Sybr premix ex taq gc dye

We collected samples continuously from the fermentation culture at four measurement points (12, 24, 36, and 48 h of incubation). Then, total RNA of samples was extracted using BIOZOL kits (Total RNA Extraction Regent, BioFast, China) and then transcribed reversely into cDNA with PrimeScript™ Regent Kit with DNAase (Takara, Japan). The gapA gene (glyceraldehyde-3-phosphate dehydrogenase gene, MXAN_2815) was chosen as the reference gene for normalization. The transcriptional level of epothilone gene cluster was analyzed by RT-qPCR on LightCycler^® 480 (Switzerland) with SYBR^® Premix Ex Taq™ GC Dye (Takara, Japan). All the primers used in RT-qPCR are listed in Table S3.

The mutant strains and wild type strains were cultured in 50 mL of CYE liquid medium for 20 h (OD600 was about 1.5), then transplanted into fresh CMO medium with a start OD600 of 0.04, and harvested at the early stage, middle stage and stable stage of exponential growth successively. Total RNA were extracted according to the protocol provided by the BIOZOL Toal RNA Extraction Regent (BioFast, China), and transcribed reversely into cDNA with PrimeScript™ Regent Kit with DNAase (Takara, Japan). We analyzed the relative transcriptional level of the esi gene and epothilone gene cluster by RT-qPCR on LightCycler^® 480 (Switzerland) with SYBR^® Premix Ex Taq™ GC Dye (Takara, Japan). The gapA gene (MXAN_RS13645), encoding glyceraldehyde-3-phosphate dehydrogenase, was used as the reference gene. The primers used in RT-qPCR were list in Additional file 6: Table S4.

We collected samples continuously from the fermentation culture after 48 h of incubation. Then, total RNA of the samples was extracted using BIOZOL kits (Total RNA Extraction Reagent, BioFast, China) and then transcribed reversely into cDNA with the PrimeScript™ reagent kit with DNAase (Takara, Japan). The gapA gene (glyceraldehyde-3-phosphate dehydrogenase gene, MXAN_2815) was chosen as the reference gene for normalization. The transcriptional level of the epothilone gene cluster was analyzed by RT-qPCR on the LightCycler®480 system (Switzerland) with SYBR® Premix Ex Taq™ GC dye (Takara, Japan). All the primers used in RT-qPCR are listed in Supplementary Table S2.