Construction And Optimization Of An In Vitro Multi-Enzyme Biocatalvtic System For Producing L-Lactate With D-Glucose As Substrate

In vitro synthetic biology is an important part of synthetic biology.in vitro synthetic biology contains a number of substrates,enzymes,enzyme complexes and coenzymes in a single reaction systems for the production of desired products from cheap substrates.In vitro synthetic biology has some obvious advantages compared with in vivo synthetic and metabolic engineering,such as high product yield,fast reaction rates,high tolerance in extreme environments,easy product separation,broad reaction conditions and easy optimization.In vitro synthetic biology has great potential application for energy,chemicals,food and drugs.1 We constructed a cofactor balanced and ATP-free in vitro synthetic enzymatic biosystem containing only five thermophilic enzymes for the synthesis of lactate from D-glucose.2 After the optimization of enzyme expression and purification,all recombinant enzyme can be expressed well in E.coli BL21(DE3).3 Through determining the activity,the dihydroxy acid dehydratase has low activity of converting glycerate to pyruvate,The low activity of dihydroxy acid dehydratase limits the application of this pathway.4 1 m L reaction system contains 100 mmol/L of HEPES buffer(p H7.0),5mmol/L of magnesium chloride,5 mmol/L of NAD+,27.75 mmol/L,1 U/m L of D-glucose dehydrogenase,dihydroxy acid dehydratase,2-keto-3-deoxygluconate aldolase,glyceraldehyde dehydrogenase and L-lactate,respectively.At 50 ?,when the reaction time is 20 hour,the lactate yield is very low,only 56.7 %.This may be at initial condition,D-glucose can't be converted to lactate,Maillard reaction between D-glucose and proteins can be easily happened at high temperature,so enzymes in the reaction system lost the catalytic activity.5 In order to enhance the L-lactate yield,we optimized the enzyme ratio,the concentration of coenzyme and buffer,respectively.1 m L reaction system contains200 mmol/L of HEPES buffer(p H7.0),5 mmol/L of magnesium chloride,5 mmol/L of NAD+,27.75 mmol/L,1 U/m L of D-glucose dehydrogenase,2-keto-3-deoxygluconate aldolase,glyceraldehyde dehydrogenase and L-lactate,2 U/m L dihydroxy acid dehydratase,respectively.Comparing with the initialreaction condition,two main parameters of the optimal condition were altered: the loading of DHAD was raised from 1.0 U/m L to 2.0 U/m L,and the concentration of HEPES-Na OH buffer was raised from 100 m M to 200 m M.These two factors might account for the low yield of L-lactate under the initial condition.After the optimization of reaction conditions,the product yield of lactate was increased from56.7% to 90%.This study suggests that the new pathway can convert D-glucose to lactate and has great potential application for industrialization.