Study On The Catalysis Of P450_BS?on The ?-hydroxylation Of Short-chain Fatty Acids

?-Hydroxy fatty acids are a class of compounds with important activities,which are widely used in natural product synthesis,drug development and biomaterials.One step oxidation of renewable fatty acids to produce ?-hydroxy fatty acids is an ideal,direct and economical preparation method.However,regioselective oxidation of the C-H bonds in fatty acids is extremely challenging chemically and enzymatically in vitro,and hasn't been realized.Peroxidase P450BS?,an enzyme with poor regioselectivity,uses hydrogen peroxide to catalyze the oxidation of the abundant feedstock fatty acids to produce ?-and?-hydroxy fatty acids as well as some amount of 1-alkenes.Moreover,the substrate specificity of P450BS? is high,only accepting fatty acids with a carbon chain length greater than 10 as the substrate.Therefore,improving the regioselectivity of P450BS?,broadening the range of substrates,and thus establishing a biosynthetic method that uses fatty acids as substrates to directly generate ?-hydroxylated fatty acids has great theoretical and application significance.In our laboratory's recent research on the fatty acid decarboxylation activity of P450BS?,it was found that engineered P450BS?decarboxylase mutant had a broad substrate scope and increased selectivity towards the production of 1-alkenes.Based on these results,we aim to achieve a P450BS?variant that is able to efficiently ?-hydroxylate short-chain fatty acids by directed evolution.Our first strategy is to conduct a combinatory saturation mutation screening of the obtained P450BS? decarboxylase mutant based on its broad substrate scope,hoping to change its favor of the decarboxylation pathway to that of ?-hydroxylation pathway.However,after several rounds of saturation mutation,the problem of suppressing 1-alkenes production could not be solved.Therefore,we changed our directed evolution strategy and used the wild-type P450BS? as the parent to screen the variant that can accept the C6 fatty acid and regioselectively produce the corresponding ?-hydroxy fatty acid.We selected the P450BS? residues within 8 A from the C1-C8 of the substrate in the crystal structure of the P450BS?-palmitic complex,and found that changes in the five residues of V74,L78,Q85,V170 and G290 were able to increase the ?-hydroxylation activity.After several rounds of saturation mutation screening,we obtained the P450BS?-L78I/Q85H/G290I variant,which can catalyze saturated and unsaturated natural fatty acids with C5-C18 chain length and non-natural fatty acid substrates with various functional groups.The conversion of the most of the substrates is above 90%with the regioselectivity above 75%.This study successfully broadens the substrate scope of P450BS?,and paves the way for the synthetic application of this P450 peroxygenase.