Transcriptional Organization of the Erythromycin Biosynthetic Gene Cluster of Saccharopolyspora erythraea

Abstract

The transcriptional organization of the erythromycin biosynthetic gene (ery) cluster of Saccharopolyspora erythraea has been examined by a variety of methods, including S1 nuclease protection assays, Northern blotting, Western blotting, and bioconversion analysis of erythromycin intermediates. The analysis was facilitated by the construction of novel mutants containing a S. erythraea transcriptional terminator within the eryAI, eryAIII, eryBIII, eryBIV, eryBV, eryBVI, eryCIV, and eryCVI genes and additionally by an eryAI ؊10 promoter mutant. All mutant strains demonstrated polar effects on the transcription of downstream ery biosynthetic genes. Our results demonstrate that the ery gene cluster contains four major polycistronic transcriptional units, the largest one extending approximately 35 kb from eryAI to eryG. Two overlapping polycistronic transcripts extending from eryBIV to eryBVII were identified. In addition, seven ery cluster promoter transcription start sites, one each beginning at eryAI, eryBI, eryBIII, eryBVI, and eryK and two beginning at eryBIV, were determined.

Phenotypes and gene expression profiles of Saccharopolyspora erythraea rifampicin-resistant (rif) mutants affected in erythromycin production

“…More interestingly, all genes of the ery cluster except eryBI (SACE 0732) showed a similar gene expression profile, being down-regulated in the rif6 mutant and not significantly affected in the rif1 compared to the wild type (Figure 4A , left panel). In contrast, eryBI , which is transcribed monocistronically with the transcript start site facing toward that of eryE [ 21 ], exhibited an opposite behavior, e.g. being up-regulated in the rif6 mutant and not significantly affected in the rif1 compared to the wild type.…”

“…Extensive genetic studies have provided some insight into the genes involved in erythromycin biosynthesis [ 19 , 20 ]. The erythromycin gene cluster contains 20 genes arranged in four major polycistronic units [ 21 ]. Evidence for regulatory genes has been missing for a long time hampering efforts to enhance erythromycin production other than by medium manipulation, random mutagenesis and selection.…”

Heterologous erythromycin production across strain and plasmid construction

“…Comprised of >20 genes and spanning 50 kbp of DNA, the pathway posed several issues to conventional E. coli expression plasmids which were generally designed for single genes. 14,[18][19][20][21][22] Further complicating the reconstitution process was the significant size (10 kb each) of the polyketide synthase genes. 19,20 The initial approach to deal with the transfer of a complex modular polyketide system such as erythromycin was to rebuild existing expression plasmids (from the pET family) with constructed operons containing the erythromycin pathway genes.…”

Improved heterologous erythromycin A production through expression plasmid re‐design

“…11,12 However, it has previously been determined that this gene is unnecessary for erythromycin A biosynthesis and, instead, most likely plays a role in antibiotic resistance and isoflavone glucoside bioconversion. [13][14][15][16][17][18] As a result, eryBI was eliminated from the operon design of pJM2. The result was expected to further reduce the metabolic burden of final coordinated gene expression.…”