Construction Of E.coli Strains Producing Glycolate From Glucose

Glycolate（HOCH₂COOH）is the simplestα-hydroxy diacid.Because of its unique properties of both alcohol and acid,it is widely used in textile,food processing,and pharmaceutical industries.Compared with chemical synthesis of glycolate,biological production of glycolate has the advantages of environmental friendliness and low energy consumption,and has become a research hotspot in recent years.In vivo,glyoxylate is the precursor for the synthesis of glycolate,which is reduced to glycolate under the catalysis of glyoxylate reductase（EC1.1.1.29,cofactor is NADPH）.To create a glycolate synthesis pathway in E.coli,there are problems of mismatched reducing power and maximizing carbon metabolic flux to glyoxylate.In view of these two key issues,in order to achieve the improvement of glycolate yield and conversion rate,the following researches were carried out in this study:（1）Introducing exogenous glyceraldehyde-3-phosphate dehydrogenase Gap C solved the problem of mismatch of reducing equivalent.NADPH is the reducing equivalent required for the production of glycolate by glyoxylate reductase,while the reducing equivalent produced by glycolysis is NADH.To balance the cofactor distribution,The gap C gene encoding the NADP⁺-dependent GAPDH（Gap C）from C.acetobutylicum was integrated by replacing the native gap A gene in E.coli ATCC 8739,then strain NZ-G400 was obtained.A low glycolate titer of 0.04 g/L was produced during aerobic shake flask fermentation,while the parental strain did not produce glycolate.（2）Inactivating the isocitrate dehydrogenase（ICDH）.Isocitrate,lies at the junction between the TCA cycle and the glyoxylate shunt.It can enter the TCA via ICDH to generateα-ketoglutarate,or enter the glyoxylate shunt via isocitrate lyase,which cleaves it to produce glyoxylate.However,most of the carbon metabolism fluxes to the TCA cycle instead of forming glyoxylate in vivo.Here,the native icd A gene（encoding ICDH）of strain NZ‐G400was completely inactivated to obtain strain NZ-G416.The glycolate titer of strain NZ‐G416increased to 1.24 g/L,with the yield increasing to 0.5 mol/mol glucose during aerobic shake flask fermentation.（3）Knocking out by-product synthesis and competing pathways.Inactivating enzymes which involved in competing pathway reactions,such as lactate dehydrogenase（Ldh A）,malate synthase（Ace B）,glycolate dehydrogenase（Glc DEF）,aldehyde dehydrogenase（Ald A）,pyruvate oxidase（Pox B）,formazan of glyoxal synthase（Mgs A）and acetate kinase（Ack A-pta）.Then strain NZ-G426 was obtained.During aerobic shake flask fermentation,the glycolate titer of strain NZ‐G426 increased to 2.36 g/L,with the yield increasing to 0.92mol/mol glucose.（4）Optimizing key enzymes to further improve glycolate production.In the synthetic pathway,isocitrate lyase（Ace A）and glyoxylate reductase（Ycd W）are the key enzymes for glycolate production.The enzyme activities of these directly affect the yield and conversion rate.In this study,starting from strain NZ-G416,we used strategies such as strong promoter,multi-copy integration and RBS library regulation to improve the enzymatic activities of Ycd W and Ace A.The strain with modulated ycd W and ace A genes,designated as NZ-G456,produced 3.17 g/L glycolate and the glycolate yield was 1.19 mol/mol glucose.（5）Inactivation of Sth A to further improve the NADPH supply.This result suggested that eliminating the interconversion between NADPH and NADH was essential to ensure a high NADPH supply in the reduction of glyoxylate to glycolate.Thus,the sth A gene was deleted,resulting in strain NZ-G466.The glycolate yield increased to 1.89 mol/mol glucose and the titer increased to 5.3 g/L.（6）Production of glycolate in batch fermentation.Batch fermentation of strain NZ-G466was performed for glycolate production.The initial concentration of glucose was 60 g/L.After 60 h,41 g/L of glycolate was produced,corresponding to a yield of 1.87 mol/mol glucose.In conclusion,through the research in this study,the glycolate production pathway was successfully created in Escherichia coli.Solving the problem of the imbalance of reducing equivalent in the pathway and optimize the key genes of the metabolic pathway.These metabolic engineering strategies can be applied in the biosynthesis of a wide range of valuable industrial chemicals.