Identification Of Internal Transcriptional Regulators Of Epothilone Gene Cluster In Myxococcus Xanthus

Epothilones are a class of macrolide compound with antitumor activity produced by the Sorangium cellulosum.It is considered an updated replacement product for the anticancer drug paclitaxel.The epothilone biosynthetic gene cluster is 56 kb in length and consists of seven genes.Our laboratory successfully expressed the epothilone gene cluster in the model bacteria Myxococcus xanthus.We found that the transcription level of the downstream gene of internal promoter DP was significantly lower than other genes.DP promoter became the short board of gene cluster transcription.In the meanwhile,it could not activate transcription by the CRISPRa system.Therefore,we speculated that the DP promoter was subject to strict transcriptional regulation in the Myxococcus xanthus,which may be an invisible rate-limiting step of epothilone biosynthesis.The laboratory carried out DNA pull down experiment using the DP promoter in Myxococcus xanthus DZ2,and identified a MXAN₅547 gene annotated as a transcriptional factor of TetR family.Overexpression of MXAN₅547 genes in ZE10（ZE series strains-heterologous expression of epothilone gene cluster in DZ2）,the transcription level of the epothilone gene increased,and the yield of epothilone increased by 1.5 times,indicating that MXAN 5547 could regulate the transcription of epothilone gene cluster.But the regulatory mechanism was unclear.This paper focuses on the analysis of the regulatory mechanism of MXAN₅547,and mainly achieves the following results:1.Identification of DP,the promoter inside the epothilone gene cluster.The software predicts that the DP promoter is 1000 bp in length,containing two possible core promoter regions and a binding site of.the developmentally relevant regulator Mrpc.According to the prediction results,the DP promoter was gradually truncated and the transcriptional activity of the truncated promoter was detected.Finally,the core region of the DP promoter was determined to be between 205～412 bp.2.Expression and purification of the transcriptional factor MXAN₅547.Vectors carrying His,His-sumo,MBP,GST protein tags were used to construct MXAN₅547 expression plasmids.After induced,the results showed that His-5547 recombinant protein was solublely expressed,but the protein was too unstable to obtain sufficient protein after affinity chromatography and ion exchange chromatography.Soluble Hissumo-5547 and GST-5547 recombinant proteins cannot be produced after induction;Soluble MBP-5547 recombinant protein was expressed,and finally we obtained sufficient MBP-5547.3.In vitro binding verification of MBP-5547 and DP promoter.Band shift assay experiments verified that MBP-5547 specifically bound to the full-length DP promoter,indicating that MBP-5547 deletion could bind to DP and play a regulatory role.By analyzing the binding Mars of MBP-5547 and truncated promoters of different lengths,the binding region of MBP-5547 was reduced to the range of 362～412 bp.Further analysis of this DNA sequence found that there were more reverse repeats in the interval of 362～396 bp,and the specific binding sites needed to be verified.4.The transcriptional activation effect on the DP promoter of MBP-5547 was identified by heterologous experiments in vivo.The DP promoter activity reporter vector pSWU19-DP-GFP and the expression vector pMAL-c5X-5547 of the MBP-5547 protein were introduced into E.coli DH5α,and a simple two-plasmid reporter system was constructed.After the addition of IPTG to successfully induce the expression of MBP-5547 protein,the fluorescence value of the strain increased significantly,indicating that the expression of MBP-5547 protein alone could activate the transcription of DP promoter.In summary,this paper identifies the internal promoter DP,the transcription shortboard of the epothilone gene cluster,and confirms that the MBP-5547 protein can bind to the DP promoter and activate reporter gene transcription through experiments in vitro.MXAN₅547 is the first epothilone transcriptional activator reported so far,which further deepens the understanding of the biosynthesis mechanism of epothilone and provides theoretical guidance for subsequent strain modification and yield optimization.