Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wroclaw, Poland1
Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK2
Department of Molecular Microbiology, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK3
Author for correspondence: Katarzyna Kuczek. Tel: +48 71 3732274. Fax: +48 71 373 2587. e-mail: kuczek{at}immuno.iitd.pan.wroc.pl
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ABSTRACT |
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Keywords: thioesterase type II, Streptomyces fradiae, disruption mutant complementation, S1 nuclease mapping
Abbreviations: PKS, polyketide synthase; TE, thioesterase
The GenBank accession number for the sequence reported in this paper is AF109727.
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INTRODUCTION |
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Many type I PKSs, and also non-ribosomal peptide synthetase clusters, contain additional TE genes located adjacent to the PKS genes within the cluster of antibiotic biosynthetic genes (Weissman et al., 1998 ; Schneider & Marahiel, 1998
; Shaw-Reid et al., 1999
; August et al., 1998
; Xue et al., 1998
; Heathcote et al., 2001
). The products of such genes are discrete proteins called type II thioesterases (TE IIs) to distinguish them from chain-terminating thioesterase (TE I) domains (Gokhale et al., 1999
). In fatty acid synthase complexes, TE IIs are alternative chain-terminating enzymes exhibiting hydrolase activity towards medium-chain-length acyl thioesters (Smith, 1994
). There is no obvious role for the TE IIs associated with multienzyme PKS complexes, nor is the mechanism of their action known.
The function of TE IIs is predicted from gene-disruption analysis, complementation studies, and determination of their substrate specificities (Weissman et al., 1998 ; Butler et al., 1999
; Heathcote et al., 2001
). Polyketide production is drastically reduced, by 90% or more, in strains with a deleted TE II gene (Xue et al., 1998
; Butler et al., 1999
; Doi-Katayama et al., 2000
), indicating an important function, proposed to involve editing of aberrant intermediates (Butler et al., 1999
) during the course of polyketide biosynthesis. More recently, this hypothesis has been confirmed and the mechanism clarified. Thus, the TylO protein displayed hydrolytic activity in vitro towards short-chain acyl-CoAs, indicating that the enzyme could remove aberrantly decarboxylated (and, therefore, non-reactive) extender acyl chains from the PKS during polyketide biosynthesis (Heathcote et al., 2001
). By hydrolytic release of such aberrant acyl groups, TE II was proposed to unblock PKS modules and restore overall efficiency of the complex enzyme.
The proposed common role for TE IIs in PKS multienzyme systems raises the question of whether specific TE IIs might be replaceable by other TE IIs normally associated with other PKS complexes. Based on the TE II substrate-specificity studies published to date (Weissman et al., 1998 ; Heathcote et al., 2001
), such enzymes do not seem to select the structure of the acyl substrates.
Genetic engineering studies allow assembly of novel polyketide chains following fusion, swapping or repositioning of catalytic domains, modules or whole peptides within PKS polypeptides (Hutchinson & Fujii, 1995 ; Ranganathan et al., 1999
; Tang et al., 2000
). In engineered PKSs, co-expression of TE IIs in addition to other PKS proteins might help in achieving elevated levels of the polyketide products.
We were studying a gene cluster for the new PKS type I in Streptomyces coelicolor A3(2) (Kuczek et al., 1997 ; K. Pawlik, M. Kotowska & K. Kuczek, unpublished data; GenBank accession numbers U88833, AF109727 and AF 202898) located in a previously unmapped region of the chromosome, between cosmids 2H4 and 10H5 (Redenbach et al., 1996
), now covered by cosmids 2C4, 1G7, BAC8D1 and IF3 (http://www.sanger.ac.uk/Projects/S_coelicolor/). During our studies, we found a new gene, encoding a putative TE II (ScoT). The gene is located within the putative polyketide biosynthetic gene cluster. In this paper, we describe this gene. When expressed in the heterologous host, Streptomyces fradiae, scoT functionally complemented disruption of the native TE II gene, tylO.
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METHODS |
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Construction of a recombinant plasmid for complementation analysis.
An S. fradiae tylO-disruption strain was complemented with cloned S. coelicolor A3(2) DNA containing scoT, using the conjugative vector pLST9828 (Butler et al., 1999 ). This is a derivative of pSET152 (Bierman et al., 1992
) that contains the powerful constitutive promoter ermEp* to drive expression of inserted DNA fragments following integration into the chromosomal
C31 attB site. A DNA fragment containing scoT was ligated into pLST9828 in a two-step process. First, the DNA containing the C-terminal part of ScoT was PCR-amplified from the pBSK(-) plasmid clone template (Fig. 1c
) using M13 reverse primer and the TE-Rev primer with an engineered XbaI site (underlined): 5'-TTTTCTAGATGTCGTACGTACACGGA-3'. The PCR product was purified using the Qiaex II DNA purification kit, digested, and ligated into pLST9828 using the BamHI and XbaI sites. Then, a second PCR product containing the N-terminal part of ScoT was obtained from the template of another pBSK(+) construct (Fig. 1b
) using the T7 universal primer and the TE-Fw primer with an engineered BamHI site (underlined): 5'-TTTTTTGGATCCGATGGGAAGTGACTGGTT-3'. The 50 µl reaction mixture contained 5 µl 10x PCR DyNAzyme buffer (Finnzymes), 1 µl 10 mM deoxynucleoside triphosphate mixture, 50 pmol each oligonucleotide, about 10 ng template DNA and 1 µl DyNAzyme II DNA polymerase (Finnzymes). Cycling was as follows: a hot start at 96 °C for 6 min, 1 min at 80 °C (adding of the enzyme), 31 cycles with denaturation at 95 °C for 1 min, annealing at 6365 °C for 1 min and extension at 72 °C for 1·5 min, followed by a final extension at 72 °C for 5 min. The product was digested with BamHI and ligated into the pLST9828 derivative obtained in the first step of the cloning procedure. Gentamicin (15 µg ml-1) was used for the selection of E. coli DH5
transformants. The authenticity and orientation of the cloned fragments were confirmed by automated sequence analysis. The gene cloned in pLST9828 was introduced into S. fradiae C373.1, a tylosin-producing strain, by transconjugation from E. coli S17-1 as described elsewhere (Butler et al., 1999
).
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S1 nuclease mapping.
For each S1 nuclease reaction, 30 or 40 µg RNA was hybridized in NaTCA buffer [Murray, 1986 ; solid NaTCA (Aldrich) dissolved to 3 M final concentration in 50 mM PIPES, 5 mM EDTA, pH 7·0) to about 0·002 pmol (approx. 104 Cerenkov c.p.m.] of the probe. The oligonucleotide 5'-GTCGAACTCGGGCGTCAGCTC-3' was uniquely labelled at its 5' end with [32P]ATP using T4 polynucleotide kinase, and was used in the PCR with the unlabelled oligonucleotide 5'-CCTCGGCGGCGGAGAGAAT-3', which anneals upstream of the scoT promoter, to generate a 430 bp probe. The PCR used M145 total DNA as a template. Subsequent steps were as described by Strauch et al. (1991
).
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RESULTS |
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Comparison of the scoT sequence (GenBank AF109727) with others in the databases revealed extensive similarities with TEs from various actinomycetes and other bacteria, and also with rat S-acyl fatty acid synthase complex. The greatest similarity was found with TE IIs (Pfam00975), i.e. 43% identity with a TE II (AF040570) from the rifamycin biosynthetic gene cluster of Amycolatopsis mediterranei, 43% identity with a TE II (X60379) associated with 6-deoxyerythronolide B synthase from Saccharopolyspora erythraea, and 40% identity with TylO (U08223), the TE II involved in tylosin biosynthesis in Streptomyces fradiae. ScoT is predicted to belong to the well-known alpha/beta hydrolase family (Pfam00975).
The ATG start codon of the scoT gene is preceded by a potential RBS sequence (AAGGGG) ending 8 bp before the start (nucleotides complementary to the 3' end of the 16S rRNA from Streptomyces lividans are underlined) (Strohl, 1992 ) (see Fig. 4b). The ORF ends with a TGA stop codon. The amino acid motif GxSxG (x=any amino acid) characteristic of acyltransferases and TEs, with Ser-90 as the active-site residue, is present within the deduced sequence of the ScoT protein. A second conserved amino acid which might also be involved in catalysis is His-224 (Fig. 2
). Usage of AGC as the codon for the active-site serine is typical of TE IIs [AGY for TE II and TCN for TE I, where Y=(C, T), N=(A, C, G, T)] (Smith, 1994
).
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Thus, the tylO disruptant was complemented by scoT, as measured by the yield of desmycosin in fermentations analysed by HPLC with desmycosin as the internal standard (Fig. 3ac
). Desmycosin production was restored to up to 48% of the level of macrolide produced by the wild-type strain (Fig. 3c
). Because of the close similarity in the molar absorbance coefficients of tylosin and desmycosin, the amount of desmycosin produced as a fermentation product by a mutant strain was directly compared with the amount of tylosin produced by the wild-type strain. Control fermentation of the non-complemented, tylO-disrupted strain yielded only minimal amounts of desmycosin (Fig. 3b
). These results showed that the TE II gene, scoT, from S. coelicolor A3(2) could, by complementation, restore macrolide production to a significant level in the tylO-disrupted strain of S. fradiae.
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DISCUSSION |
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The amino acid sequences of all the known TE IIs are strongly conserved, especially in implicated catalytically active sites. The sequence data suggest that scoT encodes a TE II which, presumably, is catalytically active. However, in the absence of data relating to its substrate specificity, the probability that ScoT might be capable of functioning in association with a heterologous PKS system could not be predicted.
ScoT complements the natural function of TE II in S. fradiae
ScoT was able to replace TylO, the TE II of the tylosin pathway, by restoring the efficiency of macrolide biosynthesis in the tylO-disrupted strain. The two enzymes appear, therefore, to be equivalent in their catalytic function. Interestingly, in their natural context, these enzymes are apparently associated with PKS enzymes that differ in module number and in the nature of the extender acyl units incorporated by the respective modules. The primary product of the tylosin PKS in S. fradiae, a linear polyketide derived from condensation of five propionate units, two acetate units and one butyrate unit (Baltz & Seno, 1988 ), undergoes cyclization into a 16-atom lactone ring, tylactone. This means that in tylactone biosynthesis, malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA are used as extender units. Although the polyketide product of the modular PKS with which ScoT putatively associates has not yet been determined, the sequences of the acyltransferase domains in each of the five extension modules of the PKS (Kuczek et al., 1997
; K. Pawlik, M. Kotowska & K. Kuczek, unpublished data) suggest that all five should use malonyl-CoA extender units according to the consensus sequence motifs correlated with the substrate specificity of the acyltransferase domains (Haydock et al., 1995
).
In any event, if ScoT is active as an editing enzyme with both the tylosin PKS and its natural PKS partner, it must be able to hydrolyse thioester bonds irrespective of the length of the extender units employed or the size of the nascent polyketide intermediates, since ScoT can apparently cooperate in the biosynthesis of a product longer than that of its native PKS partner.
We suggest that a mechanism other than substrate selectivity, probably based on differences in kinetic rates of hydrolysis by ScoT and polyketide condensation, is involved in competition between these two reactions. To clarify this, however, further studies which involve kinetic measurements of the enzyme activity are needed.
Transcription of scoT occurs in a growth-phase-dependent manner
S1 nuclease mapping of the scoT promoter region in S. coelicolor A3(2) showed that transcription was growth-phase dependent. It was detectable only during late transition phase, indicating the operation of a regulatory system that prevents expression of the gene throughout most of the growth cycle. Since production of secondary metabolites by Streptomyces is commonly initiated during transition phase, onset of scoT expression might be correlated with expression of the gene cluster encoding type I PKS, located close to scoT. Our results on this will be published in due course.
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ACKNOWLEDGEMENTS |
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Received 9 November 2001;
revised 28 January 2002;
accepted 11 February 2002.