Characterization of Streptomyces nogalater genes encoding enzymes

Ó Springer-Verlag 1997

Mol Gen Genet (1997) 256: 203±209

SHORT COMMUNICATION

S. Torkkell á K. Ylihonko á J. Hakala á M. Skurnik P. MaÈntsaÈlaÈ

Characterization of Streptomyces nogalater genes encoding enzymes involved in glycosylation steps in nogalamycin biosynthesis

Received: 26 November 1996 / Accepted: 5 June 1997

Abstract The sno gene cluster in Streptomyces nogalater ATCC 27451 contains the nogalamycin biosynthesis genes. A set of plasmid constructions carrying fragments of the sno cluster that lie downstream of snoD were used to complement the S. galilaeus mutant H039, which is blocked in rhodosamine and 2-deoxyfucose biosynthesis in the aclacinomycin pathway. Sequence analysis of this cluster revealed three contiguous open reading frames (ORFs) that were designated snoF, snoG, and snoH. Only those plasmid constructs that expressed SnoG were able to complement H039. SnoG shows similarity to GalE, a UDP-glucose-4-epimerase catalyzing the epimerization of UDP-glucose to UDP-galactose. The putative SnoF protein is similar to 3,5-epimerases involved in rhamnose biosynthesis. The deduced product of snoH is a 489-amino acid polypeptide. It is similar to the product of dau ORF3 found in the daunomycin cluster. However its function is still unclear. Based on the complementation experiments and sequence analysis, this part of the sno cluster is suggested to be involved in the biosynthesis of the sugar portion of nogalamycin. Interestingly, SnoA, a transcriptional activator for the sno minimal polyketide synthase, is also needed to express this cluster. Key words Streptomyces nogalater á Anthracycline á Nogalamycin á 3,5-Epimerase á 4-Epimerase

Communicated by A. Kondorosi S. Torkkell á K. Ylihonko (&) á P. MaÈntsaÈlaÈ Department of Biochemistry and Food Chemistry, University of Turku, Arcanum, Vatselantie 2, FIN-20014 Turku, Finland Fax: +358-2-333-6860; e-mail: kylihonk@®nabo.abo.® J. Hakala Galilaeus Oy, P.O. Box 113, FIN-20781 Kaarina, Finland M. Skurnik Centre for Biotechnology, Biocity, TykistoÈkatu 6, FIN-20520 Turku

Introduction Nogalamycin (Fig. 1) is an antitumour anthracycline antibiotic, which was discovered in 1965 by Bhuyan and Dietz. The structure of this extraordinarily glycosylated anthracycline was determined by Wiley et al. (1977). Nogalamycin di€ers from anthracyclines of the daunomycin group (e.g. Fujiwara et al. 1986) in the aglycone moiety, in positions of glycosylation and in its attached sugar residues, which are nogalamine and nogalose. Studies on anthracycline biosynthesis have mainly focused on the daunomycin group. Based on the use of well documented mutants blocked in daunomycin biosynthesis (Bartel et al. 1990) and the techniques of molecular cloning in Streptomyces, the biosynthetic pathway for daunomycin, particularly with respect to the aglycone moiety, has been almost completely resolved. Daunomycin gene clusters have been cloned from S. peucetius (Grimm et al. 1994; Otten et al. 1995; Madduri and Hutchinson 1995) and from the mutant Streptomyces sp. C5 (Ye et al. 1994; Dickens et al. 1995, 1996), and the part of the cluster involved in the biosynthesis of the sugar portion of daunomycin has been isolated from S. griseus (KruÈgel et al. 1993). Almost all the genes responsible for forming of daunomycin have been identi®ed. We are carrying out molecular genetic studies on the biosynthesis of anthracyclines with a view to the rational construction of strains that can produce hybrid anthracyclines (Niemi et al. 1994; Ylihonko et al. 1996a). In these studies, mutants derived from the aclacinomycin (Fig. 1) producer S. galilaeus (Ylihonko et al. 1994) are used as host strains. Biosynthetic genes for the nogalamycin chromophore were recently reported (Ylihonko et al. 1996b). Here, we describe the analysis of a 4-kb DNA fragment from the sno cluster that most probably encodes enzymes involved in biosynthesis of the sugar portion of nogalamycin. A putative scheme for the biosynthetic route from NDP-glucose precursors to the sugars found in anthracyclines is outlined in Fig. 1.

204 Fig. 1 A The structures of nogalamycin, aclacinomycinA, and the H039 product (Akv-(Rho)2±3). B Hypothetical scheme for the formation of aclacinomycin sugars from a dNDP-glucose precursor

Materials and methods Microbial strains, plasmids, and culture conditions The bacterial strains and plasmids used in this study are described in Table 1. The conditions used for growth of the bacteria were as described by Sambrook et al. (1989) and Hopwood et al. (1985). E1 medium, containing 2% glucose, 2% soluble starch, 0.5% Farmamedia (Trader's Mill Proteins), 0.25% yeast extract, 0.1% K2HPO4, 0.1% MgSO4 á 7H2O, 0.3% NaCl and 0.3% CaCO3, was used to culture cells for anthracycline production (Ylihonko et al. 1994). DNA isolation and cloning S. nogalater DNA fragments propagated in E. coli were cloned either in pIJ486, in pIJE486 or in pSYL1 and introduced by transformation into TK24. Plasmid constructions were then isolated from TK24 and introduced into S. galilaeus. Streptomyces spp. were transformed as described by Hopwood et al. (1985) with minor modi®cations. DNA isolation and manipulation were carried out by standard procedures (Sambrook et al. 1989; Hopwood et al. 1985).

Sequencing and sequence analysis The Qiagen Plasmid Midi kit was used to isolate the plasmids from E. coli. DNA sequencing was performed using the automatic ABI DNA sequenator (Perkin-Elmer) according to the manufacturer's instructions. In sequencing reactions, (i) denaturation for 10 min at 100° C, (ii) deazaG and deazaA and (iii) an annealing temperature of 45° C instead of 37° C were used to alleviate problems of compression caused by the high GC content of Streptomyces DNA. Sequence analyses were done using the GCG sequence analysis software package (Version 8; Genetics Computer Group, Madison, Wis., USA). The translation table was modi®ed to accept also GTG as a start codon. Codon usage was analysed using published data (Wright and Bibb 1992). Expression cloning and analysis of anthracycline production Restriction fragments of the sno cluster were cloned into plasmids pIJ486, pIJE486, and pSYL1 and introduced into H039 by transformation. Transformants were streaked on ISP4 agar supplemented with thiostrepton (50 lg/ml). E1 medium was used for anthracycline production and the products were extracted from the culture with toluene:methanol (1:1) at pH 7. HPLC was used to characterize the products. An octadecyl reverse phase column

205 Table 1 Microbial strains and plasmids Strain or plasmid

Characteristics

Source or reference

Streptomyces nogalater ATCC 27451 Streptomyces lividans TK24 Streptomyces galilaeus ATCC 31615 Streptomyces galilaeus H039 Escherichia coli XL2-Blue pUC19

Producer of nogalamycin

ATCC Bhuyan and Dietz (1965) John Innes Centre, Norwich Hopwood et al. (1985) ATCC Oki et al. (1975) Ylihonko et al. (1994)

Cloning host Producer of aclacinomycins (AcmA, AcmB, AcmY) Producer of Akv-(Rho)2±3 Cloning host E. coli cloning vector

pSL1180

E. coli cloning vector

pIJ486

Streptomyces cloning vector

pIJE486

ermE promoter (Bibb et al. 1985) cloned into polylinker of pIJ486 The snoA gene (Ylihonko et al. 1996b), encoding an activator, cloned into pIJ486 A 10-kb EcoRI fragment of the sno cluster cloned into pIJ486 A 12-kb BglII fragment of the sno cluster cloned into pIJ486 A 2-kb BglII-BamHI fragment of the sno cluster cloned into pIJ486 A 1.5-kb EcoRI-BamHI fragment of the sno cluster cloned into pIJ486 A 2-kb EcoRI-MluI fragment of the sno cluster cloned into pIJ486 A 4-kb NotI fragment of the sno cluster cloned into pIJ486 A 3-kb StyI fragment of the sno cluster cloned into pIJ486

pSYL1 pSY10 pSY15 pSY27a a

pSY30

pSY32a pSY31a a

pSY36 a

Stratagene Bullock et al. (1987) Pharmacia Yanisch-Perron et al. (1985) Pharmacia Brosius (1989) John Innes Centre, Norwich Ward et al. (1986) Ylihonko et al. (1996b) This work Unpublished Ylihonko et al. (1996a) This work This work This work This work This work

Corresponding constructions made in pSYL1 and pSYE486 are designated as pSYL27, pSYE27 etc, respectively.

(RP-18) was used with acetonitrile:60 mM potassium dihydrogen phosphate pH 3 (7:3 v/v), as a solvent system. The compounds were detected at 254 nm and at 420 nm. The products from the cultures of wild-type S. galilaeus and the H039 mutant were used as controls to analyze the anthracyclines produced by H039 clones.

Results Localization of the genes for the biosynthesis of the nogalamycin sugar residues in the sno cluster A gene cluster, sno, involved in nogalamycin biosynthesis has been previously cloned and the genes encoding the nogalamycin chromophore were characterized (Ylihonko et al. 1996a, b). Subclones from the cloned region were transferred into S. galilaeus mutants blocked in the aclacinomycin pathway in order to study their ability to complement the mutations. pSY10, carrying a 10-kb DNA fragment (SY10) from the sno cluster cloned in pIJ486, complemented the mutant H039, accumulating aklavinone-rhodinoses (Akv-Rho; see Fig. 6) for aclacinomycin production. In contrast,

pSY15, which contains an insert that overlaps pSY10 with 8 kb, failed to restore aclacinomycin production in H039. For this reason, we focused our analysis on a 4-kb region in pSY10 (see Fig. 2), which we had not previously characterized and which is not completely included in pSY15. The right end of SY15 containing the genes for the minimal PKS of nogalamycin is not present in SY10. Sequence analysis of the sno genes The 4-kb EcoRI-BamHI fragment of pSY10 was sequenced and downstream of the 3¢ end of snoE three complete ORFs designated snoFGH were found. The 5¢ end of snoE has already been sequenced (Ylihonko et al. 1996b). Based on the amino acid sequence similarities, the three genes encode the following functions. (i) The sequenced region contained the previously unsequenced 3¢ terminus of snoE, which had been suggested to encode an aromatase, sometimes termed a

206

Fig. 2 Restriction map of the nogalamycin gene cluster showing the region which is suggested to be involved in nogalamine/nogalose biosynthesis. The thick arrows show ORFs and the direction of transcription. The fragments used for expression cloning are designated as SY fragments. SY15 and SY10 cover the region not shown herein. The bolder line shows the sno cluster sequenced here. The EcoRI site shown in parentheses is derived from the kEMBL4 vector and is not present in the genomic DNA

best similarity and identity scores were with griseusin (70% and 53%) from S. griseus (Yu et al. 1994) and with the cyclase/dehydrase in the daunomycin pathway (69% and 53%; Grimm et al. 1994). (ii) SnoF is a 201-amino acid polypeptide. The similarity search suggests that it may function as a 3,5epimerase. The 3,5-epimerases have been demonstrated to catalyze the epimerizations of the hydroxyl and methyl groups of dTDP-4-dehydrorhamnose in positions 3 and 5, respectively (Liu and Thorson 1994). The alignment of the deduced amino acid sequences is shown in Fig. 3. (iii) snoG is translationally coupled to snoF, overlapping it by four bases. Its putative product, SnoG, is a 329-amino acid polypeptide. SnoG is similar to 4-epimerases and 4,6-dehydratases. However, the conserved regions TG(A,G)AGFIG and NNYGP(Y,R)Q(H,F)P present in 4,6-dehydratases (Decker et al. 1996) were not found in SnoG. Nevertheless, an NAD-binding motif (GxxGxxG; Macpherson et al. 1994) was found between positions 12 and 20 (12-GATGCVG-20). NAD+ is an essential component for the catalytic activity of UDP-glucose-4-isomerase, and the conserved sequences are found in the alignment of SnoG with 4-epimerases (Fig. 4). (iv) snoH is translationally coupled to snoG. The deduced peptide consists of 489 amino acids. SnoH is similar to DauORF3 cloned from Streptomyces sp. C5

cyclase/dehydrase. This conclusion was based on sequence similarity to known aromatases or cyclases involved in the synthesis of the aromatic polyketides. As expected, the C-terminal sequence of SnoE supported its deduced function as an aromatase. SnoE turns out to be a 324-amino acid peptide showing over 60% similarity to known aromatases of the polyketide pathway. The

Fig. 3 Alignment by the PILEUP program of the SnoF sequence with those of 3,5-epimerases. The proteins aligned are: RfbD from Shigella ¯exneri (Macpherson et al. 1994) and RfbF from Yersinia enterocolitica (Zhang et al. 1993) from the dTDP-rhamnose and dTDP-6-deoxy-L-altrose pathways of lipopolysaccharide biosynthesis, respectively; SnoF from the S. nogalater nogalamycin pathway, DauORF4 from the S. griseus daunomycin pathway (KruÈgel et al. 1993) and StrM from the S. griseus streptomycin pathway (Pissowotzki et al. 1991)

207

(Dickens et al. 1996), which is suggested to be involved in daunosamine biosynthesis. However, its function has not been resolved. Complementation studies The fragments SY27, SY30, SY32, SY31 and SY36 (Fig. 2) were cloned in the pIJ486-based vectors to study their ability to restore the production of aclacinomycin in H039. None of the subclones, when cloned into pIJ486, altered the glycosylation behaviour of H039. For this reason, we used two other vectors derived from pIJ486: pIJE486 carrying the ermE promoter (Bibb et al. 1985) and pSYL1 containing snoA, a trans-acting genetic element encoding a 665-amino acid protein, which was previously demonstrated to promote the expression of minimal PKS genes for nogalamycin (Ylihonko et al. 1996b). Indeed, if the fragments were cloned in the plasmid pSYL1 containing snoA, the subclone pSYL31 caused the accumulation of aclacinomycins containing rhodosamine, as in the S. galilaeus wild type, even though the fraction of aminosugars was smaller than in the culture of H039 carrying pSY10 (Fig. 5). The fragment SY31 contains snoE for aromatase and three ORFs, snoF, snoG and snoH, involved in glycosylation. This fragment was then cloned in pIJE486, such that the ORFs were transcribed from ermE promoter. In this case H039 was complemented both by pSYE31 and by pSYE36. The aminoglycosides were identical to those in the culture of H039 carrying pSYL31. Two complete ORFs, snoF and snoG, are present in pSYE36, suggesting that one of them is required for complementa-

Fig. 4 Alignment by the PILEUP program of the SnoG sequence with those of 4-epimerases. The proteins aligned are: GalE from S. lividans (Adams et al. 1988), ExoB from Rhizobium mellioti (Buendia et al. 1991), SnoG from S. nogalater and DauORF5 from S. griseus (KruÈgel et al. 1993)

tion. Since pSY15, which contains snoF, failed to complement H039, these results suggest that snoG is responsible for restoring aclacinomycin production in H039. Complementation of H039 with the plasmids pSY10, pSYL31, pSYE31 or pSYE36, was not, however, complete. The degree of complementation was estimated from the ability of the clones to accumulate aclacinomycins (AcmA, AcmY, and AcmB), which are the major products in the wild-type S. galilaeus. H039 does not produce aclacinomycins; instead the whole sugar fraction consists of rhodinose. H039 carrying pSY10 produced both aclacinomycins and Akv-(Rho)2±3. The HPLC pro®les of the products of the wild type, H039 and H039/pSY10 are shown in Fig. 5. When any one of the subclones pSYE31, pSYL31, pSYE36 was used for complementation, the relative size of the aminoglycoside fraction was smaller than that in H039 carrying pSY10.

Discussion S. galilaeus (ATCC 31615) produces aklavinone triglycoside (AcmN) containing rhodosamine (Rhn), 2-deoxy-L-fucose (dF) and rhodinose (Rho). The last sugar residue of AcmN, Rho, is further modi®ed to

208

Fig. 5A±C HPLC analysis of products obtained from the cultures of (A) H039, (B) H039 carrying pSY10 and (C) wild-type S. galilaeus. Abbreviations: Acm, aclacinomycin; Akv, aklavinone; Rho, rhodinose

cinerulose A (CinA), aculose (Acu) or CinB, corresponding the aclacinomycins AcmA, AcmY and AcmB, respectively, which are accumulated by S. galilaeus (Oki et al. 1975). H039, the mutant derived from S. galilaeus, accumulates Akv-Rho-Rho and Akv-RhoRho-Rho, but no aclacinomycins containing Rhn or 2dF. Based on these ®ndings, H039 was suggested to have a lesion in a gene involved in the sugar biosynthesis pathway (Ylihonko et al. 1994). However, another possibility is that H039 cannot transfer either 2-dF or Rhn residues, and would thus be de®cient in a glycosyltransferase. The latter is not, however, very likely, since some S. galilaeus mutants produce aklavinone glycosides containing di€erent combinations of sugar residues,

suggesting that glycosyltransferases are ¯exible. As an example, the S. galilaeus mutant H054 accumulates Akv with Rho and 2-dF in di€erent combinations (Ylihonko et al. 1994). The sugars found in anthracyclines have been described by Fujiwara et al. (1986). Based on the patterns of H039 complementation by the subclones, it seems likely that SnoA is also involved in facilitating the expression of the glycosylation genes. As we reported earlier, SnoA activates the expression of the sno minimal PKS (Ylihonko et al. 1996b) by an unknown mechanism. None of the pIJ486-based subclones was able to complement H039, while the pIJE486based subclones pSYE31 and pSYE36 did. Complementation of H039 by pSYL36, which harbors the fragment in the snoA-containing vector pSYL1 was not, however, successful. Since H039 was complemented by pSYL31 (Fig. 2) this suggested that the promoter for this cluster is located upstream of snoE, the gene encoding aromatization of the ®rst ring in the biosynthetic pathway for anthracyclines, and that these genes are controlled by expression of snoA. That snoG is responsible for complementation of H039 was deduced from following results: (i) pSYE36, the minimal subclone that is able to complement, contains two complete ORFs (snoF and snoG) but only the 5¢-end of snoH, encoding 346 amino acids out of 489; (ii) pSY15 contains the whole of snoF (but not snoG) and did not complement H039. The deduced function of SnoG is as a 4-epimerase, the catalytic activity that converts UDP-glucose to UDP-galactose. Based on the proposed biosynthetic pathway (Fig. 1), we can imagine that this enzyme participates in biosynthesis after the 4,6-dehydratase and the 3,5-epimerase, thus suggesting that there is a keto group at position 4 in the substrate for SnoG. Even though the enzyme that is most similar to SnoG uses a reduced C4 as a substrate, it may also be a keto group in this case. Based on these results H039, which fails to synthesize 2-dF and Rhn, is suggested to be blocked in 4-epimerase activity. Nevertheless, it is not rule out, if the reaction catalyzed by SnoG is an epimerization or possibly a dehydratation of positions 2 or 3, as 4-epimerase is abundant in the gene bank and therefore easy to recover by similarity searches. The biosynthetic steps from the NDP-glucose to Rho, 2-dF and Rhn have not been elucidated. If the biosynthesis of all these three sugar residues starts from a common precursor it is possible that the biosynthesis of Rhn and 2-dF forms a separate branch from that of Rho, and that SnoG activity is only needed in the Rhn/2-dF branch. Nogalamine, the sugar residue of nogalamycin, di€ers from Rhn only by the presence of a hydroxyl group at C-2¢, thus it is not surprising that SnoG can function as an epimerase in Rhn biosynthesis. Incomplete complementation could possibly be explained by a di€ering tendencies to favor this epimerization. Acknowledgements This research was ®nancially supported by the Academy of Finland and by the EU foundation.

209

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