Research On The Biosynthesis Of Glyoxylate And In Vitro Enzymatic Synthesis Of Glycolate

Glyoxylate is the smallest in aldehyde acid and has a series of chemical reaction characteristics.At present,it is widely used in the aera of artificial spices,drug manufacturation and pesticides synthesis.Chemical synthesis is the main method to produce glyoxylate.In this study,we studied the biosynthesis of glyoxylate from biorenewalbe resources.We modified the pathway of the biosynthesis of glyoxalic acid,deleted the genes related to glyoxylic acid metabolism,including malic acid synthase genes aceB,glcB,glyoxalic acid reductase gene ycdW and hydroxy malonic aldehyde synthase gene gcL,to reduce the by-products of gene expression and to enhance the accumulation of glyoxalic acid.Whereafter we introduced the glycolic acid biosynthesis into Escherichia coli.The results showed that the rate of bacterial growth and the utilization rate of xylose decreased with the knockout genes.The strain Xg4GA was cultured 48 h,xylose was consumed up.The yield of glycolic acid and glyoxylic acid are 1.58 g/L and 0.13 g/L respectively.Further,we overexpressed glycolate oxidaseg gene glcDEF to enchance capacity of glyoxylic acid synthesis.The yield of glyoxylic acid reached to 0.56 g/L,but the growth of Xg4GA became bad.Because glycolic acid oxidase is a kind of peroxide flavoprotein,in the process of glyoxylate's biosynthesis,H2O2 was accumulated.The accumulation of H2O2 could not be consumed immediately,so excessive H2O2 lead to bacteria biomass decreased,and the synthesis of glyoxalic acid is also affected.So we overexpressed gene katE encoded catalase,to regulate the concentration of H2O2.With the over-expression of catalase,the concentration of H2O2 decreased,and the biomass of the cells increased,and the concerntration of glyoxylate increased by 36.15%,reached to 0.74 g/L.Glyoxalic acid was toxic,high concentrataion will affect cell growth,so we research synthesis of glyoxalic acid in vitro.We achieved that 0.12 g/L of glycolic acid can be obtained after reacting 12 h by using cell lysis.