The Synthesis Of Alpha-Ketoglutarate By Whole-Cell Biotransformation

Alpha-ketoglutaric acid is an important multifunctional organic acid.It not only participates in the synthesis and metabolism of amino acids,vitamins and organic acids in the body,but also plays an important role in protein synthesis,skeletal muscle development and metabolism.Then,it can reduce the body loss of postoperative patients and long-term patients.With its important physiological functions and excellent chemical properties,?-ketoglutarate has been widely used in food,medicine,cosmetics and other fields.In this paper,Corynebacterium glutamicum was used as the chassis cells for the production of ?-ketoglutarate,and the molecular engineering and metabolic engineering theory were used to transform the recombinant Corynebacterium glutamicum strain.Corynebacterium glutamicum is a food-safe industrial microorganism,which has been used in the industrial production of various amino acids such as glutamic acid,lysine and arginine,and high value-added products such as organic acids.In recent years,due to the lack of research on the production of ?-ketoglutarate by Corynebacterium glutamicum,and its mechanism for the synthesis of ?-ketoglutarate is scarce.Acid is still subject to many restrictions.Therefore,production of ?-ketoglutarate by whole-cell of Corynebacterium glutamicum has a large potential advantage.In order to reduce the damage to cells caused by hydrogen peroxide,we screened catalase from P.putida NBRC 14164,P.ananatis Yl,E.coli MG 1655,C.glutamicum ATCC13032,B.subtilis S1,Shewanella oneidensis MR-1,respectively.Co-expressing L-glutamic acid oxidase and catalase for whole cell transformation by constructing a recombinant strain can solve the problem of exogenous addition of hydrogen peroxide.In order to improve the conversion rate of ?-ketoglutarate,we constructed a double promoter and double enzyme co-expression strain to obtain pLGOX-tacKatE recombinant strain.At the same time,in order to promote the transformation efficiency of whole cell transformation,this study selected 2.5%of Triton X-100 as the surfactant,with the substrate concentration of 250 g/L,the cell concentration OD600 of 30,35?After 48 hours of whole-cell transformation under conditions,the conversion rate of?-ketoglutarate in the recombinant bacteria reached 93.7%,which was an increase of 26%compared with the original strain.To sum up,we successfully constructed a ?-KG-producing strain by the double-enzyme co-expression and enhancer promoters to achieve efficient whole-cell catalysis in ?-KG producing.Moreover,we also increased ?-KG production in a green and friendly way,which provided useful approaches and theoretical guidance for the development of industrial ?-KG cell factories in the future.