Anhydrotetracycline oxygenase activity and biosynthesis of tetracyclines in streptomyces aureofaciens

Béhal, V.; Hošťálek, Z.; Ванек, З.

doi:10.1007/bf01388144

Cited by 50 publications

(11 citation statements)

References 10 publications

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“…ATC oxygenase catalyses the formation of 5a,lla-dehydrotetracycline by hydroxyla- tion of ATC and requires NADPH. By using purified ATC substrate, cell-free lysates of a commercial strain of S. rimosus M24167 were assayed for ATC oxygenase activity by spectrophotometric methods as described previously (2). In addition, the formation of OTC during the assay was confirmed by high-pressure liquid chromatography analysis.…”

Section: Resultsmentioning

confidence: 99%

Cloning and heterologous expression in Streptomyces lividans of Streptomyces rimosus genes involved in oxytetracycline biosynthesis

1989

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The anhydrotetracycline (ATC) oxygenase enzyme which carries out the conversion of ATC to dehydrotetracycline was purified and the N-terminal amino acid sequence was determined. The sequence displays a significant similarity to that of the p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. This is consistent with the activity of the oxygenase, i.e., addition of a hydroxyl moiety to an aromatic ring structure.Oligonucleotide probes were designed and used to clone the corresponding fragment of chromosomal DNA from Streptomyces rimosus. This DNA fragment was used to screen a cosmid library, allowing the isolation of flanking DNA sequences. Surprisingly, the gene was located within the previously cloned cluster of genes involved in the synthesis of the biosynthetic intermediate ATC and not as had been expected (P. M. Rhodes, N. Winskill, E. J. Friend, and M. Warren, J. Gen. Microbiol. 124:329-338, 1981) at a separate locus on the other side of the chromosome. Subcloning of an appropriate DNA fragment from one of the cosmid clones onto pIJ916 produced Streptomyces lividans transformants which synthesized oxytetracycline.Oxytetracyline (OTC) is a commercially important broadspectrum antibiotic produced by Streptomyces rimosus. OTC is a member of the polyketide class of antibiotics which includes, among others, avermectin, monensin, erythromycin, and other tetracyclines such as 7-chlorotetracycline.The OTC biosynthetic pathway of S. rimosus has been the subject of both biochemical and genetic analysis (15). Figure 1 indicates the proposed biosynthetic pathway from malonyl coenzyme A to the final product. The isolation and characterization of mutants blocked at various points in the pathway indicated that the genes involved in OTC biosynthesis are clustered into two distinct positions mapping at diametrically opposite locations on the circular map of S. rimosius.The loci thought to be responsible for all pathway steps leading to the production of anhydrotetracycline (ATC) were assigned to the "4 o'clock" locus, whereas the genes encoding enzymes for the final part of the pathway, including those responsible for the production of a flavinlike cofactor (cosynthetic factor 1 [CSF1]) were assigned to a 10 o'clock location.The isolation (3; M. J. Butler, E. J. Friend, I. S. Hunter, F. S. Kaczmarek, D. A. Sugden, and M. Warren, Mol. Gen. Genet., in press) of two genes, otrA and otrB, involved in host resistance to OTC and the analysis of flanking DNA indicated that the 4 o'clock cluster was organized as a single group of biosynthetic genes flanked by the two resistance loci (Fig. 2). However, the genes responsible for the conversion of ATC to OTC remained unidentified. This paper describes the isolation of the gene (otcC) encoding the ATC oxygenase, the enzyme catalyzing the hydroxylation of ATC to dehydrotetracycline. otcC (7) except that S. lividans mycelium was grown for 3 days instead of 2 days in YEME medium containing glycine and MgCl2 at 0.5% and 5 mM, respectively. S. rimosus protoplast formation, regener...

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Section: Resultsmentioning

confidence: 99%

Cloning and heterologous expression in Streptomyces lividans of Streptomyces rimosus genes involved in oxytetracycline biosynthesis

1989

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“…This was attributed to the rise in phosphatase activity after the cessation of growth. Inorganic phosphate represses the synthesis of phosphatase, allowing the ATP level to remain high, which Behal et al (1979) credit for repressing secondary metabolism. The restimulation of growth phase, if phosphate is added, may give rise to feedback inhibition of secondary metabolism.…”

Section: Phosphatementioning

confidence: 99%

Bioprocess Intensification for Production of Novel Marine Bacterial Antibiotics Through Bioreactor Operation and Design

1999

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: There is a lack of research into bioreactor engineering and fermentation protocol design in the field of marine bacterial antibiotic production. Most production strategies are carried out at the shake-flask level and lack a mechanistic understanding of the antibiotic production process, offering poor prospects for successful scale-up. This review shows that data need to be collated on media and physical optima differences between the trophophase and idiophase, along with investigations into the control mechanisms for biosynthesis, to allow implementation of novel fermentation protocols. Immobilization may play a part in bioprocess intensification of marine bacterial antibiotic production, through again this area is understudied. Similarly, mass transfer and shear stress data of fermentations are needed to provide the bioreactor design requirements to intensify antibiotic biosynthesis, with process scale-up in mind. The application of bioprocess intensification methods to the production of antibiotics (and other metabolites) from marine microbes will become an important strategy for improving supply of natural products, in order to assess their suitability as chemotherapeutic drugs.

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“…Methylation of the C-4 amino group proceeds in two steps by transmethylation from methionine and these reactions can be measured in cell-free systems using radiolabelled S-adenosyl methionine (Miller & Nash, 1975a). Hydroxylation at C-6 has been measured in cell-free extracts of S. aureofaciens (Behal et al, 1979); the reaction requires NADPH and atmospheric oxygen and the specific activity of the enzyme was found to be sufficient to account for whole cell tetracycline production. The final step in the biosynthesis of tetracyclines is the reduction of the 5a-l1a double bond; this is NADPH dependent and cell-free conversion of 7-chloro-5a, l1a-dehydrotetracycline to a bioactive product (chlortetracycline) which can be monitored by using Staphylococcus aureus (Miller & Nash, 1975b).…”

Section: Tetracyclinesmentioning

confidence: 99%

Enzymatic and Microbial Transformation of Antibiotics and Steroids

O'Sullivan

1990

Microbial Enzymes and Biotechnology

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Anhydrotetracycline oxygenase activity and biosynthesis of tetracyclines in streptomyces aureofaciens

Cited by 50 publications

References 10 publications

Cloning and heterologous expression in Streptomyces lividans of Streptomyces rimosus genes involved in oxytetracycline biosynthesis

Cloning and heterologous expression in Streptomyces lividans of Streptomyces rimosus genes involved in oxytetracycline biosynthesis

Bioprocess Intensification for Production of Novel Marine Bacterial Antibiotics Through Bioreactor Operation and Design

Enzymatic and Microbial Transformation of Antibiotics and Steroids

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