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Eric Cundliffe - One of the best experts on this subject based on the ideXlab platform.

  • regulation of tylosin production and morphological differentiation in Streptomyces fradiae by tylp a deduced gamma butyrolactone receptor
    Molecular Microbiology, 2002
    Co-Authors: George Stratigopoulos, Atul R Gandecha, Eric Cundliffe
    Abstract:

    During promoter-probe analysis carried out in Streptomyces lividans, the TylP protein powerfully inhibited reporter gene expression from the tylP promoter, raising the likelihood that tylP is autoregulated in its native host, Streptomyces fradiae. Also in S. lividans, TylP negatively controlled the tylQ promoter, even though tylQ could still be switched off in S. fradiae strains specifically disrupted in tylP. Under the latter conditions, tylosin production was brought forward and enhanced, whereas overexpression of tylP resulted in reduced levels of the antibiotic, accompanied by barely detectable transcription from multiple genes of the tylosin biosynthetic cluster. Unexpectedly, overexpression of tylP reduced transcription of tylS, a transcriptional activator essential for tylosin production. This was probably a direct effect, as TylP also reduced expression from the tylS promoter in S. lividans. For these several reasons, we conclude that TylP acts as a repressor during tylosin biosynthesis. In addition, TylP influences morphological differentiation in S. fradiae. On solid media, strains in which tylP was disrupted sporulated significantly earlier than wild type and, in liquid culture, displayed hyperfragmentation.

  • differential roles of two sarp encoding regulatory genes during tylosin biosynthesis
    Molecular Microbiology, 2002
    Co-Authors: Neil Bate, George Stratigopoulos, Eric Cundliffe
    Abstract:

    Summary The tylosin biosynthetic gene cluster of Streptomyces fradiae is remarkable in harbouring at least five regulatory genes, two of which (tylS and tylT) encode proteins of the Streptomyces antibiotic regulatory protein (SARP) family. The aim of the present work was to assess the respective contributions of TylS and TylT to tylosin production. A combination of targeted gene disruption, fermentation studies and gene expression analysis via reverse transcriptase‐polymerase chain reaction (RT‐PCR) suggests that tylS is essential for tylosin production and controls the expression of tylR (previously shown to be a global activator of the biosynthetic pathway) plus at least one other gene involved in polyketide metabolism or regulation thereof. This is the first demonstration of a SARP acting to control another regulatory gene during antibiotic biosynthesis. In contrast, tylT is not essential for tylosin production.

  • The tylosin-biosynthetic genes of Streptomyces fradiae
    Antonie van Leeuwenhoek, 2001
    Co-Authors: Eric Cundliffe, Atul R Gandecha, Neil Bate, Andrew Butler, Steven Fish, Louise Merson-davies
    Abstract:

    The tylosin-biosynthetic ( tyl ) gene cluster occupies about 1% of the genome of Streptomyces fradiae and includes at least 43 open reading frames. In addition to structural genes required for tylosin production, the tyl cluster contains three resistance determinants and several regulatory genes. Tylosin production is evidently controlled by pathway-specific and pleiotropic regulators with the likely involvement of γ-butyrolactone signalling factors. Accumulation of the polyketide aglycone is controlled by glycosylated macrolides and optimal performance of the complex polyketide synthase enzyme requires the activity of an editing thioesterase.

  • multiple regulatory genes in the tylosin biosynthetic cluster of Streptomyces fradiae
    Chemistry & Biology, 1999
    Co-Authors: Neil Bate, Atul R Gandecha, Andrew R. Butler, Eric Cundliffe
    Abstract:

    Background The macrolide antibiotic tylosin is composed of a polyketide lactone substituted with three deoxyhexose sugars. In order to produce tylosin efficiently, Streptomyces fradiae presumably requires control mechanisms that balance the yields of the constituent metabolic pathways together with switches that allow for temporal regulation of antibiotic production. In addition to possible metabolic feedback and/or other signalling devices, such control probably involves interplay between specific regulatory proteins. Prior to the present work, however, no candidate regulatory gene(s) had been identified in S. fradiae. Results DNA sequencing has shown that the tylosin biosynthetic gene cluster, within which four open reading frames utilise the rare TTA codon, contains at least five candidate regulatory genes, one of which ( tylP ) encodes a γ-butyrolactone signal receptor for which tylQ is a probable target. Two other genes ( tylS and tylT ) encode pathway-specific regulatory proteins of the Streptomyces antibiotic regulatory protein (SARP) family and a fifth, tylR , has been shown by mutational analysis to control various aspects of tylosin production. Conclusions The tyl genes of S. fradiae include the richest collection of regulators yet encountered in a single antibiotic biosynthetic gene cluster. Control of tylosin biosynthesis is now amenable to detailed study, and manipulation of these various regulatory genes is likely to influence yields in tylosin-production fermentations.

  • impact of thioesterase activity on tylosin biosynthesis in Streptomyces fradiae
    Chemistry & Biology, 1999
    Co-Authors: Andrew R. Butler, Neil Bate, Eric Cundliffe
    Abstract:

    Abstract Background: The polyketide lactone tylactone is produced in Streptomycesfradiae by the TyIG complex of five multifunctional proteins. As with other type I polyketide synthases, the enzyme catalysing the final elongation step (TyIGV) possesses an integral thioesterase domain that is believed to be responsible for chain termination and ring closure to form tylactone, which is then glycosylated to yield tylosin. In common with other macrolide producers, S. fradiae also possesses an additional thioesterase gene ( orf5 ) located within the cluster of antibiotic biosynthetic genes. The function of the Orf5 protein is addressed here. Results: Disruption of orf5 reduced antibiotic accumulation in S. fradiae by atleast 85%. Under such circumstances, the strain accumulated desmycosin (demycarosyl-tylosin) due to a downstream polar effect on the expression of orf6 , which encodes a mycarose biosynthetic enzyme. High levels of desmycosin production were restored in the disrupted strain by complementation with intact orf5 , or with the corresponding thioesterase gene, nbmB , from S. narbonensis , but not with DNA encoding the integral thioesterase domain of TyIGV. Conclusions: Polyketide metabolism in S. fradiae is strongly dependent on thethioesterase activity encoded by orf5 ( tylO ). It is proposed that the TyIG complex might operate with a significant error frequency and be prone to blockage with aberrant polyketides. A putative editing activity associated with TyIO might be essential to unblock the polyketide synthase complex and thereby promote antibiotic accumulation.

Neil Bate - One of the best experts on this subject based on the ideXlab platform.

  • differential roles of two sarp encoding regulatory genes during tylosin biosynthesis
    Molecular Microbiology, 2002
    Co-Authors: Neil Bate, George Stratigopoulos, Eric Cundliffe
    Abstract:

    Summary The tylosin biosynthetic gene cluster of Streptomyces fradiae is remarkable in harbouring at least five regulatory genes, two of which (tylS and tylT) encode proteins of the Streptomyces antibiotic regulatory protein (SARP) family. The aim of the present work was to assess the respective contributions of TylS and TylT to tylosin production. A combination of targeted gene disruption, fermentation studies and gene expression analysis via reverse transcriptase‐polymerase chain reaction (RT‐PCR) suggests that tylS is essential for tylosin production and controls the expression of tylR (previously shown to be a global activator of the biosynthetic pathway) plus at least one other gene involved in polyketide metabolism or regulation thereof. This is the first demonstration of a SARP acting to control another regulatory gene during antibiotic biosynthesis. In contrast, tylT is not essential for tylosin production.

  • The tylosin-biosynthetic genes of Streptomyces fradiae
    Antonie van Leeuwenhoek, 2001
    Co-Authors: Eric Cundliffe, Atul R Gandecha, Neil Bate, Andrew Butler, Steven Fish, Louise Merson-davies
    Abstract:

    The tylosin-biosynthetic ( tyl ) gene cluster occupies about 1% of the genome of Streptomyces fradiae and includes at least 43 open reading frames. In addition to structural genes required for tylosin production, the tyl cluster contains three resistance determinants and several regulatory genes. Tylosin production is evidently controlled by pathway-specific and pleiotropic regulators with the likely involvement of γ-butyrolactone signalling factors. Accumulation of the polyketide aglycone is controlled by glycosylated macrolides and optimal performance of the complex polyketide synthase enzyme requires the activity of an editing thioesterase.

  • multiple regulatory genes in the tylosin biosynthetic cluster of Streptomyces fradiae
    Chemistry & Biology, 1999
    Co-Authors: Neil Bate, Atul R Gandecha, Andrew R. Butler, Eric Cundliffe
    Abstract:

    Background The macrolide antibiotic tylosin is composed of a polyketide lactone substituted with three deoxyhexose sugars. In order to produce tylosin efficiently, Streptomyces fradiae presumably requires control mechanisms that balance the yields of the constituent metabolic pathways together with switches that allow for temporal regulation of antibiotic production. In addition to possible metabolic feedback and/or other signalling devices, such control probably involves interplay between specific regulatory proteins. Prior to the present work, however, no candidate regulatory gene(s) had been identified in S. fradiae. Results DNA sequencing has shown that the tylosin biosynthetic gene cluster, within which four open reading frames utilise the rare TTA codon, contains at least five candidate regulatory genes, one of which ( tylP ) encodes a γ-butyrolactone signal receptor for which tylQ is a probable target. Two other genes ( tylS and tylT ) encode pathway-specific regulatory proteins of the Streptomyces antibiotic regulatory protein (SARP) family and a fifth, tylR , has been shown by mutational analysis to control various aspects of tylosin production. Conclusions The tyl genes of S. fradiae include the richest collection of regulators yet encountered in a single antibiotic biosynthetic gene cluster. Control of tylosin biosynthesis is now amenable to detailed study, and manipulation of these various regulatory genes is likely to influence yields in tylosin-production fermentations.

  • impact of thioesterase activity on tylosin biosynthesis in Streptomyces fradiae
    Chemistry & Biology, 1999
    Co-Authors: Andrew R. Butler, Neil Bate, Eric Cundliffe
    Abstract:

    Abstract Background: The polyketide lactone tylactone is produced in Streptomycesfradiae by the TyIG complex of five multifunctional proteins. As with other type I polyketide synthases, the enzyme catalysing the final elongation step (TyIGV) possesses an integral thioesterase domain that is believed to be responsible for chain termination and ring closure to form tylactone, which is then glycosylated to yield tylosin. In common with other macrolide producers, S. fradiae also possesses an additional thioesterase gene ( orf5 ) located within the cluster of antibiotic biosynthetic genes. The function of the Orf5 protein is addressed here. Results: Disruption of orf5 reduced antibiotic accumulation in S. fradiae by atleast 85%. Under such circumstances, the strain accumulated desmycosin (demycarosyl-tylosin) due to a downstream polar effect on the expression of orf6 , which encodes a mycarose biosynthetic enzyme. High levels of desmycosin production were restored in the disrupted strain by complementation with intact orf5 , or with the corresponding thioesterase gene, nbmB , from S. narbonensis , but not with DNA encoding the integral thioesterase domain of TyIGV. Conclusions: Polyketide metabolism in S. fradiae is strongly dependent on thethioesterase activity encoded by orf5 ( tylO ). It is proposed that the TyIG complex might operate with a significant error frequency and be prone to blockage with aberrant polyketides. A putative editing activity associated with TyIO might be essential to unblock the polyketide synthase complex and thereby promote antibiotic accumulation.

Atul R Gandecha - One of the best experts on this subject based on the ideXlab platform.

  • regulation of tylosin production and morphological differentiation in Streptomyces fradiae by tylp a deduced gamma butyrolactone receptor
    Molecular Microbiology, 2002
    Co-Authors: George Stratigopoulos, Atul R Gandecha, Eric Cundliffe
    Abstract:

    During promoter-probe analysis carried out in Streptomyces lividans, the TylP protein powerfully inhibited reporter gene expression from the tylP promoter, raising the likelihood that tylP is autoregulated in its native host, Streptomyces fradiae. Also in S. lividans, TylP negatively controlled the tylQ promoter, even though tylQ could still be switched off in S. fradiae strains specifically disrupted in tylP. Under the latter conditions, tylosin production was brought forward and enhanced, whereas overexpression of tylP resulted in reduced levels of the antibiotic, accompanied by barely detectable transcription from multiple genes of the tylosin biosynthetic cluster. Unexpectedly, overexpression of tylP reduced transcription of tylS, a transcriptional activator essential for tylosin production. This was probably a direct effect, as TylP also reduced expression from the tylS promoter in S. lividans. For these several reasons, we conclude that TylP acts as a repressor during tylosin biosynthesis. In addition, TylP influences morphological differentiation in S. fradiae. On solid media, strains in which tylP was disrupted sporulated significantly earlier than wild type and, in liquid culture, displayed hyperfragmentation.

  • The tylosin-biosynthetic genes of Streptomyces fradiae
    Antonie van Leeuwenhoek, 2001
    Co-Authors: Eric Cundliffe, Atul R Gandecha, Neil Bate, Andrew Butler, Steven Fish, Louise Merson-davies
    Abstract:

    The tylosin-biosynthetic ( tyl ) gene cluster occupies about 1% of the genome of Streptomyces fradiae and includes at least 43 open reading frames. In addition to structural genes required for tylosin production, the tyl cluster contains three resistance determinants and several regulatory genes. Tylosin production is evidently controlled by pathway-specific and pleiotropic regulators with the likely involvement of γ-butyrolactone signalling factors. Accumulation of the polyketide aglycone is controlled by glycosylated macrolides and optimal performance of the complex polyketide synthase enzyme requires the activity of an editing thioesterase.

  • multiple regulatory genes in the tylosin biosynthetic cluster of Streptomyces fradiae
    Chemistry & Biology, 1999
    Co-Authors: Neil Bate, Atul R Gandecha, Andrew R. Butler, Eric Cundliffe
    Abstract:

    Background The macrolide antibiotic tylosin is composed of a polyketide lactone substituted with three deoxyhexose sugars. In order to produce tylosin efficiently, Streptomyces fradiae presumably requires control mechanisms that balance the yields of the constituent metabolic pathways together with switches that allow for temporal regulation of antibiotic production. In addition to possible metabolic feedback and/or other signalling devices, such control probably involves interplay between specific regulatory proteins. Prior to the present work, however, no candidate regulatory gene(s) had been identified in S. fradiae. Results DNA sequencing has shown that the tylosin biosynthetic gene cluster, within which four open reading frames utilise the rare TTA codon, contains at least five candidate regulatory genes, one of which ( tylP ) encodes a γ-butyrolactone signal receptor for which tylQ is a probable target. Two other genes ( tylS and tylT ) encode pathway-specific regulatory proteins of the Streptomyces antibiotic regulatory protein (SARP) family and a fifth, tylR , has been shown by mutational analysis to control various aspects of tylosin production. Conclusions The tyl genes of S. fradiae include the richest collection of regulators yet encountered in a single antibiotic biosynthetic gene cluster. Control of tylosin biosynthesis is now amenable to detailed study, and manipulation of these various regulatory genes is likely to influence yields in tylosin-production fermentations.

  • analysis of four tylosin biosynthetic genes from the tyllm region of the Streptomyces fradiae genome
    Gene, 1997
    Co-Authors: Atul R Gandecha, Sarah L Large, Eric Cundliffe
    Abstract:

    Abstract The tylLM region of the tylosin biosynthetic gene cluster of Streptomyces fradiae contains four open reading frames (orfs1 * –4 * ). The function of the orf1 * product is not known. The product of orf2 * ( tylM2 ) is the glycosyltransferase that adds mycaminose to the 5-hydroxyl group of tylactone, the polyketide aglycone of tylosin (Ty). A methyltransferase, responsible for 3- N -methylation during mycaminose production, is encoded by orf3 * ( tylM1 ). The product of orf4 * ( ccr ) is crotonyl-CoA reductase, which converts acetoacetyl-CoA to butyryl-CoA for use as a 4C extender unit during tylactone production.

Jonathan B. Spencer - One of the best experts on this subject based on the ideXlab platform.

  • The neomycin biosynthetic gene cluster of Streptomyces fradiae NCIMB 8233: characterisation of an aminotransferase involved in the formation of 2-deoxystreptamine.
    Organic & biomolecular chemistry, 2005
    Co-Authors: Fanglu Huang, Stephen F. Haydock, Tatiana Mironenko, Dieter Spiteller, Jonathan B. Spencer
    Abstract:

    The biosynthetic gene cluster of the 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotic neomycin has been cloned for the first time by screening of a cosmid library of Streptomyces fradiae NCIMB 8233. Sequence analysis has identified 21 putative open reading frames (ORFs) in the neomycin gene cluster (neo) with significant protein sequence similarity to gene products involved in the biosynthesis of other DOS-containing aminoglycosides, namely butirosin (btr), gentamycin (gnt), tobramycin (tbm) and kanamycin (kan). Located at the 5'-end of the neo gene cluster is the previously-characterised neomycin phosphotransferase gene (apH). Three genes unique to the neo and btr clusters have been revealed by comparison of the neo cluster to btr, gnt, tbm and kan clusters. This suggests that these three genes may be involved in the transfer of a ribose moiety to the DOS ring during the antibiotic biosynthesis. The product of the neo-6 gene is characterised here as the L-glutamine : 2-deoxy-scyllo-inosose aminotransferase responsible for the first transamination in DOS biosynthesis, which supports the assignment of the gene cluster.

Huimin Zhao - One of the best experts on this subject based on the ideXlab platform.

  • heterologous production of fosfomycin and identification of the minimal biosynthetic gene cluster
    Chemistry & Biology, 2006
    Co-Authors: Ryan Woodyer, Zengyi Shao, Joshua A V Blodgett, Wilfred A Van Der Donk, Paul M. Thomas, Neil L. Kelleher, William W Metcalf, Huimin Zhao
    Abstract:

    Summary Fosfomycin is a clinically utilized, highly effective antibiotic, which is active against methicillin- and vancomycin-resistant pathogens. Here we report the cloning and characterization of a complete fosfomycin biosynthetic cluster from Streptomyces fradiae and heterologous production of fosfomycin in S. lividans . Sequence analysis coupled with gene deletion and disruption revealed that the minimal cluster consists of fom1-4 , fomA-D . A LuxR-type activator that was apparently required for heterologous fosfomycin production was also discovered ∼13 kb away from the cluster and was named fomR . The genes fomE and fomF , previously thought to be involved in fosfomycin biosynthesis, were shown not to be essential by gene disruption. This work provides new insights into fosfomycin biosynthesis and opens the door for fosfomycin overproduction and creation of new analogs via biomolecular pathway engineering.

  • heterologous production of fosfomycin and identification of the minimal biosynthetic gene cluster
    Chemistry & Biology, 2006
    Co-Authors: Ryan Woodyer, Zengyi Shao, Joshua A V Blodgett, Paul M. Thomas, Neil L. Kelleher, William W Metcalf, Wilfred A Van Der Donk, Huimin Zhao
    Abstract:

    Fosfomycin is a clinically utilized, highly effective antibiotic, which is active against methicillin- and vancomycin-resistant pathogens. Here we report the cloning and characterization of a complete fosfomycin biosynthetic cluster from Streptomyces fradiae and heterologous production of fosfomycin in S. lividans. Sequence analysis coupled with gene deletion and disruption revealed that the minimal cluster consists of fom1-4, fomA-D. A LuxR-type activator that was apparently required for heterologous fosfomycin production was also discovered approximately 13 kb away from the cluster and was named fomR. The genes fomE and fomF, previously thought to be involved in fosfomycin biosynthesis, were shown not to be essential by gene disruption. This work provides new insights into fosfomycin biosynthesis and opens the door for fosfomycin overproduction and creation of new analogs via biomolecular pathway engineering.