Inhibition of erythromycin synthesis by disruption of malonyl-coenzyme A decarboxylase gene eryM in Saccharopolyspora erythraea

Abstract

Malonyl-coenzyme A (malonyl-CoA) decarboxylase is widely distributed in prokaryotes and eukaryotes.However, the biological function of this enzyme has not been established in any organism. To elucidate the structure and function of this enzyme, the malonyl-CoA decarboxylase gene from Saccharopolyspora erythraea (formerly Streptomyces erythreaus) was cloned and sequenced. This gene would encode a polypeptide of 417 amino acids. The deduced amino acid sequence matched the experimentally determined amino acid sequences of 25 N-terminal residues each of the enzyme and of an internal peptide obtained by proteolysis of the purified enzyme. This decarboxylase showed homology with aminoglycoside N6'-acetyltransferases of Pseudomonas aeruginosa, Serratia marcescens, and Kiebsiella pneumoniae. Northern (RNA) blot analysis revealed a single transcript. The transcription initiation site was 220 bp upstream of the start codon. When expressed in Escherichia coli, the S. erythraea malonyl-CoA decarboxylase gene yielded a protein that cross-reacted with antiserum prepared against S. erythraea malonyl-CoA decarboxylase and catalyzed decarboxylation of [3-14C]malonyl-CoA to acetyl-CoA and 14CO2. The S. erythraea malonyl-CoA decarboxylase gene was disrupted by homologous recombination using an integrating vector pVVHM3. The gene-disrupted transformant did not produce immunologically cross-reacting 45-kDa decarboxylase, lacked malonyl-CoA decarboxylase activity, and could not produce erythromycin. Exogenous propionate restored the ability to produce erythromycin.These results strongly suggest that the decarboxylase provides propionyl-CoA for erythromycin synthesis probably via decarboxylation of methylmalonyl-CoA derived from succinyl-CoA, and therefore the malonylCoA decarboxylase gene is designated eryM. The gene disrupted mutants also did not produce pigments.

A novel transmembrane serine/threonine protein kinase gene from a rifamycin SV‐producing Amycolatopsis mediterranei U32

“…The Nde I/ Hin dIII fragment from pZK1 was released, and the 1.8‐kb fragment was recovered from the gel with QIAquick Gel Extraction Kit (Qiagen) and end‐filled with Klenow enzyme, the blunt‐end fragment was then cloned into pWHM3 under the promoter of the erm * gene [21], the clone with correct orientation was chosen by restriction enzyme digestion. S. cinnamonensis protoplasts were prepared and transformed according to the methods described by Hopwood et al .…”

Enhancing of erythromycin production by Saccharopolyspora erythraea with common and uncommon oils

“…They have been used as antifoams [2], sole carbon sources [24], auxiliary carbon sources [9], to provide precursors for antibiotic synthesis [6] and to remove the antibiotic from bacterial access and reduce its suppressive effect on antibiotic production [10]. Oils can serve as sources of precursors for the biosynthesis of 6-erythronolide B [7,13,14]. Recently, the efficiency of rapeseed oil in the production of erythromycin by S. erythraea was shown [9,22].…”

Physical-genetic map of the erythromycin-producing organism Saccharopolyspora erythraea

“…Functions for the majority of the predicted genes have been proposed (Donadio et al, 1993;Summers et al, 1997). Several additional genes thought to be involved in erythromycin biosynthesis have been identified and characterized (Hsieh & Kolattukudy , 1994 ;Linton et With the recent development of high-resolution PFGE, physical mapping of large ( > 5 Mb) bacterial chromosomes has become feasible. Recently, the physical maps of the -8 Mb chromosomes of several Streptomyces spp., which are closely related to Saccharopolyspora, have been reported (Kieser et al, 1992;LeBlond et al, 1993LeBlond et al, , 1996Lezhava et al, 1995;Pandza et al, 1997).…”