Screening,Clonging,Expression And Application Of Baeyer-Villiger Monooxygenase

Baeyer-Villiger monooxygenase（BVMO）is a high-value and green enzyme that can complete Baeyer-Villiger oxidation reaction and converts the carbonyl groups contained in the substrate into ester groups.It has been applied to the synthesis pathway of odd-chain fatty acids in Escherichia coli,but no relevant studies have been conducted in eukaryotes,and there are few known BVMO that can be used for the catalysis of long-chain fatty acids.This paper is for the purpose of screened the key limiting enzyme BVMO in the synthesis pathway from oleic acid to odd-chain fatty acids,and established this pathway in Saccharomyces cerevisiae to achieve the synthesis of odd-chain fatty acids in eukaryotes.The research contents and results are as follows:（1）Through literature searching and NCBI pBLAST,14 kinds of BVMOs with catalytic ability for linear ketones with chain length at C10-C18 were obtained.（2）When BVMOs were expressed in E.coli,it was found that the soluble expression ability of different BVMOs was very different.The soluble protein of Afl706BVMO accounted for 65%,while the soluble protein of AmBVMO accounted for less than 10%.（3）The enzyme activities of purified BVMOs were determined.Among all BVMOs,PsaBVMO showed excellent enzyme activity,conversion rate and regioselectivity for substrates such as 3-octanone,2-decanone,2-dodecanone and 2-tetractetracenone.The conversion and regioselectivity of Af2BVMO to long chain acid such as 10-ketopalmitic acid and 10-ketostearic acid were more than 90%.（4）The results showed that eight kinds of BVMOs could be used for the transformation of long chain acids such as 10-ketopalmitic acid and 10-ketostearic acid.Then these BVMOs was applied to the synthesis of odd-chain fatty acids from oleic acid.After fermentation,it was found that Pp_E6C302LBVMO,Af2BVMO,Um BVMO and Bo BVMO could achieve the synthesis pathway,Af2BVMO had the best performance.Finally,5.37 mg/L of9-hydroxynonanoic acid was successfully measured in the engineered strain.In this study,through the expression and determination of BVMO,the enzyme activity,conversion rate and regioselectivity of 15 kinds of BVMO to linear ketone substrates were compared.Finally,8 kinds of BVMO could be used to catalyse long-chain fatty acids.The synthesis pathway from oleic acid to odd-chain fatty acids was successfully established in Saccharomyces cerevisiae as microbial factory,and the optimization of pathway enzyme BVMO was realized.