Heterologous Expression And Catalytic Mechanism Of Halogenase SclT In Streptomyces Thermolilacinus SPC6

Halides are important materials in agriculture,pharmaceutical and chemical industry.The compounds containing halogen substituents often have new activities or strong pharmacological effects.But the traditional chemical synthesis pathway has the disadvantages of unfriendly environment,harsh reaction conditions,poor regional selectivity and low synthesis efficiency.Because of its high selectivity and specificity,enzymatic method is more in line with the demand of green production and social development.Among them,flavindependent halogenase(FDH)is one of the key enzymes in the natural halide biosynthesis pathway,which is widely distributed,has substrate specificity and halogenation site selectivity,and is the most promising halogenase.A chlorine-containing antibiotic streptochlorin is obtained from Streptomyces thermolilacinus SPC6 isolated from the Badain Gillan Desert.Previous studies have found that halogenase Scl T is one of the core synthetases in the biosynthesis of streptochlorin.Therefore,in this study,the hidden Markov model is used to predict the diversity of FDH in Streptomyces from known isolated sources reported so far.The enzymatic properties of halogenase Scl T in Streptomyces thermolilacinus SPC6 is analyzed by bioinformatics methods.Halogenase Scl T and its coenzymes are heterologous expressed to achieve in vitro enzymatic reaction.Halogenase Scl T gene knockout mutant is constructed in Streptomyces thermolilacinus SPC6 to determine the substrate of halogenase Scl T.The main research results are as follows:1.We predicted the FDH sequences in 374 Streptomyces spp.isolated from known sources using a self-built model(muti-FDH),and a total of 5305 FDH sequences were obtained.The highest number of FDH sequences assigned to Streptomyces isolated from desert and polluted environments.Morevore,the Streptomyces spp.Lived in desert environments contained all the FDH types Therefore,Streptomyces derived from desert environment have a variety of halide synthesis potential worth of investigation.2.The model prediction based on multiple substrates showed that halogenase Scl T in SPC6 belongs to FDH family.Phylogenetic analysis revealed that halogenase Scl T posses some special characteristics in comparison with other known FDHs.Further analysis showed that Scl T is an intracellular soluble protein with multiple phosphorylation sites.The secondary structure of Scl T is composed of α helix,irregular curling alternately,and uniform distribution of extended chains.3.To perform in vitro enzymatic reaction of halogenase Scl T,we recombined the plasmids of Scl T and its coenzymes,which were further heterologous expressed in Escherichia coli BL21(DE3).As a rsults,we obtained and purified Scl T protein with high concentration.The protein was sequenceed and the sequence coverage was 99.7%,indicating that Scl T was expressed accurately in E.coli.The coenzyme activity assays indicated that it could reduce flavin.This reaction layed a foundation for the normal in vitro enzymatic reaction of halogenase Scl T.Based on the sequence characteristics and the chamical structure of streptochlorin,we selected8 compounds which were commonly used as the substrates of FDH in porvious studies for in vitro enzymatic reaction.The results showed no enzymatic reaction,suggesting that halogenase Scl T did not use these simple compounds as the substrates.Our results showed that there were great differences in substrates between Scl T and the similar FDHs,suggesting that Scl T may be a new type of FDH.4.To further verify the catalytic mechanism of halogenase Scl T,we constructed a scl T gene knockout strain(Δscl T)from SPC6.HPLC metabolic fingerprint analysis showed thatΔscl T mutant had multiple unresponsive streptochlorin synthetic intermediates compared with wild type(wt).The fermentation browth of Δscl T mutant was used as substrate of halogenase Scl T for in vitro enzymatic reaction.The HPLC fingerprints before and after the reaction were compared and the result showed that the peak of streptochlorin was decreased,and several compound signal peaks which did not appear in the controls were produced.,suggesting that Scl T catalyzes a variety of intermediates to synthesize streptochlorin,and Scl T has certain substrate breadth.This results broadened the study on the catalytic activity of FDH.In summary,our results elaborated the diversity of flavin-dependent halogenase in Streptomyces and explored the catalytic mechanism of halogenase Scl T in Streptomyces thermolilacinus SPC6.This study laid a foundation for further research on the extraction of flavin-dependent halogenase in Streptomyces and the synthesis mechanism of antibiotic streptochlorin.