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Peter F. Leadlay - One of the best experts on this subject based on the ideXlab platform.
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Complete genome sequence of the erythromycin-producing bacterium Saccharopolyspora erythraea NRRL23338
Nature biotechnology, 2007Co-Authors: Markiyan Oliynyk, Markiyan Samborskyy, J. B. Lester, Tatiana Mironenko, Nataliya Scott, Shilo Dickens, Stephen F. Haydock, Peter F. LeadlayAbstract:Saccharopolyspora erythraea is used for the industrial-scale production of the antibiotic erythromycin A, derivatives of which play a vital role in medicine. The sequenced chromosome of this soil bacterium comprises 8,212,805 base pairs, predicted to encode 7,264 genes. It is circular, like those of the pathogenic actinomycetes Mycobacterium tuberculosis and Corynebacterium diphtheriae, but unlike the linear chromosomes of the model actinomycete Streptomyces coelicolor A3(2) and the closely related Streptomyces avermitilis. The S. erythraea genome contains at least 25 gene clusters for production of known or predicted secondary metabolites, at least 72 genes predicted to confer resistance to a range of common antibiotic classes and many sets of duplicated genes to support its saprophytic lifestyle. The availability of the genome sequence of S. erythraea will improve insight into its biology and facilitate rational development of strains to generate high-titer producers of clinically important antibiotics.
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identification of a phosphopantetheinyl transferase for erythromycin biosynthesis in Saccharopolyspora erythraea
ChemBioChem, 2004Co-Authors: Kira J Weissman, Markiyan Oliynyk, Hui Hong, Alexis P Siskos, Peter F. LeadlayAbstract:: Phosphopantetheinyl transferases (PPTases) catalyze the essential post-translational activation of carrier proteins (CPs) from fatty acid synthases (FASs) (primary metabolism), polyketide synthases (PKSs), and non-ribosomal polypeptide synthetases (NRPSs) (secondary metabolism). Bacteria typically harbor one PPTase specific for CPs of primary metabolism ("ACPS-type" PPTases) and at least one capable of modifying carrier proteins involved in secondary metabolism ("Sfp-type" PPTases). In order to identify the PPTase(s) associated with erythromycin biosynthesis in Saccharopolyspora erythraea, we have used the genome sequence of this organism to identify, clone, and express (in Escherichia coli) three candidate PPTases: an ACPS-type PPTase (S. erythraea ACPS) and two Sfp-type PPTases (a discrete enzyme (SePptII) and another that is integrated into a modular PKS subunit (SePptI)). In vitro analysis of these recombinant PPTases, with an acyl carrier protein-thioesterase (ACP-TE) didomain from the erythromycin PKS as substrate, revealed that only SePptII is active in phosphopantetheinyl transfer with this substrate. SePptII was also shown to provide complete modification of ACP-TE and of an entire multienzyme subunit from the erythromycin PKS in E. coli. The efficiency of the SePptII in phosphopantetheinyl transfer in E. coli makes it an attractive alternative to other Sfp-type PPTases for co-expression experiments with PKS proteins.
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A New Modular Polyketide Synthase in the Erythromycin Producer Saccharopolyspora erythraea
Journal of molecular microbiology and biotechnology, 2004Co-Authors: Steven Boakes, James Staunton, Markiyan Oliynyk, Jesus Cortes, Ines U Böhm, Brian A.m. Rudd, W P Revill, Peter F. LeadlayAbstract:A previously unidentified set of genes encoding a modular polyketide synthase (PKS) has been sequenced in Saccharopolyspora erythraea, producer of the antibiotic erythromycin. This new PKS gene cluster (pke) contains four adjacent large open reading frames (ORFs) encoding eight extension modules, flanked by a number of other ORFs which can be plausibly assigned roles in polyketide biosynthesis. Disruption of the pke PKS genes gave S. erythraea mutant JC2::pSBKS6, whose growth characteristics and pattern of secondary metabolite production did not apparently differ from the parent strain under any of the growth conditions tested. However, the pke PKS loading module and individual pke acyltransferase domains were shown to be active when used in engineered hybrid PKSs, making it highly likely that under appropriate conditions these biosynthetic genes are indeed expressed and active, and synthesize a novel polyketide product.
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Heterologous expression in Saccharopolyspora erythraea of a pentaketide synthase derived from the spinosyn polyketide synthase
Organic & biomolecular chemistry, 2003Co-Authors: Christine Martin, Máire C. Timoney, Rose M. Sheridan, Steven G. Kendrew, Barrie Wilkinson, James Staunton, Peter F. LeadlayAbstract:A truncated version of the spinosyn polyketide synthase comprising the loading module and the first four extension modules fused to the erythromycin thioesterase domain was expressed in Saccharopolyspora erythraea. A novel pentaketide lactone product was isolated, identifying cryptic steps of spinosyn biosynthesis and indicating the potential of this approach for the biosynthetic engineering of spinosyn analogues. A pathway for the formation of the tetracyclic spinosyn aglycone is proposed.
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a defined system for hybrid macrolide biosynthesis in Saccharopolyspora erythraea
Molecular Microbiology, 2000Co-Authors: Sabine Gaisser, James Staunton, James Reather, Gabriele Wirtz, Laurenz Kellenberger, Peter F. LeadlayAbstract:The biological activity of polyketide antibiotics is often strongly dependent on the presence and type of deoxysugar residues attached to the aglycone core. A system is described here, based on the erythromycin-producing strain of Saccharopolyspora erythraea, for detection of hybrid glycoside formation, and this system has been used to demonstrate that an amino sugar characteristic of 14-membered macrolides (d-desosamine) can be efficiently attached to a 16-membered aglycone substrate. First, the S. erythraea mutant strain DM was created by deletion of both eryBV and eryCIII genes encoding the respective ery glycosyltransferase genes. The glycosyltransferase OleG2 from Streptomyces antibioticus, which transfers l-oleandrose, has recently been shown to transfer rhamnose to the oxygen at C-3 of erythronolide B and 6-deoxyerythronolide B. In full accordance with this finding, when oleG2 was expressed in S. erythraea DM, 3-O-rhamnosyl-erythronolide B and 3-O-rhamnosyl-6-deoxyerythronolide B were produced. Having thus validated the expression system, endogenous aglycone production was prevented by deletion of the polyketide synthase (eryA) genes from S. erythraea DM, creating the triple mutant SGT2. To examine the ability of the mycaminosyltransferase TylM2 from Streptomyces fradiae to utilise a different amino sugar, tylM2 was integrated into S. erythraea SGT2, and the resulting strain was fed with the 16-membered aglycone tylactone, the normal TylM2 substrate. A new hybrid glycoside was isolated in good yield and characterized as 5-O-desosaminyl-tylactone, indicating that TylM2 may be a useful glycosyltransferase for combinatorial biosynthesis. 5-O-glucosyl-tylactone was also obtained, showing that endogenous activated sugars and glycosyltransferases compete for aglycone in these cells.
Michael E. Bushell - One of the best experts on this subject based on the ideXlab platform.
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Decreasing the hyphal branching rate of Saccharopolyspora erythraea NRRL 2338 leads to increased resistance to breakage and increased antibiotic production
Biotechnology and bioengineering, 2002Co-Authors: John N. Wardell, S. M. Stocks, Colin R. Thomas, Michael E. BushellAbstract:Mutation and selection for increased resistance to cell-wall synthesis inhibitors led to alterations in the hyphal branching rate of Saccharopolyspora erythraea NRRL 2338. Mutants with decreased branching frequency exhibited increased hyphal strength (estimated by in vitro micromanipulation). As the hyphal strength was increased, this led to a greater proportion of hyphal particles in liquid culture with a hyphal fragment diameter of greater than 88 μm. This, in turn, coincided with proportionately increased antibiotic production. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 141–146, 2002; DOI 10.1002/bit.10210
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Effect of hyphal micromorphology on bioreactor performance of antibiotic-producing Saccharopolyspora erythraea cultures
Microbiology, 1996Co-Authors: Steven M. Martin, Michael E. BushellAbstract:When Saccharopolyspora erythraea biomass from submerged culture was filtered (100-120 μn sintered glass filter) the antibiotic yield of the retentate (mean hyphal particle diameter 103 μm) was significantly higher [1.740 mg (mg biomass)-1] than that of the filtrate [1.286 mg (mg biomass)-1; mean hyphal particle diameter 88 μn]. A hypothesis to explain this is that there is a critical hyphal particle diameter, below which the particle is incapable of producing antibiotic. This would be a consequence of the site of antibiotic production occurring at a fixed distance from the growing hyphal tip. A protocol is proposed for calculation of the hypothetical critical hyphal diameter (88 μm in this case). The proportion of retentate (more productive) fraction of biomass varied between 60% and 30% during the course of a batch culture. Bioreactor stirrer speed significantly affected mean hyphal particle diameter (70 fim at 1500 r.p.m.; 124 μm at 750 r.p.m.) and antibiotic productivity [0.867 mg (mg biomass)-1 at 1500 r.p.m.; 0.913 mg (mg biomass)-1 at 750 r.p.m.].
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The induction of antibiotic synthesis in Saccharopolyspora erythraea and Streptomyces hygroscopicus by growth rate decrease is accompanied by a down-regulation of protein synthesis rate
FEMS Microbiology Letters, 1995Co-Authors: Giles C. Wilson, Michael E. BushellAbstract:The relationship between antibiotic production and culture growth rate in Saccharopolyspora erythraea and Streptomyces hygroscopicus was manipulated by changing the growth-limiting substrate. Carbon- and nitrogen-limited cultures were studied and antibiotic synthesis was obtained in both cases in Saccharopolyspora erythraea cultures and in nitrogen-limited Streptomyces hygroscopicus cultures. In all cultures where antibiotic was detected, onset of antibiotic production coincided with the minimal protein synthesis rate. Further investigation in Saccharopolyspora erythraea cultures indicated that this corresponded to minimum ratio of charged to uncharged tRNA, i.e. when uncharged tRNA accumulated. This latter phenomenon was investigated in the presence of a protein synthesis inhibitor.
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estimation of the kinetic constants and elucidation of trends in growth and erythromycin production in batch and continuous cultures of Saccharopolyspora erythraea using curve fitting techniques
Enzyme and Microbial Technology, 1993Co-Authors: J F Mcdermott, G Lethbridge, Michael E. BushellAbstract:The kinetics of erythromycin production were dependent on the identity of the growth rate-limiting nutrient during batch cultures of Saccharopolyspora erythraea. Semilogarithmic linear regression provided a single estimate of growth rate during the exponential phase, but partial cubic spline curve fit derivatives provided time-dependent specific growth and production rate profiles. Non-growth-linked product formation was observed when the medium was glucose- or phosphate-limited. However, growth-linked product formation was observed in a nitrate-limited medium. The kinetics observed in nitrate-limited chemostat culture provided evidence that Saccharopolyspora erythraea may be subject to noncompetitive inhibition by a growth-linked product under these conditions. A mathematical model was used to test this theory. The model simulation fitted the observed data very closely and was used to calculate estimates of the kinetic parameters involved: Ypx[erythromycinbiomass] = 1.98 × 10−3; Yps[erythromycinglucose] = 1.85 × 10−3; ki = 1.56 mg l−1; ks[glucose] = 1.5 × 10−2 mg ml−1; μmax(chemostat) = 0.066 h−1
J.mark Weber - One of the best experts on this subject based on the ideXlab platform.
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Application of In Vitro Transposon Mutagenesis to Erythromycin Strain Improvement in Saccharopolyspora erythraea.
Methods in molecular biology (Clifton N.J.), 2016Co-Authors: J.mark Weber, William H. Cernota, Andrew Reeves, Roy K. WesleyAbstract:Transposon mutagenesis is an invaluable technique in molecular biology for the creation of random mutations that can be easily identified and mapped. However, in the field of microbial strain improvement, transposon mutagenesis has scarcely been used; instead, chemical and physical mutagenic methods have been traditionally favored. Transposons have the advantage of creating single mutations in the genome, making phenotype to genotype assignments less challenging than with traditional mutagens which commonly create multiple mutations in the genome. The site of a transposon mutation can also be readily mapped using DNA sequencing primer sites engineered into the transposon termini. In this chapter an in vitro method for transposon mutagenesis of Saccharopolyspora erythraea is presented. Since in vivo transposon tools are not available for most actinomycetes including S. erythraea, an in vitro method was developed. The in vitro method involves a significant investment in time and effort to create the mutants, but once the mutants are made and screened, a large number of highly relevant mutations of direct interest to erythromycin production can be found.
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Random transposon mutagenesis of the Saccharopolyspora erythraea genome reveals additional genes influencing erythromycin biosynthesis.
FEMS microbiology letters, 2015Co-Authors: Andrij Fedashchin, William H. Cernota, Melissa C. Gonzalez, Benjamin I. Leach, Noelle Kwan, Roy K. Wesley, J.mark WeberAbstract:A single cycle of strain improvement was performed in Saccharopolyspora erythraea mutB and 15 genotypes influencing erythromycin production were found. Genotypes generated by transposon mutagenesis appeared in the screen at a frequency of ∼3%. Mutations affecting central metabolism and regulatory genes were found, as well as hydrolases, peptidases, glycosyl transferases and unknown genes. Only one mutant retained high erythromycin production when scaled-up from micro-agar plug fermentations to shake flasks. This mutant had a knockout of the cwh1 gene (SACE_1598), encoding a cell-wall-associated hydrolase. The cwh1 knockout produced visible growth and morphological defects on solid medium. This study demonstrated that random transposon mutagenesis uncovers strain improvement-related genes potentially useful for strain engineering.
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Engineering of the methylmalonyl-CoA metabolite node of Saccharopolyspora erythraea for increased erythromycin production.
Metabolic engineering, 2007Co-Authors: Andrew Reeves, William H. Cernota, Melissa C. Gonzalez, Benjamin I. Leach, Igor A. Brikun, J.mark WeberAbstract:Abstract Engineering of the methylmalonyl-CoA (mmCoA) metabolite node of the Saccharopolyspora erythraea wild-type strain through duplication of the mmCoA mutase (MCM) operon led to a 50% increase in erythromycin production in a high-performance oil-based fermentation medium. The MCM operon was carried on a 6.8 kb DNA fragment in a plasmid 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 a strain 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.
Buchang Zhang - One of the best experts on this subject based on the ideXlab platform.
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GlnR-mediated regulation of nitrogen metabolism in the actinomycete Saccharopolyspora erythraea
Applied microbiology and biotechnology, 2014Co-Authors: Li-li Yao, Ying Zhou, Chengheng Liao, Sébastien Rigali, Gang Huang, Buchang ZhangAbstract:Nitrogen source sensing, uptake, and assimilation are central for growth and development of microorganisms which requires the participation of a global control of nitrogen metabolism-associated genes at the transcriptional level. In soil-dwelling antibiotic-producing actinomycetes, this role is played by GlnR, an OmpR family regulator. In this work, we demonstrate that SACE_7101 is the ortholog of actinomycetes’ GlnR global regulators in the erythromycin producer Saccharopolyspora erythraea. Indeed, the chromosomal deletion of SACE_7101 severely affects the viability of S. erythraea when inoculated in minimal media supplemented with NaNO3, NaNO2, NH4Cl, glutamine, or glutamate as sole nitrogen source. Combination of in silico prediction of cis-acting elements, subsequent in vitro (through gel shift assays) and in vivo (real-time reverse transcription polymerase chain reaction) validations of the predicted target genes revealed a very large GlnR regulon aimed at adapting the nitrogen metabolism of S. erythraea. Indeed, enzymes/proteins involved in (i) uptake and assimilation of ammonium, (ii) transport and utilization of urea, (iii) nitrite/nitrate, (iv) glutamate/glutamine, (v) arginine metabolism, (vi) nitric oxide biosynthesis, and (vii) signal transduction associated with the nitrogen source supplied have at least one paralog gene which expression is controlled by GlnR. Our work highlights a GlnR-binding site consensus sequence (t/gna/cAC-n6-GaAAc) which is similar although not identical to the consensus sequences proposed for other actinomycetes. Finally, we discuss the distinct and common features of the GlnR-mediated transcriptional control of nitrogen metabolism between S. erythraea and the model organism Streptomyces coelicolor.
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SACE_0012, a TetR-family transcriptional regulator, affects the morphogenesis of Saccharopolyspora erythraea.
Current microbiology, 2013Co-Authors: Xiaojuan Yin, Xunduan Huang, Li Yuan, Buchang ZhangAbstract:Saccharopolyspora erythraea, a mycelium-forming actinomycete, produces a clinically important antibiotic erythromycin. Extensive investigations have provided insights into erythromycin biosynthesis in S. erythraea, but knowledge of its morphogenesis remains limited. By gene inactivation and complementation strategies, the TetR-family transcriptional regulator SACE_0012 was identified to be a negative regulator of mycelium formation of S. erythraea A226. Detected by quantitative real-time PCR, the relative transcription of SACE_7115, the amfC homolog for an aerial mycelium formation protein, was dramatically increased in SACE_0012 mutant, whereas erythromycin biosynthetic gene eryA, a pleiotropic regulatory gene bldD, and the genes SACE_2141, SACE_6464, SACE_6040, that are the homologs to the sporulation regulators WhiA, WhiB, WhiG, were not differentially expressed. SACE_0012 disruption could not restore its defect of aerial development in bldD mutant, and also did not further accelerate the mycelium formation in the mutant of SACE_7040 gene, that was previously identified to be a morphogenesis repressor. Furthermore, the transcriptional level of SACE_0012 had not markedly changed in bldD and SACE_7040 mutant over A226. Taken together, these results suggest that SACE_0012 is a negative regulator of S. erythraea morphogenesis by mainly increasing the transcription of amfC gene, independently of the BldD regulatory system.
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Identification of SACE_7040, a member of TetR family related to the morphological differentiation of Saccharopolyspora erythraea.
Current microbiology, 2011Co-Authors: Shu Han, Ping Song, Ting Ren, Xunduan Huang, Cheng Cao, Buchang ZhangAbstract:SACE_7040 is presumed to be a member of the TetR family of transcriptional regulators in Saccharopolyspora erythraea, but its biological function is unknown. It was shown that the SACE_7040 gene knockout mutant formed aerial mycelium earlier than its original strain, and this phenotype could be restored by complementation of a single copy of SACE_7040 gene, demonstrating that SACE_7040 is an important regulator of the morphological differentiation of Sac. erythraea. When SACE_7040 gene was disrupted in the bldD mutant, we intriguingly found that the defect in aerial development exhibited by the bldD mutant could be overcome, suggesting a crosstalk between SACE_7040 and BldD in Sac. erythraea morphogenesis. These findings provide novel insights toward the Sac. erythraea developmental biology.
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Study on relationship between length of homologous sequences and chromosomic recombination rate in Saccharopolyspora erythraea
Sheng wu gong cheng xue bao = Chinese journal of biotechnology, 2003Co-Authors: Buchang Zhang, Yiguang Wang, Zhi-hu Zhao, Chuanxuan LiuAbstract:In order to study the relationship between lengths of homologous fragments and chromsomic recombination rate in Saccharopolyspora erythraea, three homologous sequences, with mutant loci and different flanking sequences, (26bp + 27bp), (500bp + 576bp) and (1908bp + 1749bp), were synthesized by chemical reaction or PCR amplification, and cloned into pWHM3 to construct homologous recombination plasmids, pWHM1113, pWHM1116 and pWHM1119. When the plasmids were transformed into protoplast of Saccharopolyspora erythraea A226 under PEG mediated, on an average 30, 69 and 170 transformants grew on each plate for the three plasmids respectively, but chromosomic integration frequency were 0, 2% and 19% among corresponding transformants. Both pWHM1116 and pWHM1119 could take double crossover recombination, and exchange the mutant loci in the chromosome. It was concluded that when the flanking sequences were equal or more than (500bp + 576bp), they could take effective single and double recombination with Saccharopolyspora erythraea chromosome.
Roy K. Wesley - One of the best experts on this subject based on the ideXlab platform.
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Application of In Vitro Transposon Mutagenesis to Erythromycin Strain Improvement in Saccharopolyspora erythraea.
Methods in molecular biology (Clifton N.J.), 2016Co-Authors: J.mark Weber, William H. Cernota, Andrew Reeves, Roy K. WesleyAbstract:Transposon mutagenesis is an invaluable technique in molecular biology for the creation of random mutations that can be easily identified and mapped. However, in the field of microbial strain improvement, transposon mutagenesis has scarcely been used; instead, chemical and physical mutagenic methods have been traditionally favored. Transposons have the advantage of creating single mutations in the genome, making phenotype to genotype assignments less challenging than with traditional mutagens which commonly create multiple mutations in the genome. The site of a transposon mutation can also be readily mapped using DNA sequencing primer sites engineered into the transposon termini. In this chapter an in vitro method for transposon mutagenesis of Saccharopolyspora erythraea is presented. Since in vivo transposon tools are not available for most actinomycetes including S. erythraea, an in vitro method was developed. The in vitro method involves a significant investment in time and effort to create the mutants, but once the mutants are made and screened, a large number of highly relevant mutations of direct interest to erythromycin production can be found.
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Random transposon mutagenesis of the Saccharopolyspora erythraea genome reveals additional genes influencing erythromycin biosynthesis.
FEMS microbiology letters, 2015Co-Authors: Andrij Fedashchin, William H. Cernota, Melissa C. Gonzalez, Benjamin I. Leach, Noelle Kwan, Roy K. Wesley, J.mark WeberAbstract:A single cycle of strain improvement was performed in Saccharopolyspora erythraea mutB and 15 genotypes influencing erythromycin production were found. Genotypes generated by transposon mutagenesis appeared in the screen at a frequency of ∼3%. Mutations affecting central metabolism and regulatory genes were found, as well as hydrolases, peptidases, glycosyl transferases and unknown genes. Only one mutant retained high erythromycin production when scaled-up from micro-agar plug fermentations to shake flasks. This mutant had a knockout of the cwh1 gene (SACE_1598), encoding a cell-wall-associated hydrolase. The cwh1 knockout produced visible growth and morphological defects on solid medium. This study demonstrated that random transposon mutagenesis uncovers strain improvement-related genes potentially useful for strain engineering.
