Complex organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase

MacNeil, Douglas J.; Occi, James; Gewain, Keith M.; MacNeil, Tanya; Gibbons, Patrice H.; Ruby, Carolyn L.; Danis, Susan J.

doi:10.1016/0378-1119(92)90549-5

Cited by 185 publications

(85 citation statements)

References 13 publications

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“…and Leadlay, EF., unpublished results) for the acetate-specific AT domain from the second module of the avermectin-producing polyketide synthase in Streptomyces avermitilis, and the sequence is an excellent match to the derived acetate-specific motif. AT sequences have also been reported for modules seven and nine of the PKS from Streptomyces cyaneogriseus that synthesises the avermectin analogue nemadectin [14]. These modules are both predicted to be propionate-specific, and in agreement with this both AT sequences convincingly match the propionate consensus.…”

Section: Starter Atsmentioning

confidence: 53%

“…Rap 1-14 indicate the different AT domains present in modules 1-14 of the PKS for rapamycin biosynthesis [7]. Ery 1 6, Ave 2, Ole 5 and 6, Nem 6 and 9, and Sor 5, indicate AT domains present in the corresponding modules of the modular PKSs for erythromycin [3,4], avermectin, oleandomycin [13], nemadectin [14] and soraphen A [15] biosynthesis respectively. MSAS represents the 6-methylsalicylic acid synthase AT [16], and MAS that of mycocerosic acid synthase [17].…”

Section: Starter Atsmentioning

confidence: 99%

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Divergent sequence motifs correlated with the substrate specificity of (methyl)malonyl‐CoA:acyl carrier protein transacylase domains in modular polyketide synthases

et al. 1995

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The amino acid sequences of a large number of polyketide synthase domains that catalyse the transacylation of either methylmalonyl-CoA or malonyl-CoA onto acyl carrier protein (ACP) have been compared. Regions were identified in which the acyltransferase sequences diverged according to whether they were specific for malonyl-CoA or methylmalonyl-CoA. These differences are sufficiently clear to allow unambiguous assignment of newly-sequenced acyltransferase domains in modular polyketide synthases. Comparison with the recently-determined structure of the malonyltransferase from Escherichia coli fatty acid synthase showed that the divergent region thus identified lies near the acyltransferase active site, though not close enough to make direct contact with bound substrate.

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Section: Starter Atsmentioning

confidence: 53%

Section: Starter Atsmentioning

confidence: 99%

Divergent sequence motifs correlated with the substrate specificity of (methyl)malonyl‐CoA:acyl carrier protein transacylase domains in modular polyketide synthases

et al. 1995

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“…The foundation of this approach has been based on the tenet that the genes involved in the biosynthesis of polyketide natural products such as tetracenomycin [29], erythromycin [lo, 301, actinorhodin [31], avermectin [32] and others [33] are clustered. With the availability of the gene encoding the FKMT protein, we have examined the clustering of the FK-506 and immunomycin biosynthetic genes.…”

Section: Discussionmentioning

confidence: 99%

Enzymology of FK‐506 biosynthesis

Shafiee

Motamedi

Chen

1994

European Journal of Biochemistry

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FK-506 is a macrolide antibiotic with immunosuppressant activity. Structurally, this compound contains three methylated hydroxyl groups at C13, C15 and C31. Previous biosynthetic studies using stable isotope-feeding experiments have established methionine as the source of the methyl for these methylated hydroxyl groups. Based on this information and also the availability of the 31-0-desmethylFK-506, a metabolic precursor for the biosynthesis of FK-506, a S-adenosyl-L-methionine-dependent enzyme assay was developed and the enzyme 3 1 -0-desmethylFK-506 0 :methyltransferase was isolated from an extract of Streptomyces sp. MA 6858 and purified to near homogeneity. 31-0-DesmethylFK-506 0 :methyltransferase is a monomeric protein with an apparent molecular mass of 30000 Da and a PI of 4.4. The first 38 N-terminal amino acids have been sequenced and are H,N-SDVVETLRLPNGATVAHVNAGEAQFIYREIFTDRXYLRH. Functionally, this enzyme has a requirement for Mg" with an optimum temperature of 34°C and a pH of 7.4 for full activity. Moreover, it catalyses the methylation of 31-0-desmethylimmunomycin as efficiently as its own natural substrate, 31 -0-desmethylFK-506. Additionally, FKMT catalyzes the C31 transmethylation reaction of 13,31-0-bis-desmethyl-, 15,31-O-bisdesmethyl-, 13,15,31-0-trisdesmethyl-and 31 -0-19,22-cyclic-hemiketalimmunomycins, which are all structural analogues of FK-506. The reaction is, however, completely blocked if the vicinal hydroxyl which is present at the C-32 position of the 31-0-desmethylFK-506 structure is replaced with azide, phosphate or other substituents. Finally, evidence is presented indicating the close similarity of FKMT and DIMT, a 31-0-desmethylimmunomycin : 0 methyltransferase, previously isolated from a cell-free extract of Streptomyces hygroscopicus var ascomyceticus, an immunomycin (ascomycin/FK-520) producer. in numerous laboratories searching for compounds having novel structures with useful pharmacological activities. In the mean time, extensive biochemical and genetic studies are being pursued in order to unravel the biosynthetic pathways for the formation of these complex structures, hoping, through gene manipulations [6], to improve the yield [7], biosynthesize modified [8] and hybrid [9] structures andor develop mutants [lo]. To these ends, a program was initiated in our laboratories to carry out biochemical and genetic studies on a newly discovered immunosuppressant agent, FK-506. This compound, which is currently undergoing clinical trial for the prevention of organ transplant rejection [ll], is a macrocyclic polyketide produced by Streptomyces tsukubaensis, first reported in 1987 [12]. We have also discovered an FK-506-producing strain of Streptomyces, identified as MA 6858 (ATCC No. 55098). Precursor-feeding studies of this strain by Byrne et al. [13] have established that acetate, propionate, butyrate, pipecolic acid, shikimic acid and methionine participate in the biosynthesis of FK-506. The methionine has been shown to be the source of the methyl groups for the methylation ...

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“…The EGFP gene was obtained from Clontech as pEGFP. Plasmids were constructed in Escherichia coli strain DH5α (Hanahan, 1983) using standard procedures (Sambrook et al, 1989), and introduced into the methylationdeficient E. coli strain ET12567 (dam dcm hsdS ; MacNeil et al, 1992) containing the helper plasmid pUZ8002 (Paget et al, 1999a) prior to conjugation into S. coelicolor M600. Integration of pIJ8600 derivatives at the φC31 chromosomal attachment site of M600 was confirmed by Southern hybridization.…”

Section: Methodsmentioning

confidence: 99%

Green fluorescent protein as a reporter for spatial and temporal gene expression in Streptomyces coelicolor A3(2) This paper is dedicated to the memory of Kathy Kendrick, whose devotion to understanding the biology of Streptomyces was unsurpassed.

et al. 1999

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The enhanced green fluorescent protein (EGFP) gene is a modified version of the green fluorescent protein gene of the jellyfish Aequorea victoria with a codon usage that corresponds well to that found in many GC-rich streptomycete genes. Here the use of EGFP as a reporter for the analysis of spatially and temporally regulated gene expression in Streptomyces coelicolor A3(2) is demonstrated. The EGFP gene was inserted into plasmids that can replicate in Escherichia coli, greatly facilitating the construction of EGFP gene fusions. The plasmids can be transferred readily to S. coelicolor by conjugation, whereupon two of them (pIJ8630 and pIJ8660) integrate at the chromosomal attachment site for the temperate phage φC31. These vectors were used to analyse the spatial and temporal expression of sigF, which encodes a σ factor required for spore maturation, and of redD, a pathway-specific regulatory gene for the production of undecylprodigiosin, one of the four antibiotics made by S. coelicolor. While transcription of sigF appeared to be confined to developing and mature spore chains, transcription of redD occurred only in ageing substrate mycelium. A further plasmid derivative (pIJ8668) was made that lacks the φC31 attachment site, allowing the EGFP gene to be fused transcriptionally to genes of interest at their native chromosomal locations.

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Complex organization of the Streptomyces avermitilis genes encoding the avermectin polyketide synthase

Cited by 185 publications

References 13 publications

Divergent sequence motifs correlated with the substrate specificity of (methyl)malonyl‐CoA:acyl carrier protein transacylase domains in modular polyketide synthases

Divergent sequence motifs correlated with the substrate specificity of (methyl)malonyl‐CoA:acyl carrier protein transacylase domains in modular polyketide synthases

Enzymology of FK‐506 biosynthesis

Green fluorescent protein as a reporter for spatial and temporal gene expression in Streptomyces coelicolor A3(2) This paper is dedicated to the memory of Kathy Kendrick, whose devotion to understanding the biology of Streptomyces was unsurpassed.

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