Isolation And Identification Of Epothilone Synthesis Related Transcription Factors From Sorangium Cellulosum So0157-2

Sorangium celluosum is a genius bacteria which can produce various secondary metabolite. Among them epothilone is the most impressive which has the same anti-tumor activity as the well-known paclitaxel as they both can stabilize microtubule.Further more epothilone has many advandages over paclitaxel such as smaller molecular weight, simpler structure, having activity in cell lines with paclitaxel resistance, more water soluble, less side effects which makes it a promising agents against tumor as a successor to paclitaxel.The low synthetic efficiency of the producer Sorangium cellulosum, however, has been a great encumbrance for further research. Besides due to lacking of genetic tools, the study of the epothilone biosynthetic regulation and genetic modification is not available. In order to understand the regulation mechanism of the epothilone biosynthesis and improve the epothilone production we separate and identify the epothilone biosynthetic regulator of the Sorangium cellulosum So0157-2.We conducted the DNA pull-down assay which used the noncoding upstream sequence of the epothilone biosynthetic gene clusters as the bait and the magnetic beads based on the biotin-streptomycin interaction. We chosed two different culture conditions,M26culture and synthetic culture, which had an obvious different epothilone yield. We used the full-length upstream sequence of epothilone biosynthetic gene clusters which is about1332bp so as to get the most probable regulate proteins.The mixture of the proteins which had been pulled down from the assay were analysed by the SDS-PAGE.The proteins which exist in both cultures from the synthetic culture were removed and shot-gun mass spectrometry(MS) was used to detect the rest proteins.The result of the MS was aligned to the So ce56and the So0157-2database separately.Sixteen transcription factors were found aligning the So ce56database and five So0157-2specific transcription factors from the So0157-2database respectively. Three proteins among those proteins were chosen after bioinformatics analysis which were named Etf、Etf2、Etf3.The three proteins heterogeneous expression vectors were conducted. The pure proteins were obtained after heterogeneous expression and purification. The EMSA(Electrophoretic mobility shift assay) was used to prove the DNA binding activity. The in vivo regulation of the specific regulation factor couldn’t be detected in the initial strains as the deficiency of Sorangium cellulosum So0157-2’s genetic manipulation system, so we cloned the regulation factor and the promoter region into two separate compatible plasmids and cotransformed E. coli Top10F’ Three different upstream sequences of epothilone biosynthetic gene clusters USE(full-length of upstream sequence of epothilone biosynthetic gene clusters,1332bp),CUSE(conserved USE,425bp) and PUSE(putative USE,158bp)were connected to EGFP(enhanced green fluorescent protein) and cloned into pET-28a named as pWLUSE01,pWLUSE02,pWLUSE03respectively.Genes of three chosen regulation factors were cloned into pBAD33under arabinose promoter so the expression can be induced by arabinose named as pWLETF01, pWLETF02,pWLETF03.The EMSA results showed Etfl can specific bind to the regulatory sequences with USE and CUSE which can form a clear shift band. Non-specific competitive DNA polydldC was added to the reaction solution and retardant bands emerged which proved that the interaction between Etfl and regulatory sequence was specific. The salt concentration in Etfl and regulatory sequence reaction solution had very little influence on the interaction.The interaction between Etf2, Etf3and USE was rather weak as the shift band was smear.We use Etfl to verify the in vivo system.The transcription of Etf1was verified by RT-PCR in the first place,then the expression was confirmed by westernblot. The fluorescence signal values of E.coli Topi OF’ which was cotransformed by pWLETF01and pWLUSE01,pWLUSE02,pWLUSE03were detected by fluorescence spectrophotometer. The fluorescence intensity of the Top10F’which were cotransformed by pWLETF01and pWLUSE01,pWLUSE02was increasing during incubation under non-induced condition and stoped increasing when inducer arabinose added.No fluorescence signal was detected of the ToplOF’which were cotransformed by pWLETF01and pWLUSE03.We conclude that the regulatory sequence USE and CUSE can be recognized by the transcription system of E.coli whereas PUSE can’t. Furthermore Etfl is a negative transcription factor which can bind the regulatory sequence USE and CUSE and repress the transcription of the downstream genes. Meanwhile the in vivo system has been proven. We used the in vivo system to verify the in vivo activity of Etf2and Etf3,however, there was no changes of the fluorescence signal. As Etf2and Etf3had a weak DNA binding activity in vivo,this result showed that the in vivo activity was weak too.