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Yuqing Tian - One of the best experts on this subject based on the ideXlab platform.
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Identification of a butenolide signaling system that regulates Nikkomycin biosynthesis in Streptomyces
Journal of Biological Chemistry, 2018Co-Authors: Wenxi Wang, Jihui Zhang, Yue Li, Dong Li, Yuqing TianAbstract:: Butenolides are an emerging family of signaling molecules in Streptomyces. They control complex physiological traits, such as morphological differentiation and antibiotic production. However, how butenolides regulate these processes is poorly investigated because of obstacles in obtaining these signaling molecules. This study reports the identification of a butenolide-type signaling system for Nikkomycin biosynthesis in Streptomyces ansochromogenes with distinct features. We identified a gene cluster, sab, consisting of three genes, sabAPD, for butenolide biosynthesis and two regulator genes, sabR1 and sabR2, and characterized three butenolides (SAB1, -2, and -3) by heterologous expression of sabAPD. sabA disruption abolished Nikkomycin production, which could be restored by the addition of SABs or by deletion of sabR1 in ΔsabA. Electrophoretic mobility-shift assays and transcriptional analyses indicated that SabR1 indirectly represses the transcription of Nikkomycin biosynthetic genes, but directly represses sabA and sabR1 In the presence of SABs, the SabR1 transcriptional regulator dissociated from its target genes, verifying that SabR1 is the cognate receptor of SABs. Genome-wide scanning with the conserved SabR1-binding sequence revealed another SabR1 target gene, cprC, whose transcription was strongly repressed by SabR1. Intriguingly, CprC positively regulated the pleiotropic regulatory gene adpA by binding to its promoter and, in turn, activated Nikkomycin biosynthesis. This is the first report that butenolide-type signaling molecules and their cognate receptor SabR1 can regulate adpA via a newly identified activator, CprC, to control Nikkomycin production. These findings pave the way for further studies seeking to unravel the regulatory mechanism and functions of the butenolide signaling system in Streptomyces.
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Engineering and heterologous expression of a Nikkomycin biosynthetic gene cluster
Acta Microbiologica Sinica, 2015Co-Authors: Lu Wang, Jine Li, Deyao Du, Yuqing TianAbstract:OBJECTIVE: We expressed a Nikkomycin biosynthetic gene cluster in the well-characterized surrogate Streptomyces coelicolor M1146. METHODS: By using PCR-targeting method, we replaced the promoters of sanG and sanF in pNIK, which contains Nikkomycin biosynthetic gene cluster, with the hrdB promoter to generate pNIKm. We transferred pNIK and pNIKm into S. coelicolor M1146 by intergeneric conjugation and obtained M1146-NIK and M1146-NIKm, respectively. We then evaluated expression of the gene cluster in the heterologous host by RT-PCR. Furthermore, we also compared the antifugal activity and Nikkomycin production of M1146-NIK and M1146-NIKm by bioassay against Alternaria longipes and HPLC analysis. RESULTS: M1146-NIK and M1146-NIKm exhibited antifungal activity, and they can produce a trace amount of Nikkomycin X, Nikkomycin Z and pseudo-Z. There was a substantial accumulation of uridine in M1146-NIK, whereas substantial accumulations of uridine, ribofuranosyl-4-formyl-4-imidazolone and pyridylhomothreonine were observed in M1146-NIKm. CONCLUSION: We successfully expressed the Nikkomycin biosynthetic gene cluster in the heterologous host and identified Nikkomycins and some of its key biosynthetic intermediates. This study will provide the basis for enzymatic reaction of the condensation between the two Nikkomycin moieties and for the generation of hybrid antibiotics by combinatorial biosynthesis.
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Improvement of gougerotin and Nikkomycin production by engineering their biosynthetic gene clusters
Applied Microbiology and Biotechnology, 2013Co-Authors: Deyao Du, Yuqing TianAbstract:Nikkomycins and gougerotin are peptidyl nucleoside antibiotics with broad biological activities. The Nikkomycin biosynthetic gene cluster comprises one pathway-specific regulatory gene (sanG) and 21 structural genes, whereas the gene cluster for gougerotin biosynthesis includes one putative regulatory gene, one major facilitator superfamily transporter gene, and 13 structural genes. In the present study, we introduced sanG driven by six different promoters into Streptomyces ansochromogenes TH322. Nikkomycin production was increased significantly with the highest increase in engineered strain harboring hrdB promoter-driven sanG. In the meantime, we replaced the native promoter of key structural genes in the gougerotin (gou) gene cluster with the hrdB promoters. The heterologous producer Streptomyces coelicolor M1146 harboring the modified gene cluster produced gougerotin up to 10-fold more than strains carrying the unmodified cluster. Therefore, genetic manipulations of genes involved in antibiotics biosynthesis with the constitutive hrdB promoter present a robust, easy-to-use system generally useful for the improvement of antibiotics production in Streptomyces.
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SabR enhances Nikkomycin production via regulating the transcriptional level of sanG, a pathway-specific regulatory gene in Streptomyces ansochromogenes
BMC Microbiology, 2011Co-Authors: Linqi Wang, Xihong He, Yuqing TianAbstract:sabR is a pleiotropic regulatory gene which has been shown to positively regulate the Nikkomycin biosynthesis and negatively affect the sporulation of Streptomyces ansochromogenes. In this study, we investigate the mechanism of SabR on modulating Nikkomycin production in Streptomyces ansochromogenes. The transcription start point of sabR was determined by high-resolution S1 nuclease mapping and localized at the nucleotide T at position 37 bp upstream of the potential sabR translation start codon (GTG). Disruption of sabR enhanced its own transcription, but retarded the Nikkomycin production. Over-expression of sabR enhanced Nikkomycin biosynthesis in Streptomyces ansochromogenes. EMSA analysis showed that SabR bound to the upstream region of sanG, but it did not bind to the upstream region of its encoding gene (sabR), sanF and the intergenic region between sanN and sanO. DNase 1 footprinting assays showed that the SabR-binding site upstream of sanG was 5'-CTTTAAGTCACCTGGCTCATTCGCGTTCGCCCAGCT-3' which was designated as SARE. Deletion of SARE resulted in the delay of Nikkomycin production that was similar to that of sabR disruption mutant. These results indicated that SabR modulated Nikkomycin biosynthesis as an enhancer via interaction with the promoter region of sanG, and expanded our understanding about regulatory cascade in Nikkomycin biosynthesis.
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Hybrid antibiotics with the Nikkomycin nucleoside and polyoxin peptidyl moieties
Metabolic Engineering, 2011Co-Authors: Jine Li, Lei Li, Yuqing TianAbstract:Acting as competitive inhibitors of chitin synthase, Nikkomycins and polyoxins are potent antibiotics against pathogenic fungi. Taking advantage of the structural similarities between these two peptidyl nucleoside antibiotics, genes required for the biosynthesis of the dipeptidyl moiety of polyoxin from Streptomyces cacaoi were introduced into a Streptomyces ansochromogenes mutant producing the nucleoside moiety of Nikkomycin X. Two hybrid antibiotics were generated. One of them was identified as polyoxin N, and the other, a novel compound, was named polynik A. The hybrid antibiotics exhibited merits from both parents: they had better inhibitory activity against phytopathogenic fungi than polyoxin B, and were more stable under different pH and temperature conditions than Nikkomycin X. This study demonstrates the use of the combinatorial biosynthetic approach to produce valuable and novel hybrid antibiotics with improved properties.
Christiane Bormann - One of the best experts on this subject based on the ideXlab platform.
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A putative enolpyruvyl transferase gene involved in Nikkomycin biosynthesis.
The Journal of Antibiotics, 2000Co-Authors: Bettina Lauer, Dietmar Kaiser, Roderich D. Süssmuth, Günther Jung, Christiane BormannAbstract:The nikO gene encoding a putative enolpyruvyl transferase has been identified within the Streptomyces tendae Tu901/8c Nikkomycin gene cluster. nikO encodes a deduced protein of 471 amino acid residues which exhibits significant sequence similarity to UDP-N-acetylglucosamine enolpyruvyl transferase and 5-enol-pyruvylshikimate 3-phosphate synthase from various origin. The nikO gene was inactivated by inserting a kanamycin resistance cassette; the mutant did not produce biologically active Nikkomycins I, J, X, and Z nor the nucleoside moieties, Nikkomycins Cx and Cz, but accumulated the novel component RT 2.0. RT 2.0 has been isolated from culture filtrate and its structure was determined by using mass spectrometry and NMR analyses as ribofuranosyl-4-formyl-4-imidazolone which represents a novel nucleoside. The putative activity of the nikO gene product in Nikkomycin biosynthesis will be discussed.
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Molecular characterization of two genes from Streptomyces tendae Tü901 required for the formation of the 4-formyl-4-imidazolin-2-one-containing nucleoside moiety of the peptidyl nucleoside antibiotic Nikkomycin.
European journal of biochemistry, 2000Co-Authors: Bettina Lauer, Roland Russwurm, Christiane BormannAbstract:The genes nikQ and nikR were identified by sequencing DNA of the Nikkomycin biosynthetic gene cluster from Streptomyces tendae Tu901/8c. The nikQ gene encodes a P450 cytochrome, and the predicted NikR gene product shows 48–56% sequence identity with uracil phosphoribosyltransferases from eukaryotic organisms. The nikQ and nikR genes were inactivated separately by insertion of a kanamycin-resistance cassette. Inactivation of the nikQ gene abolished synthesis of Nikkomycins containing 4-formyl-4-imidazolin-2-one as the base (Nikkomycins X and I), whereas production of Nikkomycins containing uracil (Nikkomycins Z and J) was not affected. Nikkomycin X and I production could be restored by feeding 4-formyl-4-imidazolin-2-one to the nikQ mutants, indicating that NikQ is responsible for its formation from l-histidine. Disruption of the nikR gene resulted in formation of decreased amounts of Nikkomycins X and I, whereas Nikkomycins Z and J were synthesized at wild-type levels. A fluorouracil-resistant nikR mutant lacking uracil phosphoribosyltransferase (UPRTase) activity did not synthesize Nikkomycins X and I and accumulated 4-formyl-4-imidazolin-2-one in its culture filtrate, whereas formation of Nikkomycins Z and J was unimpaired. The mutant was complemented to Nikkomycin X and I production by nikR expressed from the mel promoter of plasmid pIJ702. The nikR gene expressed in Escherichia coli led to the production of UPRTase activity. Our results indicate that NikR converts 4-formyl-4-imidazolin-2-one to yield 5′-phosphoribosyl-4-formyl-4-imidazolin-2-one, the precursor of Nikkomycins containing this base.
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Molecular characterization of co-transcribed genes from Streptomyces tendae Tü901 involved in the biosynthesis of the peptidyl moiety of the peptidyl nucleoside antibiotic Nikkomycin
Molecular Genetics and Genomics, 1999Co-Authors: Christina Bruntner, Bettina Lauer, V. Möhrle, W. Schwarz, Christiane BormannAbstract:Six genes (nikP1, nikP2, nikS, nikT, nikU, and nikV) from Streptomyces tendae Tu901 were identified by analysis of the nucleotide sequence of the Nikkomycin gene cluster. These genes, together with the previously described nikQ and nikR, span 9.39 kb and are transcribed as a polycistronic mRNA in a growth-phase-dependent manner. The nikP1 gene encodes a non-ribosomal peptide synthase consisting of an adenylation domain, a thiolation domain, and an N-terminal 70-residue segment of unknown function. The amino acid sequence encoded by the nikP2 gene displays similarity to the sequences of thioesterases, and the nikS product belongs to a superfamily of proteins characterized by a specific ATP-binding fold. The N-terminal 70 amino acids of the predicted nikT gene product show significant sequence similarity to acyl carrier proteins, and the C-terminal 330 amino acids to aminotransferases. The sequences of the deduced proteins NikU and NikV exhibit similarity to components S and E, respectively, of glutamate mutase from Clostridium. Disruption of the nikP1, nikS, nikT, or nikV gene by insertion of a kanamycin resistance cassette abolished formation of Nikkomycins I, J, X, and Z, all of which contain hydroxypyridylhomothreonine as the peptidyl moiety. The nikP1 mutants, and the nikS and nikT mutants accumulated the nucleoside moieties Nikkomycin Cz, and Nikkomycins Cx and Cz, respectively. The nikV mutants formed Nikkomycins Ox and Oz, which contain 2-amino-4-hydroxy-4-(3'-hydroxy-6'-pyridyl) butanoic acid as the peptidyl moiety. The nikP2 mutants synthesized Nikkomycins I, J, X, and Z, but amounts of Nikkomycins I and X, which contain formylimidazolone as the base, were lower. Feeding formylimidazolone to nikP2 mutants restored the ability to form Nikkomycins I and X. Our results indicate that nikU and nikV are required for the synthesis of hydroxypyridylhomothreonine, the genes nikP1, nikP2 and nikS are required for the assembly of Nikkomycins, and nikT is required for both pathways. The putative activities of each of their products are discussed.
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production of Nikkomycins bx and bz by mutasynthesis with genetically engineered streptomyces tendae tu901
The Journal of Antibiotics, 1999Co-Authors: Christiane Bormann, A Kalmanczhelyi, Roderich D. Süssmuth, Guenther JungAbstract:The previously described Streptomyces tendae nikC::aph mutant was used to mutasynthesize Nikkomycins Bx and Bz. The mutant is deficient in L-lysine 2-aminotransferase, which transaminates lysine to form piperideine 2-carboxylate, the precursor of the peptidyl side chain of the biologically active Nikkomycins I, J, X, and Z, and is therefore unable to produce these Nikkomycins. The mutant accumulates the biologically inactive biosynthetic nucleoside precursors Nikkomycins Cx and Cz. Resting cell cultures of the mutant fed with benzoic acid produced the biologically active Nikkomycins Bx and Bz, which contain 2-amino-4-hydroxy-3-methyl-4-(4'-hydroxyphenyl)butanoic acid as the peptidyl side chain. The structures of Nikkomycins Bx and Bz were confirmed by mass spectrometry and NMR. Nikkomycins Bx and Bz exhibit significantly higher pH stability than their analogues Nikkomycins X and Z.
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The Streptomyces tendae Tü901 L-lysine 2-aminotransferase catalyzes the initial reaction in Nikkomycin D biosynthesis
FEBS Journal, 1998Co-Authors: Christina Bruntner, Christiane BormannAbstract:: Protein P8 was previously identified as a putative Nikkomycin biosynthesis protein. The gene (nikC) encoding protein P8 was cloned from the Streptomyces tendae Tu901 Nikkomycin gene cluster and sequenced. The nikC gene was inactivated by inserting a kanamycin resistance cassette; the mutant did not produce the biologically active Nikkomycins I, J, X, and Z, but accumulated the nucleoside moieties Nikkomycins C(X) and C(Z). The mutant was complemented to Nikkomycin production (I, J, X, Z) by nikC expressed from the mel promoter of the vector pIJ702. Furthermore, the Nikkomycin-negative phenotype was reversed by the addition of picolinic acid, a precursor of the peptidyl moiety of Nikkomycins (Nikkomycin D), into the culture medium. The nikC gene was expressed in Escherichia coli and identified and characterized at the enzyme level. NikC encodes an L-lysine 2-aminotransferase, and the activity was exclusively detected in Nikkomycin producers and its presence correlated to Nikkomycin production. The nikC-inactivated mutant grew with L-lysine as sole source of nitrogen and carbon, indicating that L-lysine 2-aminotransferase is not required for lysine catabolism. Our results identified the nikC-encoded L-lysine 2-aminotransferase as the Nikkomycin biosynthetic enzyme that catalyzes the initial reaction in Nikkomycin D biosynthesis. The NikC protein belongs to a novel family of pyridoxamine or pyridoxal-phosphate-dependent dehydrases and aminotransferases, some of which are involved in dideoxy- and deoxyaminosugar biosynthesis.
Christina Bruntner - One of the best experts on this subject based on the ideXlab platform.
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Molecular characterization of co-transcribed genes from Streptomyces tendae Tü901 involved in the biosynthesis of the peptidyl moiety of the peptidyl nucleoside antibiotic Nikkomycin
Molecular Genetics and Genomics, 1999Co-Authors: Christina Bruntner, Bettina Lauer, V. Möhrle, W. Schwarz, Christiane BormannAbstract:Six genes (nikP1, nikP2, nikS, nikT, nikU, and nikV) from Streptomyces tendae Tu901 were identified by analysis of the nucleotide sequence of the Nikkomycin gene cluster. These genes, together with the previously described nikQ and nikR, span 9.39 kb and are transcribed as a polycistronic mRNA in a growth-phase-dependent manner. The nikP1 gene encodes a non-ribosomal peptide synthase consisting of an adenylation domain, a thiolation domain, and an N-terminal 70-residue segment of unknown function. The amino acid sequence encoded by the nikP2 gene displays similarity to the sequences of thioesterases, and the nikS product belongs to a superfamily of proteins characterized by a specific ATP-binding fold. The N-terminal 70 amino acids of the predicted nikT gene product show significant sequence similarity to acyl carrier proteins, and the C-terminal 330 amino acids to aminotransferases. The sequences of the deduced proteins NikU and NikV exhibit similarity to components S and E, respectively, of glutamate mutase from Clostridium. Disruption of the nikP1, nikS, nikT, or nikV gene by insertion of a kanamycin resistance cassette abolished formation of Nikkomycins I, J, X, and Z, all of which contain hydroxypyridylhomothreonine as the peptidyl moiety. The nikP1 mutants, and the nikS and nikT mutants accumulated the nucleoside moieties Nikkomycin Cz, and Nikkomycins Cx and Cz, respectively. The nikV mutants formed Nikkomycins Ox and Oz, which contain 2-amino-4-hydroxy-4-(3'-hydroxy-6'-pyridyl) butanoic acid as the peptidyl moiety. The nikP2 mutants synthesized Nikkomycins I, J, X, and Z, but amounts of Nikkomycins I and X, which contain formylimidazolone as the base, were lower. Feeding formylimidazolone to nikP2 mutants restored the ability to form Nikkomycins I and X. Our results indicate that nikU and nikV are required for the synthesis of hydroxypyridylhomothreonine, the genes nikP1, nikP2 and nikS are required for the assembly of Nikkomycins, and nikT is required for both pathways. The putative activities of each of their products are discussed.
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The Streptomyces tendae Tü901 L-lysine 2-aminotransferase catalyzes the initial reaction in Nikkomycin D biosynthesis
FEBS Journal, 1998Co-Authors: Christina Bruntner, Christiane BormannAbstract:: Protein P8 was previously identified as a putative Nikkomycin biosynthesis protein. The gene (nikC) encoding protein P8 was cloned from the Streptomyces tendae Tu901 Nikkomycin gene cluster and sequenced. The nikC gene was inactivated by inserting a kanamycin resistance cassette; the mutant did not produce the biologically active Nikkomycins I, J, X, and Z, but accumulated the nucleoside moieties Nikkomycins C(X) and C(Z). The mutant was complemented to Nikkomycin production (I, J, X, Z) by nikC expressed from the mel promoter of the vector pIJ702. Furthermore, the Nikkomycin-negative phenotype was reversed by the addition of picolinic acid, a precursor of the peptidyl moiety of Nikkomycins (Nikkomycin D), into the culture medium. The nikC gene was expressed in Escherichia coli and identified and characterized at the enzyme level. NikC encodes an L-lysine 2-aminotransferase, and the activity was exclusively detected in Nikkomycin producers and its presence correlated to Nikkomycin production. The nikC-inactivated mutant grew with L-lysine as sole source of nitrogen and carbon, indicating that L-lysine 2-aminotransferase is not required for lysine catabolism. Our results identified the nikC-encoded L-lysine 2-aminotransferase as the Nikkomycin biosynthetic enzyme that catalyzes the initial reaction in Nikkomycin D biosynthesis. The NikC protein belongs to a novel family of pyridoxamine or pyridoxal-phosphate-dependent dehydrases and aminotransferases, some of which are involved in dideoxy- and deoxyaminosugar biosynthesis.
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Cloning and heterologous expression of the entire set of structural genes for Nikkomycin synthesis from Streptomyces tendae Tü901 in Streptomyces lividans.
Journal of Bacteriology, 1996Co-Authors: Christiane Bormann, V. Möhrle, Christina BruntnerAbstract:A genomic library from Streptomyces tendae raised in shuttle cosmid vector pKC505 was screened with a previously isolated 8-kb DNA fragment containing the orfP1 gene, which is involved in Nikkomycin biosynthesis. The entire set of structural genes for Nikkomycin synthesis was heterologously expressed in S. lividans TK23 by introducing recombinant cosmids p24/32 and p9/43-2, carrying inserts of about 31 and 27 kb, respectively, overlapping by 15 kb. S. lividans transformants synthesized Nikkomycins X, Z, I, and J, which were identified by high-pressure liquid chromatography analyses of culture filtrates.
V. Möhrle - One of the best experts on this subject based on the ideXlab platform.
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Molecular characterization of co-transcribed genes from Streptomyces tendae Tü901 involved in the biosynthesis of the peptidyl moiety of the peptidyl nucleoside antibiotic Nikkomycin
Molecular Genetics and Genomics, 1999Co-Authors: Christina Bruntner, Bettina Lauer, V. Möhrle, W. Schwarz, Christiane BormannAbstract:Six genes (nikP1, nikP2, nikS, nikT, nikU, and nikV) from Streptomyces tendae Tu901 were identified by analysis of the nucleotide sequence of the Nikkomycin gene cluster. These genes, together with the previously described nikQ and nikR, span 9.39 kb and are transcribed as a polycistronic mRNA in a growth-phase-dependent manner. The nikP1 gene encodes a non-ribosomal peptide synthase consisting of an adenylation domain, a thiolation domain, and an N-terminal 70-residue segment of unknown function. The amino acid sequence encoded by the nikP2 gene displays similarity to the sequences of thioesterases, and the nikS product belongs to a superfamily of proteins characterized by a specific ATP-binding fold. The N-terminal 70 amino acids of the predicted nikT gene product show significant sequence similarity to acyl carrier proteins, and the C-terminal 330 amino acids to aminotransferases. The sequences of the deduced proteins NikU and NikV exhibit similarity to components S and E, respectively, of glutamate mutase from Clostridium. Disruption of the nikP1, nikS, nikT, or nikV gene by insertion of a kanamycin resistance cassette abolished formation of Nikkomycins I, J, X, and Z, all of which contain hydroxypyridylhomothreonine as the peptidyl moiety. The nikP1 mutants, and the nikS and nikT mutants accumulated the nucleoside moieties Nikkomycin Cz, and Nikkomycins Cx and Cz, respectively. The nikV mutants formed Nikkomycins Ox and Oz, which contain 2-amino-4-hydroxy-4-(3'-hydroxy-6'-pyridyl) butanoic acid as the peptidyl moiety. The nikP2 mutants synthesized Nikkomycins I, J, X, and Z, but amounts of Nikkomycins I and X, which contain formylimidazolone as the base, were lower. Feeding formylimidazolone to nikP2 mutants restored the ability to form Nikkomycins I and X. Our results indicate that nikU and nikV are required for the synthesis of hydroxypyridylhomothreonine, the genes nikP1, nikP2 and nikS are required for the assembly of Nikkomycins, and nikT is required for both pathways. The putative activities of each of their products are discussed.
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Cloning and heterologous expression of the entire set of structural genes for Nikkomycin synthesis from Streptomyces tendae Tü901 in Streptomyces lividans.
Journal of Bacteriology, 1996Co-Authors: Christiane Bormann, V. Möhrle, Christina BruntnerAbstract:A genomic library from Streptomyces tendae raised in shuttle cosmid vector pKC505 was screened with a previously isolated 8-kb DNA fragment containing the orfP1 gene, which is involved in Nikkomycin biosynthesis. The entire set of structural genes for Nikkomycin synthesis was heterologously expressed in S. lividans TK23 by introducing recombinant cosmids p24/32 and p9/43-2, carrying inserts of about 31 and 27 kb, respectively, overlapping by 15 kb. S. lividans transformants synthesized Nikkomycins X, Z, I, and J, which were identified by high-pressure liquid chromatography analyses of culture filtrates.
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Identification of cellular proteins involved in Nikkomycin production in Streptomyces tendae Tü901.
Molecular Microbiology, 1995Co-Authors: V. Möhrle, Ulrich Roos, Christiane BormannAbstract:Summary Expression of genes involved in Nikkomycin production in Streptomyces tendae was investigated by two-dimensional gel electrophoresis of cellular proteins. Ten gene products (P1–P10) were identified that were synthesized when Nikkomycin was produced; these proteins were not detected in non-producing mutants. N-terminal sequences of six of the 10 proteins were obtained by microsequencing of protein spots excised from preparative two-dimensional gels. Protein P8 was identified as l-histidine amino-transferase (HisAT), which has been previously correlated with Nikkomycin production. By using oligo-nucleotide probes deduced from the N-terminal sequences of protein P2 and P6, we isolated an 8 kb Bam HI fragment and a 6.5 kb Pvu II fragment, respectively, from the genome of Streptomyces tendae Tu901. Restriction analyses revealed that both fragments overlapped within a region of 1.5 kb. Mapping of the oligonucleotide probe hybridizing sites indicated that the genes encoding protein P2 and P6 are closely spaced on the 8 kb Bam HI fragment, and the latter is located on the overlapping region. DNA sequence analysis revealed that proteins P1 and P2 are encoded by a single gene, orfP1, that is translated at two initiation codons. The orfP1 gene was interrupted by homologous recombination using the integrating vector pWHM3. The gene-disrupted transformants did not produce Nikkomycin, indicating that proteins P1 and P2 are essential for Nikkomycin production. The data presented show that reverse genetics was successfully used to isolate genes Involved in Nikkomycin production.
Haihua Yang - One of the best experts on this subject based on the ideXlab platform.
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Cloning, reassembling and integration of the entire Nikkomycin biosynthetic gene cluster into Streptomyces ansochromogenes lead to an improved Nikkomycin production
Microbial Cell Factories, 2010Co-Authors: Guojian Liao, Jine Li, Haihua Yang, Lei Li, Yuqing TianAbstract:Background: Nikkomycins are a group of peptidyl nucleoside antibiotics produced by Streptomyces ansochromogenes. They are competitive inhibitors of chitin synthase and show potent fungicidal, insecticidal, and acaricidal activities. Nikkomycin X and Z are the main components produced by S. ansochromogenes. Generation of a high-producing strain is crucial to scale up Nikkomycins production for further clinical trials. Results: To increase the yields of Nikkomycins, an additional copy of Nikkomycin biosynthetic gene cluster (35 kb) was introduced into Nikkomycin producing strain, S. ansochromogenes 7100. The gene cluster was first reassembled into an integrative plasmid by Red/ET technology combining with classic cloning methods and then the resulting plasmid(pNIK)was introduced into S. ansochromogenes by conjugal transfer. Introduction of pNIK led to enhanced production of Nikkomycins (880 mg L -1 , 4 -fold Nikkomycin X and 210 mg L -1 , 1.8-fold Nikkomycin Z) in the resulting exconjugants comparing with the parent strain (220 mg L -1 Nikkomycin X and 120 mg L -1 Nikkomycin Z). The exconjugants are genetically stable in the absence of antibiotic resistance selection pressure. Conclusion: A high Nikkomycins producing strain (1100 mg L -1 Nikkomycins) was obtained by introduction of an extra Nikkomycin biosynthetic gene cluster into the genome of S. ansochromogenes. The strategies presented here could be applicable to other bacteria to improve the yields of secondary metabolites.
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Article URL
2010Co-Authors: Guojian Liao, Haihua Yang, Yuqing Tian, Huarong TanAbstract:PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. Cloning, reassembling and integration of the entire Nikkomycin biosynthetic gene cluster into Streptomyces ansochromogenes lead to an improved Nikkomycin productio
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Selectively improving Nikkomycin Z production by blocking the imidazolone biosynthetic pathway of Nikkomycin X and uracil feeding in Streptomyces ansochromogenes
Microbial Cell Factories, 2009Co-Authors: Guojian Liao, Jine Li, Haihua Yang, Lei Li, Yuqing TianAbstract:Background Nikkomycins are a group of peptidyl nucleoside antibiotics and act as potent inhibitors of chitin synthases in fungi and insects. Nikkomycin X and Z are the main components produced by Streptomyces ansochromogenes. Of them, Nikkomycin Z is a promising antifungal agent with clinical significance. Since highly structural similarities between Nikkomycin Z and X, separation of Nikkomycin Z from the culture medium of S. ansochromogenes is difficult. Thus, generating a Nikkomycin Z selectively producing strain is vital to scale up the Nikkomycin Z yields for clinical trials.
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Microbial Cell Factories BioMed Central
2009Co-Authors: Guojian Liao, Haihua Yang, Yuqing Tian, Huarong TanAbstract:Selectively improving Nikkomycin Z production by blocking the imidazolone biosynthetic pathway of Nikkomycin X and uracil feeding in Streptomyces ansochromogene
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A pathway-specific transcriptional regulatory gene for Nikkomycin biosynthesis in Streptomyces ansochromogenes that also influences colony development.
Molecular Microbiology, 2005Co-Authors: Yuqing Tian, Haihua YangAbstract:Summary DNA sequence analysis of a 7.5 kb Xho I DNA fragment from the region flanking the Nikkomycin biosynthesis gene cluster in Streptomyces ansochromogenes revealed one 3.3 kb open reading frame (ORF), designated sanG . The deduced product of sanG (1061 amino acids), which is similar to PimR of Streptomyces natalensis , contains an OmpR-like DNA binding domain in its N-terminal portion and A- and B-type nucleotide binding motifs in the middle of the protein. Disruption of sanG abolished Nikkomycin biosynthesis, reduced sporulation and led to brown pigment accumulation. All aspects of this complex phenotype were complemented by a single copy sanG which was integrated into the chromosome. The introduction of multiple copies of sanG resulted in increased Nikkomycin production. S1 mapping results indicated that sanG is transcribed from at least three promoters (P1, P2 and P3), P1 being strongly upregulated when production of Nikkomycins starts. Two putative transcription units for Nikkomycin biosynthesis, starting from sanN and sanO , are dependent on the expression of sanG , whereas a putative transcription unit starting from sanF was not regulated by sanG . These results suggested that sanG encodes a transcriptional activator important for Nikkomycin biosynthesis that, unusually, also has pleiotropic effects on secondary metabolism and development.