| Bacteria that produce pharmaceutical products such as antibiotics typically do so in very small quantities. A key challenge is to enhance these yields to meet drug development needs and commercial demand. A derivative of a Streptomyces coelicolor strain, which produces an erythromycin precursor (6-dEB), was found spontaneously to produce more than 10-fold higher yields than previously observed (Hu et al., 2003). Initial studies determined that the SCP2*-derived plasmid which carries the polyketide biosynthetic genes is present at about 100 copies per genome. In contrast, the original plasmid is low copy and exists as only one to five copies per genome. In addition to being high copy, the new plasmid is large, about 81 kb, and is stably maintained in the cell population. This combination of high copy number, large size, and stability has not been previously observed in Streptomyces and thus represents a breakthrough in technology to over-express antibiotic gene clusters.; This project focuses on (1) determining what genetic elements are required for high copy and stability of the large plasmid by sub-cloning specific plasmid regions, (2) determining the mechanism of high copy plasmid replication by characterizing replication gene activity, and (3) examining how high plasmid copy number leads to increased product yields by varying the copy number ratios of the transcriptional activator actII-ORF4 and the polyketide biosynthesis genes (DEBS).; Our studies reveal that a plasmid transfer gene, spd, and over-expression of two partition genes, parAB, are required for high copy and stability of the large plasmid. A 45 by deletion in the origin of replication is also required for high copy. This mutation allows for increased expression of the replication genes repI and repII by abolishing binding of the RepI protein to a site upstream of repI and suggests that replication is controlled by iterons. Experiments with the activator gene actII-ORF4 show that both DEBS and actII-ORF4 must be present at high copy number for high metabolite production. Overall, we have made significant progress in developing a novel, practical cloning vector which has the potential to over-express any polyketide gene cluster of interest in Streptomyces . |
