Conjugal transfer of cosmid DNA from Escherichia coli to Saccharopolyspora spinosa: effects of chromosomal insertions on macrolide A83543 production

Matsushima, Patti; Broughton, Mary C.; Turner, J. R.; Baltz, Richard H.

doi:10.1016/0378-1119(94)90831-1

Cited by 87 publications

(80 citation statements)

References 16 publications

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“…In comparison to transformation protocols described for Streptomyces (3), conjugation is much more efficient, and it is successful with many actinomycetes (33). It also avoids the processes of protoplasting and regeneration used during transformation procedures, which are often difficult to develop for other strains and which can cause genetic changes.…”

Section: Discussionmentioning

confidence: 99%

PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin

Gust

Challis

Fowler

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

1,345

1,554

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Streptomycetes are high G؉C Gram-positive, antibiotic-producing, mycelial soil bacteria. The 8.7-Mb Streptomyces coelicolor genome was previously sequenced by using an ordered library of Supercos-1 clones. Here, we describe an efficient procedure for creating precise gene replacements in the cosmid clones by using PCR targeting and -Red-mediated recombination. The cloned Streptomyces genes are replaced with a cassette containing a selectable antibiotic resistance and oriTRK2 for efficient transfer to Streptomyces by RP4-mediated intergeneric conjugation. Supercos-1 does not replicate in Streptomyces, but the clones readily undergo double-crossover recombination, thus creating gene replacements. The antibiotic resistance cassettes are flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and oriT RK2 to generate unmarked, nonpolar mutations. The technique has been used successfully by >20 researchers to mutate around 100 Streptomyces genes. As an example, we describe its application to the discovery of a gene involved in the production of geosmin, the ubiquitous odor of soil. The gene, Sco6073 (cyc2), codes for a protein with two sesquiterpene synthase domains, only one of which is required for geosmin biosynthesis, probably via a germacra-1 (10) E,5E-dien-11-ol intermediate generated by the sesquiterpene synthase from farnesyl pyrophosphate.FLP recombinase ͉ lambda Red recombinase ͉ oriT ͉ SCO5222 ͉ SCO6073

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Section: Discussionmentioning

confidence: 99%

PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin

Gust

Challis

Fowler

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

1,345

1,554

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“…When required, ampicillin was added to 100 g/ml, and kanamycin was added to 50 g/ml. S. spinosa strains were grown routinely at 29°C in CSM broth (10). S. spinosa transconjugants were selected on R6 medium (10) and then maintained on brain heart infusion (BHI) agar (Difco) when possible.…”

Section: Methodsmentioning

confidence: 99%

Rhamnose Biosynthesis Pathway Supplies Precursors for Primary and Secondary Metabolism in Saccharopolyspora spinosa

Madduri¹,

Waldron²,

Merlo³

2001

J Bacteriol

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Rhamnose is an essential component of the insect control agent spinosad. However, the genes coding for the four enzymes involved in rhamnose biosynthesis in Saccharopolyspora spinosa are located in three different regions of the genome, all unlinked to the cluster of other genes that are required for spinosyn biosynthesis. Disruption of any of the rhamnose genes resulted in mutants with highly fragmented mycelia that could survive only in media supplemented with an osmotic stabilizer. It appears that this single set of genes provides rhamnose for cell wall synthesis as well as for secondary metabolite production. Duplicating the first two genes of the pathway caused a significant improvement in the yield of spinosyn fermentation products.Spinosyns, the active ingredients in Dow AgroSciences' new Naturalyte line of insect control products, are produced by fermentation of the actinomycete Saccharopolyspora spinosa. Spinosyns are macrolides ( Fig. 1) consisting of a 21-carbon tetracyclic lactone to which are attached two deoxysugars: tri-O-methylated rhamnose and forosamine (6). The most active components of the spinosyn family of compounds are spinosyns A and D, which differ from each other by a single methyl substituent at position 6 of the polyketide. Other factors in this family have different levels of methylation and are significantly less active. Both the rhamnose and forosamine moieties are essential for the insecticidal activity of spinosyns (2). Spinosad is highly effective against target insects and has an excellent environmental and mammalian toxicological profile (2,13,14).Spinosyn biosynthesis occurs via the nonglycosylated intermediate, the aglycone (AGL). Rhamnose is the first sugar attached and is tri-O-methylated to yield the intermediate pseudoaglycone.Only after the rhamnose is attached can the forosamine sugar be incorporated (M. C. Broughton, M. L. B. Huber, L. C. Creemer, H. A. Kirst, and J. R. Turner, Abstr. 91st Annu. Meet. Am. Soc. Microbiol. 1991, abstr. K-58, p. 224, 1991. Both trimethyl rhamnose and forosamine are believed to be synthesized from glucose-1-phosphate via the common intermediate 4-keto-6-deoxy-D-glucose (Fig. 2). The biosynthetic pathway for rhamnose (Fig. 2) has been elucidated in enteric bacteria, where the deoxysugar is an element of surface antigens (8, 18). The first step, activation of glucose by addition of a nucleotidyl diphosphate (NDP), is catalyzed by an NDP-glucose synthase (the gtt gene product). The second step, dehydration to NDP-4-keto-6-deoxyglucose, is catalyzed by glucose dehydratase (the gdh gene product). 4-Keto-6-deoxy-D-glucose is the common intermediate to many deoxysugar biosynthetic pathways, and the enzymes encoded by the gtt and gdh genes may supply the precursors for all of them. Rhamnose synthesis requires two additional enzymes, a 3Ј5Ј epimerase (encoded by epi) and a 4Ј ketoreductase (encoded by kre), that are unique to the pathway. They convert the NDP-4-keto-6-deoxyglucose to NDP-L-rhamnose, the activated sugar that is the substrate of th...

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“…Then the 604 bp fragment was digested by HindIII (Fermentas) and XbaI (Fermentas) and ligated to pOJ260 obtaining pLu106. pLu106 was introduced into S. spinosa ATCC 49460 by conjugation from E. coli S17-1 and homologous recombination into the chromosome as described previously [28]. The plasmid was inserted into the middle rex of S. spinosa ATCC 49460 to create S. spinosa △rex (Lu106).…”

Section: Strains Mutant Construction and Growth Conditionsmentioning

confidence: 99%

Suitable extracellular oxidoreduction potential inhibit

et al. 2014

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Background: Polyketides, such as spinosad, are mainly synthesized in the stationary phase of the fermentation. The synthesis of these compounds requires many primary metabolites, such as acetyl-CoA, propinyl-CoA, NADPH, and succinyl-CoA. Their synthesis is also significantly influenced by NADH/NAD + . Rex is the sensor of NADH/NAD + redox state, whose structure is under the control of NADH/NAD + ratio. The structure of rex controls the expression of many NADH dehydrogenases genes and cytochrome bd genes. Intracellular redox state can be influenced by adding extracellular electron acceptor H 2 O 2 . The effect of extracellular oxidoreduction potential on spinosad production has not been studied. Although extracellular oxidoreduction potential is an important environment effect in polyketides production, it has always been overlooked. Thus, it is important to study the effect of extracellular oxidoreduction potential on Saccharopolyspora spinosa growth and spinosad production.

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Conjugal transfer of cosmid DNA from Escherichia coli to Saccharopolyspora spinosa: effects of chromosomal insertions on macrolide A83543 production

Cited by 87 publications

References 16 publications

PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin

PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin

Rhamnose Biosynthesis Pathway Supplies Precursors for Primary and Secondary Metabolism in Saccharopolyspora spinosa

Suitable extracellular oxidoreduction potential inhibit

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