Construction of thiostrepton-inducible, high-copy-number expression vectors for use in Streptomyces spp.

Takano, Eriko; White, Janet; Thompson, Charles J.; Bibb, Mervyn J.

doi:10.1016/0378-1119(95)00545-2

Cited by 133 publications

(111 citation statements)

References 22 publications

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“…When lipA was placed under the control of the strong and inducible tipA promoter in plasmid pIJ6021 (Takano et al, 1995), lipase activity could be clearly detected in the absence of a functional lipR gene (0n67 units per ml of supernatant obtained under inducing conditions in LB medium), indicating that, as expected, the lipR product was required for lipA transcription, and not for lipase processing or secretion. When total protein in supernatants of cultures carrying pB108 and pB110 was analysed by SDS-PAGE, a protein band in the predicted size range for the lipA-encoded lipase (around 29 kDa) was clearly visible in cultures carrying pB108, but absent from cultures carrying pB110 (Fig.…”

Section: Expression Of the Cloned S Coelicolor A3(2) Lipase Genesupporting

confidence: 57%

“…Competent cells for transformation were prepared by the method of Inoue et al (1990). In-phase cloning of the S. coelicolor lipA in expression vector pIJ6021 (Takano et al, 1995) was based on site-directed mutagenesis according to the protocol of Kunkel (1985), using an oligonucleotide with the sequence 5h-GGAC-ACCCCCCATATGCAGCAGAACCC-3h. The PCR was carried out with AmpliTaq DNA polymerase from Perkin-Elmer according to the instructions of the manufacturer.…”

Section: Methodsmentioning

confidence: 99%

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The Streptomyces coelicolor A3(2) lipAR operon encodes an extracellular lipase and a new type of transcriptional regulator The GenBank accession numbers for the sequences described in this paper are AF009336 and U03114.

et al. 1999

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Section: Expression Of the Cloned S Coelicolor A3(2) Lipase Genesupporting

confidence: 57%

Section: Methodsmentioning

confidence: 99%

The Streptomyces coelicolor A3(2) lipAR operon encodes an extracellular lipase and a new type of transcriptional regulator The GenBank accession numbers for the sequences described in this paper are AF009336 and U03114.

et al. 1999

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“…A thiostrepton resistance plasmid pIJ702 (14) (40 -300 copies per chromosome (13)) containing the melC promoter was used for DNA manipulation and gene expression in S. griseus. For production of GriI and GriH proteins in S. lividans TK21, pIJ4123 (15) containing the thiostrepton-inducible tipA promoter, was used. Escherichia coli strains JM109, JM110, and TOP10 (Invitrogen) and plasmids pUC19 and pCR4Blunt-TOPO (Invitrogen) were used for DNA manipulation.…”

Section: Methodsmentioning

confidence: 99%

Novel Benzene Ring Biosynthesis from C3 and C4 Primary Metabolites by Two Enzymes

Suzuki

Ohnishi

Furusho

et al. 2006

Journal of Biological Chemistry

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The shikimate pathway, including seven enzymatic steps for production of chorismate via shikimate from phosphoenolpyruvate and erythrose-4-phosphate, is common in various organisms for the biosynthesis of not only aromatic amino acids but also most biogenic benzene derivatives. 3-Amino-4-hydroxybenzoic acid (3,4-AHBA) is a benzene derivative serving as a precursor for several secondary metabolites produced by Streptomyces, including grixazone produced by Streptomyces griseus. Our study on the biosynthesis pathway of grixazone led to identification of the biosynthesis pathway of 3,4-AHBA from two primary metabolites. Two genes, griI and griH, within the grixazone biosynthesis gene cluster were found to be responsible for the biosynthesis of 3,4-AHBA; the two genes conferred the in vivo production of 3,4-AHBA even on Escherichia coli. In vitro analysis showed that GriI catalyzed aldol condensation between two primary metabolites, L-aspartate-4-semialdehyde and dihydroxyacetone phosphate, to form a 7-carbon product, 2-amino-4,5-dihydroxy-6-one-heptanoic acid-7-phosphate, which was subsequently converted to 3,4-AHBA by GriH. The latter reaction required Mn 2؉ ion but not any cofactors involved in reduction or oxidation. This pathway is independent of the shikimate pathway, representing a novel, simple enzyme system responsible for the synthesis of a benzene ring from the C 3 and C 4 primary metabolites.The shikimate pathway (Fig. 1B), involving seven enzymatic steps that produce chorismate via shikimate from phosphoenolpyruvate (PEP) 2 and erythrose-4-phosphate, is well established as the common pathway for the biosynthesis of aromatic amino acids in bacteria, fungi, algae, and higher plants. Not only aromatic amino acids but also most biogenic benzene derivatives, such as p-aminobenzoic acid, m-aminobenzoic acid, 2-amino-3-hydroxybenzoic acid, 2-amino-6-hydroxybenzoic acid, and many vitamins, are derived from chorismate (1, 2). The shikimate biosynthesis pathway is also employed by Archaea, although the genes encoding the first two enzymes involved in 3-dehydroquinate (DHQ) synthesis are missing in the genomic sequences of many Archaea (3). In one of Archaea, Methanocaldococcus jannaschii, DHQ is synthesized from aspartate 4-semialdehyde (ASA) and 6-deoxy-5-ketofructose-1-phosphate by two alternative enzymes and supplied to the shikimate pathway (4). Recent studies (5, 6) showed that 3-amino-5-hydroxybenzoic acid, a precursor for ansamycin antibiotics, is also synthesized through the aminoshikimate pathway, a variant of the shikimate pathway. Thus, the benzene ring as one of the primary chemical structures in nature is extensively formed through the shikimate pathway, although some benzene derivatives are formed from aliphatic acyl-CoA by polyketide synthases (7, 8).We recently isolated grixazone (Fig. 1A), a mixture of yellow pigments grixazone A and grixazone B, containing a phenoxazinone chromophore, as secondary metabolites of Streptomyces griseus (9, 10). In the present study on the grixazone biosynthesis, we ...

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“…The purified product was restricted and ligated into E. coli expression vector pET-15b, resulting in the 6708 bp plasmid pJN14. The asnO gene was excised from pJN14 by digestion with BamHI and XhoI, and ligated into restriction-digested pIJ6021 (Takano et al, 1995), resulting in the 8904 bp plasmid pJN15. The ligation products were used to transform Streptomyces lividans 1326, and positive transformants were selected by growth on medium containing kanamycin.…”

Section: Methodsmentioning

confidence: 99%

An asparagine oxygenase (AsnO) and a 3-hydroxyasparaginyl phosphotransferase (HasP) are involved in the biosynthesis of calcium-dependent lipopeptide antibiotics

et al. 2007

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Nonribosomal peptides contain a wide range of unusual non-proteinogenic amino acid residues. As a result, these complex natural products are amongst the most structurally diverse secondary metabolites in nature, and possess a broad spectrum of biological activities. b-Hydroxylation of amino acid precursors or peptidyl residues and their subsequent processing by downstream tailoring enzymes are some of the most common themes in the biosynthetic diversification of these therapeutically important peptides. Identification and characterization of the biosynthetic intermediates and enzymes involved in these processes are thus pivotal in understanding nonribosomal peptide assembly and modification. To this end, the putative asparaginyl oxygenase-and 3-hydroxyasparaginyl phosphotransferase-encoding genes hasP and asnO were separately deleted from the calcium-dependent antibiotic (CDA) biosynthetic gene cluster of Streptomyces coelicolor. Whilst the parent strains produce a number of 3-hydroxyasparagine-and 3-phosphohydroxyasparagine-containing CDAs, the DhasP mutants produce exclusively non-phosphorylated CDAs. On the other hand, DasnO mutants produce several new Asn-containing CDAs not present in the wild-type, which retain calcium-dependent antimicrobial activity. This confirms that AsnO and HasP are required for the b-hydroxylation and phosphorylation of the Asn residue within CDA. INTRODUCTIONThe calcium-dependent antibiotics (CDAs) from Streptomyces coelicolor (Hojati et al., 2002;Kempter et al., 1997) belong to the group of structurally related acidic lipopetide antibiotics, which include A54145 (Fukuda et al., 1990; Miao et al., 2006), daptomycin (Baltz et al., 2005;Miao et al., 2005;Raja et al., 2003), friulimicins and amphomycins (Vértesy et al., 2000). All of these nonribosomally biosynthesized lipopeptides contain N-terminal fatty acid side chains, which is a trans-2,3-epoxyhexanoyl moiety in the case of CDA (Fig. 1), along with decapeptide lactone or lactam cores. Within the decapeptide cores are a number of conserved amino acids, including acidic residues responsible for co-ordination of calcium ions, which are essential for antimicrobial activity. Notably, daptomycin was recently approved for clinical use, becoming the first new structural class of natural antibiotic to reach the clinic in over 30 years (Baltz et al., 2005;Raja et al., 2003). As a result of this, there has been considerable interest in establishing the biosynthetic origins of the acidic lipopeptide group of antibiotics, with the specific aim of engineering new lipopeptide variants with improved therapeutic properties. To this end we have focused on engineering the biosynthesis of CDAs, using adenylation domain active site modifications to replace Asp In addition to D-HPG, CDA contains a number of other non-proteinogenic amino acids, including L-3-methylglutamic acid (3-MeGlu; at position 10), which is also present at the same relative position in the decapeptide cores of daptomycin and A54145 (Milne et al., 2006), and a Cterminal Z-dehydro...

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Construction of thiostrepton-inducible, high-copy-number expression vectors for use in Streptomyces spp.

Abstract: Gene, 166 (1995) 133-137

Cited by 133 publications

References 22 publications

The Streptomyces coelicolor A3(2) lipAR operon encodes an extracellular lipase and a new type of transcriptional regulator The GenBank accession numbers for the sequences described in this paper are AF009336 and U03114.

The Streptomyces coelicolor A3(2) lipAR operon encodes an extracellular lipase and a new type of transcriptional regulator The GenBank accession numbers for the sequences described in this paper are AF009336 and U03114.

Novel Benzene Ring Biosynthesis from C3 and C4 Primary Metabolites by Two Enzymes

An asparagine oxygenase (AsnO) and a 3-hydroxyasparaginyl phosphotransferase (HasP) are involved in the biosynthesis of calcium-dependent lipopeptide antibiotics

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