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1.
《Molecular & general genetics : MGG》1998,258(1-2):78-88
The gene cluster (ery) governing the biosynthesis of the macrolide antibiotic erythromycin A by Saccharopolyspora erythraea contains, in addition to the eryA genes encoding the polyketide synthase, two regions containing genes for later steps in the pathway. The region 5′ of eryA that lies between the known genes ermE (encoding the erythromycin resistance methyltransferase) and eryBIII (encoding a putative S-adenosylmethionine-dependent methyltransferase), and that contains the gene eryBI (orf2), has now been sequenced. The inferred product of the eryBI gene shows striking sequence similarity to authentic β-glucosidases. Specific mutants were created in eryBI, and the resulting strains were found to synthesise erythromycin A, showing that this gene, despite its position in the biosynthetic
gene cluster, is not essential for erythromycin biosynthesis. A␣mutant in eryBIII and a double mutant in eryBI and eryBIII were obtained and the analysis of novel erythromycins produced by these strains confirmed the proposed function of EryBIII
as a C-methyltransferase. Also, a chromosomal mutant was constructed for the previously sequenced ORF19 and shown to accumulate
erythronolide B, as expected for an eryB mutant and consistent with its proposed role as an epimerase in dTDP-mycarose biosynthesis.
Received: 13 August 1997 / Accepted: 27 November 1997 相似文献
2.
S. Gaisser G. A. Böhm J. Cortés P. F. Leadlay 《Molecular genetics and genomics : MGG》1997,256(3):239-251
The gene cluster (ery) governing the biosynthesis of the macrolide antibiotic erythromycin A by Saccharopolyspora erythraea contains, in addition to the eryA genes encoding the polyketide synthase, two regions containing genes for later steps in the pathway. The region 5′ of eryA, and lying between eryA and the gene eryK, which is known to encode the C-12 hydroxylase, has been sequenced and shown to contain seven additional open reading frames (ORFs 13–19). On the basis of sequence similarities, roles are proposed for several of these ORFs in the biosynthesis of the deoxysugar mycarose and the deoxyaminosugar desosamine. A chromosomal mutant carrying a deletion in ORF15 has been constructed and shown to accumulate 3-O-mycarosyl-erythronolide B, as expected for an eryC mutant. Similarly, a chromosomal mutant carrying a deletion in ORF16 has been constructed and shown to accumulate erythronolide B, as expected for an eryB mutant. 相似文献
3.
《Molecular & general genetics : MGG》1997,256(3):239-251
The gene cluster (ery) governing the biosynthesis of the macrolide antibiotic erythromycin A by Saccharopolyspora erythraea contains, in addition to the eryA genes encoding the polyketide synthase, two regions containing genes for later steps in the pathway. The region 5′ of eryA, and lying between eryA and the gene eryK, which is known to encode the C-12 hydroxylase, has been sequenced and shown to contain seven additional open reading frames
(ORFs 13–19). On the basis of sequence similarities, roles are proposed for several of these ORFs in the biosynthesis of the
deoxysugar mycarose and the deoxyaminosugar desosamine. A chromosomal mutant carrying a deletion in ORF15 has been constructed
and shown to accumulate 3-O-mycarosyl-erythronolide B, as expected for an eryC mutant. Similarly, a chromosomal mutant carrying a deletion in ORF16 has been constructed and shown to accumulate erythronolide
B, as expected for an eryB mutant.
Received: 10 March 1997 / Accepted: 12 June 1997 相似文献
4.
K. Salah-Bey M. Doumith J.-M. Michel S. Haydock J. Cortés P. F. Leadlay M.-C. Raynal 《Molecular genetics and genomics : MGG》1998,257(5):542-553
The production of erythromycin A by Saccharopolysporaerythraea requires the synthesis of dTDP-D-desosamine and dTDP-L-mycarose, which serve as substrates for the transfer of the two sugar residues onto the macrolactone ring. The enzymatic activities involved in this process are largely encoded within the ery gene cluster, by two sets of genes flanking the eryA locus that encodes the polyketide synthase. We report here the nucleotide sequence of three such ORFs located immediately downstream of eryA, ORFs 7, 8 and 9. Chromosomal mutants carrying a deletion either in ORF7 or in one of the previously sequenced ORFs 13 and 14 have been constructed and shown to accumulate erythronolide B, as expected for eryB mutants. Similarly, chromosomal mutants carrying a deletion in either ORF8, ORF9, or one of the previously sequenced ORFs 17 and 18 have been constructed and shown to accumulate 3-α-mycarosyl erythronolide B, as expected for eryC mutants. The ORF13 (eryBIV?), ORF17 (eryCIV?) and ORF7 (eryBII?) mutants also synthesised small amounts of macrolide shunt metabolites, as shown by mass spectrometry. These results considerably strengthen previous tentative proposals for the pathways for the biosynthesis of dTDP-D-desosamine and dTDP-L-mycarose in Sac. erythraea and reveal that at least some of these enzymes can accommodate alternative substrates. 相似文献
5.
6.
Boakes S Oliynyk M Cortés J Böhm I Rudd BA Revill WP Staunton J Leadlay PF 《Journal of molecular microbiology and biotechnology》2004,8(2):73-80
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. 相似文献
7.
Vijayashankar Nataraj Poh-choo Pang Stuart M. Haslam Natacha Veerapen David E. Minnikin Anne Dell Gurdyal S. Besra Apoorva Bhatt 《PloS one》2015,10(4)
Lipooligosaccharides are glycolipids found in the cell wall of many mycobacterial species including the opportunistic pathogen Mycobacterium kansasii. The genome of M. kansasii ATCC12478 contains a cluster with genes orthologous to Mycobacterium marinum LOS biosynthesis genes. To initiate a genetic dissection of this cluster and demonstrate its role in LOS biosynthesis in M. kansasii, we chose MKAN27435, a gene encoding a putative glycosyltransferase. Using Specialized Transduction, a phage-based gene knockout tool previously used to generate null mutants in other mycobacteria, we generated a MKAN27435 null mutant. The mutant strain was found to be defective in the biosynthesis of higher LOS subspecies, viz LOS-IV, LOS-V, LOS-VI and LOS-VII. Additionally, a range of low abundance species were detected in the mutant strain and mass spectroscopic analysis indicated that these were shunt products generated from LOS-III by the addition of up to six molecules of a pentose. 相似文献
8.
Lucie Najmanova Dana Ulanova Marketa Jelinkova Zdenek Kamenik Eliska Kettnerova Marketa Koberska Radek Gazak Bojana Radojevic Jiri Janata 《Folia microbiologica》2014,59(6):543-552
The biosynthetic gene cluster of porothramycin, a sequence-selective DNA alkylating compound, was identified in the genome of producing strain Streptomyces albus subsp. albus (ATCC 39897) and sequentially characterized. A 39.7 kb long DNA region contains 27 putative genes, 18 of them revealing high similarity with homologous genes from biosynthetic gene cluster of closely related pyrrolobenzodiazepine (PBD) compound anthramycin. However, considering the structures of both compounds, the number of differences in the gene composition of compared biosynthetic gene clusters was unexpectedly high, indicating participation of alternative enzymes in biosynthesis of both porothramycin precursors, anthranilate, and branched L-proline derivative. Based on the sequence analysis of putative NRPS modules Por20 and Por21, we suppose that in porothramycin biosynthesis, the methylation of anthranilate unit occurs prior to the condensation reaction, while modifications of branched proline derivative, oxidation, and dimethylation of the side chain occur on already condensed PBD core. Corresponding two specific methyltransferase encoding genes por26 and por25 were identified in the porothramycin gene cluster. Surprisingly, also methyltransferase gene por18 homologous to orf19 from anthramycin biosynthesis was detected in porothramycin gene cluster even though the appropriate biosynthetic step is missing, as suggested by ultra high-performance liquid chromatography-diode array detection-mass spectrometry (UHPLC-DAD-MS) analysis of the product in the S. albus culture broth. 相似文献
9.
G. Blanco E. Fernández M. J. Fernández A. F. Braña U. Weissbach E. Künzel J. Rohr C. Méndez J. A. Salas 《Molecular genetics and genomics : MGG》2000,262(6):991-1000
A 2580-bp region of the chromosome of Streptomyces argillaceus, the producer of the antitumor polyketide mithramycin, was sequenced. Analysis of the nucleotide sequence revealed the presence of two genes (mtmGIII and mtmGIV?) encoding proteins that showed a high degree of similarity to glycosyltransferases involved in the biosynthesis of various antibiotics and antitumor drugs. Independent insertional inactivation of both genes produced mutants that did not synthesize mithramycin but accumulated several mithramycin intermediates. Both mutants accumulated premithramycinone, a non-glycosylated intermediate in mithramycin biosynthesis. The mutant affected in the mtmGIII gene also accumulated premithramycin A1, which contains premithramycinone as the aglycon unit and a D-olivose attached at C-12a-O. These experiments demonstrate that the glycosyltransferases MtmGIV and MtmGIII catalyze the first two glycosylation steps in mithramycin biosynthesis. A model is proposed for the glycosylation steps in mithramycin biosynthesis. 相似文献
10.
K. Salah-Bey M. Doumith J.-M. Michel S. Haydock J. Cortés P. F. Leadlay M.-C. Raynal 《Molecular & general genetics : MGG》1998,257(5):542-553
The production of erythromycin A by Saccharopolysporaerythraea requires the synthesis of dTDP-D-desosamine and dTDP-L-mycarose, which serve as substrates for the transfer of the two sugar
residues onto the macrolactone ring. The enzymatic activities involved in this process are largely encoded within the ery gene cluster, by two sets of genes flanking the eryA locus that encodes the polyketide synthase. We report here the nucleotide sequence of three such ORFs located immediately
downstream of eryA, ORFs 7, 8 and 9. Chromosomal mutants carrying a deletion either in ORF7 or in one of the previously sequenced ORFs 13 and
14 have been constructed and shown to accumulate erythronolide B, as expected for eryB mutants. Similarly, chromosomal mutants carrying a deletion in either ORF8, ORF9, or one of the previously sequenced ORFs
17 and 18 have been constructed and shown to accumulate 3-α-mycarosyl erythronolide B, as expected for eryC mutants. The ORF13 (eryBIV ), ORF17 (eryCIV ) and ORF7 (eryBII ) mutants also synthesised small amounts of macrolide shunt metabolites, as shown by mass spectrometry. These results considerably
strengthen previous tentative proposals for the pathways for the biosynthesis of dTDP-D-desosamine and dTDP-L-mycarose in
Sac. erythraea and reveal that at least some of these enzymes can accommodate alternative substrates.
Received: 29 July 1997 / Accepted: 16 October 1997 相似文献
11.
Dayu Yu Fuchao Xu Shuwei Zhang Lei Shao Siyuan Wang Jixun Zhan 《Bioorganic & medicinal chemistry letters》2013,23(20):5667-5670
The herboxidiene biosynthetic gene cluster contains a regulatory gene and six biosynthetic genes that encode three polyketide synthases (HerB, HerC and HerD) and three tailoring enzymes (HerE, HerF and HerG). Through single crossover recombination, an integrative plasmid was inserted into the genome of Streptomyces chromofuscus ATCC 49982 between herE and herF, resulting in low-level expression of herF and the downstream herG. The mutant strain produced two new compounds, 18-deoxy-25-demethyl-herboxidiene and 25-demethyl-herboxidiene. HerF was expressed in Escherichia coli and biochemically characterized as the dedicated methyltransferase in herboxidiene biosynthesis. It prefers 25-demethyl-herboxidiene to 18-deoxy-25-demethyl-herboxidiene, suggesting that C-25 methylation is the last tailoring step. 相似文献
12.
Characterization of a Gene Cluster Responsible for the Biosynthesis of Anticancer Agent FK228 in Chromobacterium violaceum No. 968
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A gene cluster responsible for the biosynthesis of anticancer agent FK228 has been identified, cloned, and partially characterized in Chromobacterium violaceum no. 968. First, a genome-scanning approach was applied to identify three distinctive C. violaceum no. 968 genomic DNA clones that code for portions of nonribosomal peptide synthetase and polyketide synthase. Next, a gene replacement system developed originally for Pseudomonas aeruginosa was adapted to inactivate the genomic DNA-associated candidate natural product biosynthetic genes in vivo with high efficiency. Inactivation of a nonribosomal peptide synthetase-encoding gene completely abolished FK228 production in mutant strains. Subsequently, the entire FK228 biosynthetic gene cluster was cloned and sequenced. This gene cluster is predicted to encompass a 36.4-kb DNA region that includes 14 genes. The products of nine biosynthetic genes are proposed to constitute an unusual hybrid nonribosomal peptide synthetase-polyketide synthase-nonribosomal peptide synthetase assembly line including accessory activities for the biosynthesis of FK228. In particular, a putative flavin adenine dinucleotide-dependent pyridine nucleotide-disulfide oxidoreductase is proposed to catalyze disulfide bond formation between two sulfhydryl groups of cysteine residues as the final step in FK228 biosynthesis. Acquisition of the FK228 biosynthetic gene cluster and acclimation of an efficient genetic system should enable genetic engineering of the FK228 biosynthetic pathway in C. violaceum no. 968 for the generation of structural analogs as anticancer drug candidates. 相似文献
13.
14.
《Gene》1997,185(1):35-41
The Gram-negative bacterium Flavobacterium sp. strain R1534 is a natural producer of zeaxanthin. A 14 kb genomic DNA fragment of this organism has been cloned and a 5.1 kb piece containing the carotenoid biosynthesis genes sequenced. The carotenoid biosynthesis cluster consists of five genes arranged in at least two operons. The five genes are necessary and sufficient for the synthesis of zeaxanthin. The encoded proteins have significant homology to the crtE, crtB, crtY, crtI and crtZ gene products of other carotenogenic organisms. Biochemical assignment of the individual gene products was done by HPLC analysis of the carotenoid accumulation in Escherichia coli host strains transformed with plasmids carrying deletions of the Flavobacterium sp. strain R1534 carotenoid biosynthesis cluster. 相似文献
15.
Knockout of the Erythromycin Biosynthetic Cluster Gene, eryBI, Blocks Isoflavone Glucoside Bioconversion during Erythromycin Fermentations in Aeromicrobium erythreum but Not in Saccharopolyspora erythraea
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Andrew R. Reeves Ramya Seshadri Igor A. Brikun William H. Cernota Melissa C. Gonzalez J. Mark Weber 《Applied microbiology》2008,74(23):7383-7390
Isoflavone glucosides are valuable nutraceutical compounds and are present in commercial fermentations, such as the erythromycin fermentation, as constituents of the soy flour in the growth medium. The purpose of this study was to develop a method for recovery of the isoflavone glucosides as value-added coproducts at the end of either Saccharopolyspora erythraea or Aeromicrobium erythreum fermentation. Because the first step in isoflavone metabolism was known to be the conversion of isoflavone glucosides to aglycones by a β-glucosidase, we chose to knock out the only β-glucosidase gene known at the start of the study, eryBI, to see what effect this had on metabolism of isoflavone glucosides in each organism. In the unicellular erythromycin producer A. erythreum, knockout of eryBI was sufficient to block the conversion of isoflavone glucosides to aglycones. In S. erythraea, knockout of eryBI had no effect on this reaction, suggesting that other β-glucosidases are present. Erythromycin production was not significantly affected in either strain as a result of the eryBI knockout. This study showed that isoflavone metabolism could be blocked in A. erythreum by eryBI knockout but that eryBI knockout was not sufficient to block isoflavone metabolism in S. erythraea. 相似文献
16.
Jianuan Zhou Yingying Cheng Mingfa Lv Lisheng Liao Yufan Chen Yanfang Gu Shiyin Liu Zide Jiang Yuanyan Xiong Lianhui Zhang 《BMC genomics》2015,16(1)
Background
Dickeya zeae is a bacterial species that infects monocotyledons and dicotyledons. Two antibiotic-like phytotoxins named zeamine and zeamine II were reported to play an important role in rice seed germination, and two genes associated with zeamines production, i.e., zmsA and zmsK, have been thoroughly characterized. However, other virulence factors and its molecular mechanisms of host specificity and pathogenesis are hardly known.Results
The complete genome of D. zeae strain EC1 isolated from diseased rice plants was sequenced, annotated, and compared with the genomes of other Dickeya spp.. The pathogen contains a chromosome of 4,532,364 bp with 4,154 predicted protein-coding genes. Comparative genomics analysis indicates that D. zeae EC1 is most co-linear with D. chrysanthemi Ech1591, most conserved with D. zeae Ech586 and least similar to D. paradisiaca Ech703. Substantial genomic rearrangement was revealed by comparing EC1 with Ech586 and Ech703. Most virulence genes were well-conserved in Dickeya strains except Ech703. Significantly, the zms gene cluster involved in biosynthesis of zeamines, which were shown previously as key virulence determinants, is present in D. zeae strains isolated from rice, and some D. solani strains, but absent in other Dickeya species and the D. zeae strains isolated from other plants or sources. In addition, a DNA fragment containing 9 genes associated with fatty acid biosynthesis was found inserted in the fli gene cluster encoding flagellar biosynthesis of strain EC1 and other two rice isolates but not in other strains. This gene cluster shares a high protein similarity to the fatty acid genes from Pantoea ananatis.Conlusion
Our findings delineate the genetic background of D. zeae EC1, which infects both dicotyledons and monocotyledons, and suggest that D. zeae strains isolated from rice could be grouped into a distinct pathovar, i.e., D. zeae subsp. oryzae. In addition, the results of this study also unveiled that the zms gene cluster presented in the genomes of D. zeae rice isolates and D. solani strains, and the fatty acid genes inserted in the fli gene cluster of strain EC1 were likely derived from horizontal gene transfer during later stage of bacterial evolution.Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1545-x) contains supplementary material, which is available to authorized users. 相似文献17.
《Gene》1996,169(1):1-7
Analysis of the gene cluster from Streptomyces hygroscopicus that governs the biosynthesis of the polyketide immuno-suppressant rapamycin (Rp) has revealed that it contains three exceptionally large open reading frames (ORFs) encoding the modular polyketide synthase (PKS). Between two of these lies a fourth gene (rapP) encoding a pipecolate-incorporating enzyme that probably also catalyzes closure of the macrolide ring. On either side of these very large genes are ranged a total of 22 further ORFs before the limits of the cluster are reached, as judged by the identification of genes clearly encoding unrelated activities. Several of these ORFs appear to encode enzymes that would be required for Rp biosynthesis. These include two cytochrome P-450 monooxygenases (P450s), designated RapJ and RapN, an associated ferredoxin (Fd) RapO, and three potential SAM-dependent O-methyltransferases (MTases), RapI, RapM and RapQ. All of these are likely to be involved in ‘late’ modification of the macrocycle. The cluster also contains a novel gene (rapL) whose product is proposed to catalyze the formation of the Rp precursor, L-pipecolate, through the cyclodeamination of L-lysine. Adjacent genes have putative roles in Rp regulation and export. The codon usage of the PKS biosynthetic genes is markedly different from that of the flanking genes of the cluster 相似文献
18.
Julia Schumacher Angélique Gautier Guillaume Morgant Lena Studt Paul-Henri Ducrot Pascal Le Pêcheur Saad Azeddine Sabine Fillinger Pierre Leroux Bettina Tudzynski Muriel Viaud 《PloS one》2013,8(1)
The gene cluster responsible for the biosynthesis of the red polyketidic pigment bikaverin has only been characterized in Fusarium ssp. so far. Recently, a highly homologous but incomplete and nonfunctional bikaverin cluster has been found in the genome of the unrelated phytopathogenic fungus Botrytis cinerea. In this study, we provided evidence that rare B. cinerea strains such as 1750 have a complete and functional cluster comprising the six genes orthologous to Fusarium fujikuroi ffbik1-ffbik6 and do produce bikaverin. Phylogenetic analysis confirmed that the whole cluster was acquired from Fusarium through a horizontal gene transfer (HGT). In the bikaverin-nonproducing strain B05.10, the genes encoding bikaverin biosynthesis enzymes are nonfunctional due to deleterious mutations (bcbik2-3) or missing (bcbik1) but interestingly, the genes encoding the regulatory proteins BcBIK4 and BcBIK5 do not harbor deleterious mutations which suggests that they may still be functional. Heterologous complementation of the F. fujikuroi Δffbik4 mutant confirmed that bcbik4 of strain B05.10 is indeed fully functional. Deletion of bcvel1 in the pink strain 1750 resulted in loss of bikaverin and overproduction of melanin indicating that the VELVET protein BcVEL1 regulates the biosynthesis of the two pigments in an opposite manner. Although strain 1750 itself expresses a truncated BcVEL1 protein (100 instead of 575 aa) that is nonfunctional with regard to sclerotia formation, virulence and oxalic acid formation, it is sufficient to regulate pigment biosynthesis (bikaverin and melanin) and fenhexamid HydR2 type of resistance. Finally, a genetic cross between strain 1750 and a bikaverin-nonproducing strain sensitive to fenhexamid revealed that the functional bikaverin cluster is genetically linked to the HydR2 locus. 相似文献
19.
Andrea M. Matter Sara B. Hoot Patrick D. Anderson Susana S. Neves Yi-Qiang Cheng 《PloS one》2009,4(9)
Many Streptomyces strains are known to produce valinomycin (VLM) antibiotic and the VLM biosynthetic gene cluster (vlm) has been characterized in two independent isolates. Here we report the phylogenetic relationships of these strains using both parsimony and likelihood methods, and discuss whether the vlm gene cluster shows evidence of horizontal transmission common in natural product biosynthetic genes. Eight Streptomyces strains from around the world were obtained and sequenced for three regions of the two large nonribosomal peptide synthetase genes (vlm1 and vlm2) involved in VLM biosynthesis. The DNA sequences representing the vlm gene cluster are highly conserved among all eight environmental strains. The geographic distribution pattern of these strains and the strict congruence between the trees of the two vlm genes and the housekeeping genes, 16S rDNA and trpB, suggest vertical transmission of the vlm gene cluster in Streptomyces with no evidence of horizontal gene transfer. We also explored the relationship of the sequence of vlm genes to that of the cereulide biosynthetic genes (ces) found in Bacillus cereus and found them highly divergent from each other at DNA level (genetic distance values≥95.6%). It is possible that the vlm gene cluster and the ces gene cluster may share a relatively distant common ancestor but these two gene clusters have since evolved independently. 相似文献
20.
Blanco G Fernández E Fernández MJ Braña AF Weissbach U Künzel E Rohr J Méndez C Salas JA 《Molecular & general genetics : MGG》2000,262(6):991-1000
A 2580-bp region of the chromosome of Streptomyces argillaceus, the producer of the antitumor polyketide mithramycin, was sequenced. Analysis of the nucleotide sequence revealed the presence of two genes (mtmGIII and mtmGIVv) encoding proteins that showed a high degree of similarity to glycosyltransferases involved in the biosynthesis of various antibiotics and antitumor drugs. Independent insertional inactivation of both genes produced mutants that did not synthesize mithramycin but accumulated several mithramycin intermediates. Both mutants accumulated premithramycinone, a non-glycosylated intermediate in mithramycin biosynthesis. The mutant affected in the mtmGIII gene also accumulated premithramycin A1, which contains premithramycinone as the aglycon unit and a D-olivose attached at C-12a-O. These experiments demonstrate that the glycosyltransferases MtmGIV and MtmGIII catalyze the first two glycosylation steps in mithramycin biosynthesis. A model is proposed for the glycosylation steps in mithramycin biosynthesis. 相似文献