Enzymatic Preparation Of Musky Macrolactones Based On Cytochrome P450 Monooxygenase

In all synthetic musks,macrolides are the structurally closest to natural musks,which have wide application in the field of cosmetics,pharmaceutical,food and etc.Currently,musk macrolides are mainly prepared by chemical methods.However,traditional chemical synthesis steps are cumbersome and often accompanied by high energy consumption and high pollution.Due to the high conversion ratio,outstanding regio-and stereo-selectivity and mild reaction condition,biocatalysts—enzymes can be used as a green alternative to the traditional chemical synthesis method.In this paper,the biosynthesis method of macrolides based on cytochrome P450 monooxygenase was studied.Using fatty acids as the initial substrate,two strategies for synthesizing macrolides,exaltolide and silvanone supra,were developed.In addition,the the activity and selectivity of key enzymes were optimized using directed evolution and rational design.Firstly,two-enzyme(combo)cascade reaction was created by coupling a P450-mu from the bacterium Bacillus megaterium or CYP153A from the Marinobacter with a lipase from Psudomonas cepacia.Using pentadecanoic acid and stearic acid as the initial substrate,we show for the first time the application of synthesis hydroxyfatty acid and then lactonization by transesterification.Finally,81 mg of exaltolide were obtained from 242 mg pentadecanoic acid,and 199 mg of silvanone supra from 256 mg palmitic acid.Secondly,a high throughput screening method for identifying P450 variants with selectivity for terminal oxidation was developed and validated.Specificfatty alcohol oxidase(FAO)from Candida tropicalis was applied to quantitatively detect the products of co-hydroxy fatty acids.Then the formation of hydrogen peroxide during the process of oxidation of ?-hydroxy fatty acids is coupled with horse radish peroxidase(HRP)for color reaction with the chromogenic substrate of 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid;ABTS).The oxidation product ABTS radical cation has specific absorbing peak at 405 nm.Based on this principle,the content of c?-hydroxy fatty acids can be detected in few minutes.The FAO-ABTS detection system can be widely employed in directed evolution to improve the activity,selectivity and stability of P450 terminal hydroxylase.Thirdly,in order to further reduce the cost of the substrate,a novel pathway for the synthesis of exaltolide and silvanone supra from stearic acid via a hydroxylation-carbonylation-esterification-hydrolysis-lactonization cascade reaction is presented.The key step was catalyzed by engineered variants of the P450 monooxygenase BM3.Compared with wild-type of P450 BM3,the optimized P450-L188Q mutant showed 4.2-fold increased yield of ?-1 hydroxy stearic acid and 4.5-fold increased yield of ?-2 hydroxy stearic acid.Based on this strategy,we demonstrate the high-value added musky flavors,exaltolide and silvanone supra,can be synthesized from stearic acid with the yield of 3.3%and 5.4%,respectively.Finally,we aimed to improve the stability of PfBVMO,a potential significant tool enzyme in synthesis route of macrolide,by site-directed mutagenesis.The PfBVMO-CtoS mutant enzyme with entire cysteine residues replaced by serine residues,showed significantly higher stability than the wild type.The result would facilitate further work on the macrolide synthesis to simplify the reaction conditions and improve the synthesis efficiency.In summary,based on the hydroxylation reaction of cytochrome P450,high value-added musky macrolides were synthesized successfully from relatively cheap fatty acids.This provides a new way for value-added utilization of fatty acids and green synthesis of musky macrolides.