A new approach for improving epothilone B yield in Sorangium cellulosum by the introduction of vgb epoF genes
A recombinant plasmid, pRP-GFP, harboring the green fluorescent protein (GFP) gene from the broad-host-range mobilizable plasmid pRK415 of the RK2 family was constructed and transferred by conjugation from Escherichia coli S17-1 to Sorangium cellulosum So ce90. The results of Southern blot analysis showed that the plasmid replicated autonomously without being integrated into the chromosome of the bacterium. In pRP-GFP, gfp was driven by a So ce90 DNA fragment called EpoPro, which is an 890-bp DNA segment spanning from -890 to -1 relative to the start codon (GTG) of epoA, a gene that encodes EPOS A, which is presumed to be involved in the initiation of epothilone biosynthesis. The GFP in the transformed S. cellulosum So ce90 was detected by fluorescence microscopy, which suggests that the EpoPro fragment had the function of promoter. Because the green fluorescent bacilli could be directly observed by fluorescence microscopy, the stable expression of GFP was rapid and convenient for conjugation screening in addition to the antibiotic resistance genes within the constructed plasmid. This is the first report on an exogenous plasmid that can be stably maintained as an extrachromosomal element in S. cellulosum.