Expression,Purification,and Functional Study Of Flavin-Dependent Halogenase Spih From The Deep-Sea Fungus Spiromastix Sp.

Halogenated compounds are a kind of important natural products,which are widely used in pharmaceutical,chemical and agricultural industry.The presence of halogen elements can enhance the bioactivity of compounds.The synthesis of halides by chemical methods requires strict reaction conditions and presents challenges in controlling regioselectivity.Many organisms in nature can produce halides,such as sponges,corals,fungi and bacteria,which means there are related halogenase in organisms.Recent studies on halogenases show that flavin-dependent halogenases have outstanding performance in substrate specificity and regioselectivity,and are expected to be practical tools for halogenations.Marine natural products are important sources of halides.A large number of polychlorinated compounds Spiromastixones B-O,Spiromastilactones A-M and Spiromastols A-C were isolated from the fungus Spiromastix sp.MCCC 3A00308.They demonstrated potent antibacterial and antiviral activity.The antibacterial or antiviral activity of some compounds was significantly stronger than that of positive control,and resistance was not easy to develop.The discovery of these compounds provides important lead compounds for the development of new drugs.We are interested in the halogenase of these bioactive compounds.Bioinformatic analysis was performed to find the putative halogenases in Spiromastix sp.One halogenase gene g3973 was assumed to be the halogenase and was identified to be responsible for the biosynthesis of Spiromastixones and Spiromastols by CRISPR/Cas9 gene editing technology.The accumulated precursor was isolated and determined to be Spiromastixone A from the knockout mutant.Therefore,the halogenase protein encoded by g3973 was named SpiH.SpiH was compared with known halogenase sequences,and AcoTAhal was the most similar to flavin-dependent halogenase with homology of 61.93%.SpiH was identified as flavin-dependent halogenase according to GGGPSG binding domain in SpiH.Subsequently,SpiH and Fre expression plasmids were constructed and purified SpiH and Fre proteins were obtained in E.coli hosts.In vitro,SpiH can catalyze the mono-,di-,tri-and tetra-chlorination of spiromastixone A.The monochlorination of Spiromastixone A was isolated,purified,and identified to be Spiromastixone B.In addition,SpiH can catalyze the bromination reaction of Spiromastixone A to form several monobrominated compounds.Furthermore,SpiH can catalyze the chlorination of flavone,isoflavone,xanthone,SNAC derivatives of 2,4-dihydroxybenzoic acid and so on.Through the isolation and identification of enzymatic products,SpiH regioselectively halogenates proximal to the bridging position,indicating the great potential of SpiH as biocatalyst with broad substrate promiscuity.