Fig. 5
Verification of fdx_0135, fdr_0130 and fdr_7100 knock-out. a Schematic diagram: PCR verification of the knock-out of three redox protein genes. The control strains contained the plasmid expressing each target gene. Knock-out of each target gene was confirmed based on positive and negative PCR verification. The upstream and downstream fragments flanking the left and right homologous arms of the knock-out gene were successfully amplified from the knock-out strains, but the internal fragments near the upstream and downstream homologous arms were amplified from only the control strains (upa, upstream homologous arm; downa, downstream homologous arm; up, the upstream fragments flanking the upstream homologous arm; down, the downstream fragments flanking the downstream homologous arm; in-up, internal validation sequence near the upstream homologous arm; in-down, internal validation sequence near the downstream homologous arm). b One-step selection method for knock-out of the three target genes. Hygromycin and sucrose were added to the electroporation plate for sacB-mediated negative selection. Positive PCR verification was conducted by successfully amplifying the up and down fragments of each gene in the knock-out strains; negative PCR verification was conducted by successfully amplifying the in-up and in-down fragments of each gene in the control strains. For fdx_0135: up, 2705 bp; down, 2319 bp; in-up, 931 bp; in-down, 1613 bp. For fdr_0130: up, 2490 bp; down, 2434 bp; in-up, 2334 bp; in-down, 936 bp. For fdr_7100: up, 2174 bp; down, 2106 bp; in-up, 665 bp; in-down, 797 bp. (in-u, in-up; in-d, in-down. PCR fragments from positive PCR verification are marked with solid red arrows; PCR fragments from negative PCR verification are marked with unfilled red arrows). c Epothilone A + B production, total production, and epothilone A + B proportion of the strains with fdx_0135, fdr_0130, and fdr_7100 knock-out compared with the respective control strains. Each strain was analysed in triplicate, and the data are presented as the means ± standard deviations of the values from three independent experiments (n = 3)