Directed Evolution Of P450_BSβ For γ-lactonization Of Fatty Acids

γ-Lactones are a class of secondary metabolites that exist in nature and are chemically classified as cyclic esters of y-hydroxyacids.They possess a series of unique and important chemical activities,which can be widely used in many fields such as food flavor,drug development,biological control and protection,and biocompatible materials.However,the synthesis of γ-lactones,especially chiral y-lactones,is subject to many limitations,such as the need for activated substrates,the economic value of substrates,numerous reaction steps,the need for precious metal catalyst and harsh reaction conditions.Therefore,the direct synthesis of y-lactones from green,biorenewable substrates is a very valuable work.Lactone synthesis is associated with fatty acid metabolism in organisms.Inspired by natural strategies,we envisage obtaining y-lactones directly from nature’s widespread,green,biorenewable fatty acids through hydroxylation and lactonization of methylene C-H bonds.We selected the hydroxylase P450BSβ of the cytochrome P450 enzyme family as the object of study.Wild-type P450BSβ can catalyze the formation of α-hydroxy-fatty acids and β-hydroxyfatty acids by hydrogen peroxide bypass pathway.We expect to obtain mutants with high regional selectivity and high stereoisomerism for oxidation at the Cγ of fatty acids by directed evolution of P450BSβ,so as to achieve one-step synthesis of chiral γ-lactones from fatty acids.In this study,based on the hydroxylation mechanism of P450 enzyme and the crystal structure of P450BSβ,we selected 38 sites in the Heme domain and substrate 7 A for iterative saturation mutation based on the directed evolution experience of the P450 family of enzymes.After five rounds of iterative saturation mutation,the final mutant P450BSβG290I/L78G/F173S/Q85F was obtained,which could catalyse the conversion of caprylic acid(C8:0)to y-octanolactone with 93%Cγ region-selectivity and 89%substrate conversion,and the product enantiomer ratio reached 95:5.The optimal H2O2 concentration for 5 mM caprylic acid was 5 mM,and the conversion number and conversion efficiency of γ-lactone reached 3278 and 364 min-1,respectively,showing a better enzyme activity.In addition,this mutant catalyzed the production of γ-octanolactone from caprylic acid in preparation stage expansion experiments,and the yield reached 70%,indicating that this mutant has the potential to be used in a large number of synthesis.In summary,the mutant enzyme can catalyze green,cheap,bioavailable caprylic acid to produce chiral(S)-γ-octanolactone with high regional selectivity,high stereoisomerism selectivity and high reactivity under mild reaction conditions using cheap hydrogen peroxide as oxidant.In this study,a new pathway of one-step synthesis of chiral y-lactone from fatty acids catalyzed by enzyme was opened up through the directed evolution of P450BSβ,which successfully expanded the application range of P450BSβ,and provided a theoretical and practical reference for the directed evolution of P450 enzyme.