Department of Biochemistry, University of Turku, Vatselantie 2, FIN-20014 Turku, Finland1
Galilaeus Oy, PO BOX 113, FIN-20781 Kaarina, Finland2
Author for correspondence: Kristiina Ylihonko. Tel: +358 2 3336879. Fax: +358 2 3336860. e-mail: kristiina.ylihonko{at}finabo.abo.fi
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ABSTRACT |
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Keywords: anthracyclines, auramycinone, biosynthesis, heterologous expression, Streptomyces
Abbreviations: AAME, aklanonic acid methyl ester; minPKS, minimal PKS; PKS, polyketide synthase
The GenBank accession number for acmA reported in this paper is AF043550.
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INTRODUCTION |
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The organization of the polyketide synthase (PKS) gene cluster of S. galilaeus has been reported recently (Fujii & Ebizuka, 1997 ) and the molecular genetics of daunomycin biosynthesis has been intensively studied by Hutchinson, Strohl and their coworkers (e.g. Madduri & Hutchinson, 1995
; Rajgarhia & Strohl, 1997
). Related work on other aromatic polyketides has added to our knowledge of biosynthetic reactions in the anthracycline pathway (e.g. Hutchinson & Fujii, 1995
; McDaniel et al., 1995
; Hopwood, 1997
; Zawada & Khosla, 1997
).
We report here a step-by-step introduction of single genes for anthracyclinone biosynthesis into a plasmid construct in the order predicated by the proposed biosynthetic pathway (Ylihonko et al., 1996b ). The compounds accumulated in a heterologous host were analysed after each step. The choice of Streptomyces lividans as the host for expression cloning was based on evidence that it does not itself accumulate anthracycline metabolites in the culture conditions used for fermentation.
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METHODS |
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Strains were cultivated in 60 ml E1 medium for 56 d (28 °C, 330 r.p.m.) to produce polyketides. Products were isolated by extracting with chloroform/methanol (3:1, v/v) for TLC and HPLC analysis.
Expression constructs.
Plasmids were made in pSY21 carrying the nogalamycin minimal PKS (minPKS) genes or in pSY15 expressing the genes for the nogalamycin chromophore (Ylihonko et al., 1996b ). MinPKS and an activator from the S. nogalater gene cluster were cloned in the Streptomyces vector pIJ486 as a 5·5 kb SacIBglII fragment to give pSY21 (Fig. 2
). A 12 kb BglII fragment from the sno cluster was cloned in pIJ486 to give pSY15 (Fig. 2
). According to Table 1
, suitable restriction sites were used for cloning. The restriction sites were made blunt-ended by treatment with Klenow polymerase to make pSY21a, pSY15a and pSY15b. Furthermore, the inserts introduced into the vector to generate pSY21b, pSY21c and pSY21d were cloned through the E. coli vector pSL1180 to add convenient restriction sites. The gene for aklanonic acid methyl ester cyclase was subcloned from pAcmA, derived from the S. galilaeus gene cluster and the gene encoding aklaviketone reductase was subcloned from pDx2 carrying a 2·2 kb DNA fragment of the daunomycin gene cluster cloned from S. peucetius. All plasmids were introduced into S. lividans strain TK24. Constructs are listed in Table 1
.
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Instrumental analysis.
1H and 13C NMR spectra using CDCl3 or DMSO-d6 as solvent were recorded on a JEOL JNM-GX400 spectrometer. NMR analysis included NON (1D proton spectra), BMC (1D carbon spectra), NOE (nuclear Overhauser effect), DEPT (distortionless enhancement by polarization transfer) and HMBC (heteronuclear multiple-bond connectivities) techniques. Spectra were internally referenced to tetramethylsilane. MS was performed on a Varian VG707E spectrometer. Metabolites were detected by HPLC (LaChrom, Merck Hitachi, pump L-7100, detector L-7400 and integrator D-7500) using a LiChroCART RP-18 column.
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RESULTS |
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A sno cluster containing the nogalamycin biosynthetic region was discovered by sequential use of the actI and acm probes (Ylihonko et al., 1996a ). The sequence is listed in GenBank under the accession number (Z48262)/AJ224512. It was used as the source of seven structural genes encoding activities needed for polyketide biosynthesis and of the gene responsible for esterification of the aklanonic acid analogue, nogalonic acid (Fig. 3
). A fragment containing the gene required for each biosynthetic step was subcloned from the sno cluster using convenient restriction sites shown in Table 1
and Fig. 2(a)
.
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A dau cluster covering about 60 kb contiguous DNA for daunomycin biosynthesis was located and cloned by hybridizing to the acm probe. A 2·2 kb DNA fragment (pDx2) of the dau cluster complemented H036, a S. galilaeus mutant accumulating aklaviketone. Sequencing and sequence analysis of pDx2 revealed one complete ORF named dauE, because the nucleotide sequence was 98% identical to the gene (dauE) previously characterized from Streptomyces sp. strain C5 (Dickens et al., 1996 ) encoding aklaviketone reductase. A SacIAvrII fragment of pDx2 (Fig. 2c
) was used to introduce the C-7 ketoreductase gene into the final construct, pSY15b (Fig. 4
).
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Plasmid pSY21 was introduced into the S. galilaeus mutant H039, which produces aklavinone with attached rhodinose residues (Ylihonko et al., 1994 ). When S. nogalater minPKS in pSY21 was expressed in H039, the hydrolysed products obtained were aklavinone and auramycinone, whereas aklavinone was obtained after hydrolysis of H039 products. Aklavinone and auramycinone differ only in the ethyl or methyl substituent at C-9, respectively, due to use of a different starter unit in building the polyketide chain (Ylihonko et al., 1996b
).
Plasmid pSY21a was introduced into the polyketide-ketoreductase-deficient S. galilaeus mutant H061 for complementation. H061 was previously complemented to produce aklavinone glycosides by snoD encoding polyketide ketoreductase (KR) for nogalamycin biosynthesis (Ylihonko et al., 1996b ). When the hydrolysed products of H061/pSY21a were analysed, the aglycones obtained were aklavinone and auramycinone, as expected. H061 accumulates a 2-OH compound exhibiting a different folding pattern from anthracyclines (Kantola et al., 1997
).
S. steffisburgensis producing steffimycin (see Fig. 1 for the structure of 8-demethoxy steffimycin) was employed to analyse the effect of co-expression of aromatase with ketoreductase in pSY21b. We recently characterized the products of S. steffisburgensis (Kunnari et al., 1997
) and found that the main product of S. steffisburgensis (ATCC 27466) was 8-demethoxy steffimycin in the conditions used for fermentation. Steffimycinone differs from nogalamycin aglycone in position 2, the site for polyketide ketoreductase action (Figs 1
and 3
) in nogalamycin biosynthesis. The absence of this step in the steffimycin pathway results in a methoxy group at C-2 in steffimycin. Expression of snoD and snoE in S. steffisburgensis caused the production of 2,8-demethoxy steffimycins (Kunnari et al., 1997
) and pSY21b generated the same products in this strain, confirming the functionality of the construct.
Plasmid pSY21c was the first construct that caused the production of stable compounds in TK24 and in the same proportion in separate fermentation batches. Structural analysis of these products revealed the aklanonic acid analogue nogalonic acid (IVA, Fig. 3) supporting the function of snoB as an oxygenase and suggesting that snoB is probably the last component in the PKS complex, as a stable compound was formed. Nevertheless, the major compound obtained (IVC) was a hybrid, perhaps caused by the concomitant action of cloned nogalamycin genes and the hosts endogenous actinorhodin biosynthesis genes. Furthermore, a compound IVB of 18 carbons was also accumulated. IVB was suggested to be a biosynthetic product derived from nine acetates, presumably derived from the flexibility of minPKS in chain elongation of a growing polyketide chain. This was, however, a minor product. We have also found that some S. galilaeus mutants accumulating intermediates in aklavinone biosynthesis produce anthracycline metabolites derived from shorter polyketide chains than expected if the typical ten building blocks were used.
Plasmid pSY21d contains the same genes for biosynthesis of the aglycone as pSY15, and the product profiles of TK24/pSY21d and TK24/pSY15 were identical, as expected. The accumulation of nogalonic acid methyl ester (VA) demonstrated that snoC is responsible for methylation of a carboxy group at C-10. The structures of the compounds produced by TK24/pSY15 are shown in Fig. 3. All the compounds, including the intermediate IVA, the shunt product IVB and the hybrid product IVC, were converted to the methylated forms by the action of SnoC, resulting in VA, VB and VC, respectively. Furthermore, the reduced compound VD was accumulated in TK24/pSY21d as in TK24/pSY15. Because the product profiles caused by pSY15 and pSY21d were identical, pSY15 was chosen as a vector for preparation of the last two constructs to allow the usage of suitable restriction sites for cloning.
Plasmid pSY15a, expressing acmA in addition to genes covering steps 15 (Fig. 3), caused the accumulation of auraviketone (VI) in TK24. Finally, the addition of the SacIAvrII fragment carrying dauE, a C-7 ketoreductase from S. peucetius, to pSY15a generated pSY15b, resulting in the production of auramycinone in S. lividans TK24. Auramycinone made up about 30% of the detectable metabolites.
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DISCUSSION |
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The host chosen for this study was S. lividans TK24 even though Streptomyces coelicolor CH999 has been frequently used in the investigation of aromatic polyketide metabolites (e.g. Zawada & Khosla, 1997 ; Fu et al., 1996
). CH999 has been genetically engineered to lack all the genes needed for actinorhodin biosynthesis (McDaniel et al., 1993
). Unfortunately, our attempts to produce metabolites with pSY15- or pSY21-based CH999 clones were not successful, since aromatic polyketides were not accumulated in the liquid cultures. Plasmids pSY21c- and pSY15-based constructs caused the formation of 20-carbon hybrid products in TK24 (Fig. 3
, IVC, VC), whereas metabolites of 16 carbons (actinorhodin is built up from eight acetate units; Fig. 1
) were not recovered. SnoA is an activator that presumably promotes expression of other polyketide genes in addition to nogalamycin biosynthetic genes. An expression plasmid comparable to pSY21c but containing the ermE promoter (Bibb et al., 1985
) in pIJ486 and not containing snoA, decreased the production of metabolites though hybrid products were not obtained in the culture extract. However, the yield was too low for structural analysis of the compounds (J. Kantola, unpublished results).
The production of stable compounds when oxygenase was present suggests that this enzyme is the last PKS component in forming the product, which is then released from the enzyme complex. The hybrid products obtained (Fig. 3, IVC, VC) sharing structural features of actinorhodin (Fig. 1
) and nogalamycin were not oxygenated, suggesting that the reaction possibly directed at the actVI locus (Fernandez-Moreno et al., 1994
; Ichinose et al., 1999
) is competitive with that directed by the oxygenase. The factor that determines condensation of the second and third rings was not, however, clarified. The presence of oxygenase (or a corresponding enzyme) with the PKS may result in the correct orientation of a polyketide chain to allow condensation of two aromatic rings (B and C), suggesting that specific cyclases are not essential. This hypothesis, that no separate gene products are needed for the cyclizations, is consistent with the minimal construct being able to cause the production of aklanonic acid for daunomycin biosynthesis (Rajgarhia & Strohl, 1997
; Gerlitz et al., 1997
). On the other hand, dpsY, recently identified in the daunomycin cluster, is probably essential for closing of the aromatic rings (Lomovskaya et al., 1998
), which is why we cannot rule out the possibility that cyclization activities were derived from TK24. Also DpsH has been mentioned to be involved in closing the second and the third rings (Gerlitz et al., 1997
), but recently published results by Lomovskaya et al. (1999)
, have suggested that DpsH is involved in daunosamine biosynthesis or its attachment to rhodomycinone. Since spontaneous cyclization reactions result in only minor amounts of products (generally less than 1%), the fact that auramycinone was a major product (30% of the whole extract) suggests that an enzymatic reaction was involved.
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ACKNOWLEDGEMENTS |
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Received 21 June 1999;
revised 10 September 1999;
accepted 24 September 1999.
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