In carbohydrate-based fermentations of Saccharopolyspora erythraea, a polar knockout of the methylmalonyl-CoA mutase (MCM) gene, mutB, improved erythromycin production an average of 126% (within the range of 102-153% for a 0.95 confidence interval). In oil-based fermentations, where erythromycin production by the wild-type strain averages 184% higher (141-236%, 0.95 CI) than in carbohydrate-based fermentations, the same polar knockout in mutB surprisingly reduced erythromycin production by 66% (53-76%, 0.95 CI). A metabolic model is proposed where in carbohydrate-based fermentations MCM acts as a drain on the methylmalonyl-CoA metabolite pool, and in oil-based fermentations, MCM acts in the reverse direction to fill the methylmalonyl-CoA pool. Therefore, the model explains, in part, how the well-known oil-based process improvement for erythromycin production operates at the biochemical level; furthermore, it illustrates how the mutB erythromycin strain improvement mutation operates at the genetic level in carbohydrate-based fermentations.

Engineering of the methylmalonyl-CoA (mmCoA) metabolite node of the Saccharopolyspora erythraea wild type strain (FL2267) through duplication of the mmCoA mutase (MCM) operon led to a 51% (range 40%-64%, 0.95 CI, N = 152) increase in erythromycin production in a highperformance oil-based fermentation medium. The MCM operon was carried on a 6.8 kb DNA fragment in plasmid pFL2212 which was inserted by homologous recombination into the S. erythraea chromosome. The fragment contained one uncharacterized gene, ORF1; three MCM related genes, mutA, mutB, meaB; and one gntR-family regulatory gene, mutR. Additional strains were constructed containing partial duplications of the MCM operon, as well as a knockout of ORF1, none of these strains showed any significant alteration in their erythromycin production profile. The combined results showed that increased erythromycin production only occurred in strain FL2385 containing a duplication of the entire MCM operon including mutR and a predicted stem-loop structure overlapping the 3′ terminus of the mutR coding sequence.

No abstract

An 8.1-kb region of the Saccharopolyspora erythraea genome, significant for its contiguity to the known genes of the erythromycin biosynthetic gene cluster, was mutationally analyzed and its DNA sequence was determined. The region lies immediately adjacent to eryCI. The newly characterized region is notable for a large, 3.0-kb segment, predicted not to be translated, followed by four probable genes: an acetyltransferase gene, a protease inhibitor gene, a methyltransferase gene, and a transposase gene. Because the probable functions of the genes in this region are not required for erythromycin biosynthesis or resistance and because a deletion of a 6.0-kb portion of this region had no effect on erythromycin biosynthesis, this region marks the outside boundary of the erythromycin gene cluster. Therefore, eryCI represents the end of the cluster. These results complete the analysis of the erythromycin gene cluster and eliminate the possibility that additional soughtafter pathway-specific structural or regulatory genes might be found within or adjacent to the cluster.

The erythromycin-biosynthetic (ery) gene cluster of Aeromicrobium erythreum was cloned and characterized. The 55.4-kb cluster contains 25 ery genes. Homologues were found for each gene in the previously characterized ery gene cluster from Saccharopolyspora erythraea. In addition, four new predicted ery genes were identified. Two of the new predicted genes, coding for a phosphopantetheinyl transferase (eryP) and a type II thioesterase (eryTII), were internal to the ery cluster. The other two new genes, coding for a thymidine 5'-diphosphate-glucose synthase (eryDI) and a MarR-family transcriptional repressor (ery-ORF25), were found at the two ends of the ery cluster. A knockout in eryDI showed it to be essential for erythromycin biosynthesis. The gene order of the two ery clusters was conserved within a core region of 15 contiguous genes, with the exception of IS1136 which was not found in the A. erythreum cluster. Beyond the core region, gene shuffling had occurred between the two sides of the cluster. The flanking regions of the two ery clusters were not alike in the type of genes found.