk have the same operon,and Pgk is a essential gene,so the discovery of new features of FbaA shed new light on the regulation of glycolytic pathway.Enzyme kinetics parameter such as Km and specific enzyme activity were measured by varing the substrate concentrations.By titration of pure enzymes to crude enzyme extract obtained from a lactate producing strain,the absolute enzyme concentration in the enzyme extract were determined to be:37.8mg/L glucokinase(Glk),107mg/Lphosphoglucose isomerase(Pgi),78.7mg/L 6-phosphofructokinase I(PfkA),262.3mg/Lfructose bisphosphate aldolase class II(FbaA),63.4mg/L triosephosphate isomerase(TpiA),1457mg/L glyceraldehyde 3-phosphate dehydrogenase(GapA),806mg/Lphosphoglycerate kinase(Pgk),6.7 mg/L pyruvate kinase II(PykA),and 316.1 mg/Llactate dehydrogenase(Ldh).By measuring relative flux changes after the addition of one enzyme the flux control coefficient(FCC)of that enzyme can be calculated.PykA and GapA had the largest FCCs,0.98 and 0.60 respectively.Therefore these two enzymes are the key enzymes in the lactate producing strain.Overexpression of PykA and GapA in the strain could further improve lactate production.