Expression And Purification Of Key Enzymes In Dithiolopyrrolone Biosynthesis And Study On Orf4 Which Upregulates Antibiotic Biosynthesis

Holomycin and thiolutin are two most famous representatives of dithiolopyrrolone antibiotics with broad spectrum antimicrobial activity and potential anti-tumor activity.To elucidate the biosynthetic pathways of these two compounds and establish catalytic reactions in vitro,we tried to overexpress and purify several enzymes which play important roles in the biosynthesis of holomycin and thiolutin.Soluble proteins of three nonribosomal peptide synthetases HlmE,AutE and ThiE,two acyltransferases AutA and ThiA2 were achieved by induced expression in E.coli;lesser soluble proteins of two acyltransferases HlmA and ThiAl,thioesterase HlmC were achieved by induced expression in E.coli,which required larger amounts of induced expression in E.coli;while thioesterase HlmK and an amide synthetase HlmL were expressed as inclusion-body proteins which cannot used for functional analysis in vitro.In order to obtain all soluble enzymes,an inducible expression system in Streptomyces based on the PmtA-NitR inducible system isolated from Rhodococcus rhodochrous J1 was constructed.Unfortunately,only small amount of target proteins and the regulator protein NitR for induction in inclusion-body form were observed,which indicated that the newly established system could not work as expected.The Orf4 protein with unknown function was isolated from Streptomyces thioluteus.Previous study found that heterologous expression of Orf4 improved production of multiple antibiotics in many Streptomyces strains.Bioinformatics analyses showed that Orf4 was a transmembrane protein with 7 transmembrane domains of N terminus and intracellular domain of C terminus.To investigate the function of Orf4,we expressed and purified the C terminal intracellular domain of Orf4(Orf4(247))and obtained the polyclonal antibody against Orf4.Pull-down assay did not find any protein interacting with Orf4.Phosphorylation detection found that Orf4 could not be phosphorylated by itself or dephosphorylated by FastAP alkaline phosphatase,which demonstrated that Orf4 was not a phosphokinase.Western blotting showed that no positive signal could be detected in the heterologous expression strain S.lividans ZX1::pZMYORF4 using ?-Orf4 antibody,indicating no expression of Orf4 in the heterologous expression strain or too low expression level is out of detection.The insert DNA of pZMYORF4 includes the entire open reading frame of orf4 and 390 bp of the downstream region comprised with 99 bp of non-coding region and partial open reading frame of orf5 encoding a cytochrome P450 oxygenase.We found that biosynthesis of undecylprodigiosin was activated by heterologous expression of the 390 bp DNA fragment instead of the separate orf4 gene.