Introduction

Polyketides are useful natural products that include important therapeutic agents, such as antibacterial, immunosuppressive, and cholesterol-lowering compounds (O’Hagan 1991). The number of cloned and sequenced genes involved in polyketide biosynthesis has increased rapidly during the past decade, and increasing knowledge on genetic information has enabled the manipulation of polyketide biosynthetic pathways to yield novel compounds. However, many of the original polyketide producers are difficult to manipulate genetically, have poor growth characteristics, or yield poor titers. The transfer of biosynthetic genes from the original producers to a more amenable and robust heterologous host therefore becomes an attractive alternative to producing high levels of desired compounds or to providing a basis for subsequent combinatorial biosynthetic approaches (Pfeifer and Khosla 2001; Rude and Khosla 2004; Wenzel and Muller 2005). The most widely used heterologous hosts for polyketide production are Streptomyces coelicolor (McDaniel et al. 1995) and Streptomyces lividans (Ziermann and Betlach 1999). In these strains, a number of metabolic pathways have been successfully engineered to increase the titer of natural products themselves and to generate a variety of novel unnatural products. Saccharopolyspora erythraea (Gaisser et al. 2000; Martin et al. 2003) and Streptomyces fradiae (Rodriguez et al. 2004; Reeves et al. 2004) have been also engineered to create strains that could be used for the production of novel polyketides through heterologous expression and manipulation of polyketide biosynthetic pathways. However, these heterologous hosts have a slow growth rate and need relatively prolonged culture periods (6–9 days) to reach the maximum production level of metabolites in the fermentation process. In contrast, Streptomyces venezuelae has been recently developed as a heterologous host (Yoon et al. 2002; Jung et al. 2003; Hong et al. 2004) that requires a short culture period (3–4 days) for metabolite production compared to other Streptomyces species (Xue and Sherman 2001). It is also amenable to genetic manipulation and has high transformation efficiency. These characteristics make S. venezuelae an alternative system for a rapid heterologous production of polyketides from benchtop genetic manipulation to product fermentation (Xue and Sherman 2001).

Tylosin polyketide synthase (Tyl PKS) encoded by tylGI–V of S. fradiae is responsible for the biosynthesis of the macrolactone, tylactone, which is further modified by oxidation at C-20 and C-23 and glycosylation of mycaminose, mycinose, and mycarose to produce tylosin (Fig. 1a). Tyl PKS has a biochemical architecture similar to that of the pikromycin (Pik) PKS of S. venezuelae (Fig. 1b). Based on this similarity between Tyl PKS and Pik PKS, the hybrid Tyl–Pik modular PKS system in S. venezuelae has been developed in a previous study (Yoon et al. 2002). The expression of several hybrid PKS resulted in the production of macrolactones with predicted structural alterations and further elaborated structures by the action of post-PKS-modifying enzymes of S. venezuelae, DesVII glycosyltransferase, and PikC hydroxylase.

Fig. 1
figure 1

Organization of Tyl PKS genes and pikromycin biosynthetic gene cluster. a Organization of Tyl PKS genes and structures of tylactone and tylosin. b Organization of pikromycin biosynthetic gene cluster and structures of narbonolide and pikromycin. Each arrow represents an open reading frame (ORF). The direction of transcription and the relative sizes of ORFs are indicated

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In this study, we describe the expression of the entire Tyl PKS from two compatible plasmids in a Pik-PKS-deleted strain of S. venezuelae in order to probe the applicability of S. venezuelae as a heterologous host for the expression of a whole PKS gene cluster. The resulting strain produced a 16-membered ring macrolactone, tylactone, in a considerably reduced culture period (4 days) compared to those of other Streptomyces heterologous hosts. Interestingly, tylactone was further modified probably by the action of flexible glycosyltransferase DesVII, leading to its desosamine-glycosylated derivative. Furthermore, the production level of tylactone was enhanced considerably through the feeding of several precursors of ethylmalonyl-CoA, which is expected to be the limiting substrate of Tyl PKS in S. venezuelae. This work demonstrates the unique capacity of S. venezuelae as a new system for the rapid expression of heterologous PKS and the creation of hybrid polyketides.

Materials and methods

Bacterial strains, culture conditions,and genetic manipulation

S. venezuelae ATCC15439 was used for the construction of mutant strains. The transformants of S. venezuelae were selected on R2YE agar plates (Kieser et al. 2000) by overlaying with an appropriate antibiotic or a combination of antibiotics (Yoon et al. 2002). SGGP liquid medium (Kieser et al. 2000) was used for the propagation of S. venezuelae. S. fradiae ATCC19609 grown in tryptic soy broth medium (Kieser et al. 2000) was used for the preparation of genomic DNA. Genetic manipulation was performed in Escherichia coli DH10B using standard protocols (Sambrook et al. 2001). Transformation of DNA into protoplasts of S. venezuelae followed standard protocols (Kieser et al. 2000). Litmus28 (New England Biolabs) and pGEM-3Zf(+) (Promega) were used for subcloning, and Southern blot hybridization was performed according to the protocol recommended by a digoxigenin labeling and detection kit (Roche).

Construction of pikA deletion mutant

A gene replacement plasmid (pYJ230) derived from pKC1139 (Bierman et al. 1992), which contains an apramycin-resistant marker, was constructed for the replacement of pikAI–IV genes by hygromycin resistance gene (Elhai and Wolk 1988). Plasmid pYJ230 was constructed by cloning the hygromycin resistance gene flanked by two 1-kb fragments homologous to the upstream and downstream regions of pikA. The following primers with engineered restriction sites (underlined) were used to generate the two 1-kb fragments by polymerase chain reaction (PCR): a HindIII–XbaI fragment containing the upstream region of pikAI(forward,5′-AAAAAGCTTGGGGATGTGGCGCCGGAGGATCTC-3′;reverse,5′-TTTTCTAGACATCCGGCTCCGTCTCCGGAAGCC-3′) and a KpnI–EcoRI fragment containing pikAV and part of desVIII (forward, 5′-AAAGGTACCGAGGGGGCGGGCAAGTGACCGACAGAC-3′; reverse, 5′-AAAGAATTCGGACAGCGGCCGGAGCCGCTCCAG-3′). The plasmid pYJ230 was introduced into a previously constructed pikAIII–IV deletion mutant of S. venezuelae YJ004 (Yoon et al. 2002), for plasmid-mediated homologous recombination, as described previously (Yoon et al. 2002). Several double crossover mutants were identified on the basis of their phenotype of hygromycin resistance and apramycin sensitivity, and their genotype by Southern blot hybridization. The resulting strain was designated DHS2001.

Expression of Tyl PKS under the controlof pikAI promoter

Tyl PKS genes were individually cloned into two compatible vectors, pDHS702 (Xue and Sherman 2000) and pDHS618 (Zhao et al. 1999), which are E.coli–Streptomyces shuttle vectors containing a pikAI promoter (Xue and Sherman 2001), SCP2* origin, and different antibiotic resistance genes. The tylGI–III genes were cloned into pDHS702 as a MunI–NsiI fragment by combining an EcoRI–SphI partial fragment of tylGI–III isolated from pSET506 (a gift from Eli Lilly Co.; GenBank accession number SFU78289), a MunI–EcoRI fragment of the 5′ region, and an SphI–NsiI fragment of the 3′ region of tylGI–III genes amplified by PCR to generate pBB155 (Fig. 1a). Both ends of tylGI–III were obtained by PCR using the following primers: a MunI–EcoRI fragment containing the 5′ end of tylGI (forward, 5′-AAACAATTGACACAGCTCGGTGACACGGCAGCC-3′; reverse, 5′-AAAGAATTCCTCGACACCGGCCGCGCCCGGCAG-3′) and an SphI–NsiI fragment containing the 3′ end of tylGIII (forward, 5′- AAAGCATGCAACGGCGCCTGAACCGGAGCGGCA-3′; reverse, 5'-AAAATGCATGTGTTTTCACCTGTGCTGTCGCTG-3′) (restriction sites are underlined). The plasmid pDHS3003 (Fig. 2b) expressing tylGIV–V under the control of the pikAI promoter was constructed as described previously (Yoon et al. 2002). Transformation of the DHS2001 strain by the plasmids pBB155 and pDHS3003 generated the mutant strain YJ005.

Fig. 2
figure 2

Maps of plasmids for the expression of Tyl PKS. a An expression plasmid pBB155 containing tylGI–III. b An expression plasmid pDHS3003 containing tylGIV–V

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Expression of Tyl PKS expression under the controlof actI promoter

An actI promoter and a pathway-specific activator gene (actII–ORF4) (McDaniel et al. 1993) were also used for the expression of Tyl PKS. A 1.43-kb PacI–KpnI fragment containing an actI/actII–ORF4 promoter–activator pair was amplified from pHGF7505 (Yu et al. 2001) by PCR and ligated to a 313-bp KpnI–EcoRI fragment corresponding to the 5′ end of tylGI amplified by PCR from the genomic DNA of S. fradiae. The resulting PacI–EcoRI fragment was ligated to the same sites of pBB155 to generate pYJ153. The following primers with engineered restriction sites (underlined) were used: a PacI–KpnI fragment (forward, 5′-AAGCTTTTAATTAAAGTTTAAACAGCTCGGATT-3′; reverse, 5′-ATCCTTGGTACCTCGATGCGTTCGTCCGGTGGT-3′) and a KpnI–EcoRI fragment (forward, 5′-GCCGCAGGTACCGTTGGCAGGCTCACCGGCACG-3′; reverse, 5′-TTCCCAGAATTCCTCGACACCGGCCGCGCCCGG-3). The EcoRI–XbaI fragment containing tylGIV–V genes from plasmid pDHS3003 was transferred to the same sites of a pRM5 derivative (McDaniel et al. 1993), which contains the actI/actII–ORF4 promoter–activator pair and apramycin resistance gene. The resulting plasmid was designated pYJ154.

Production and analysis of heterologous polyketides

All strains were cultured on R2YE solid medium (Kieser et al. 2000) at 30°C for 4 days with appropriate antibiotic selection. Agar medium production gives more reproducible results than liquid fermentation, although liquid fermentation typically supports the same level of antibiotic production. Agar-grown culture was diced and extracted with 2 vol of methanol. The extract was washed with water, extracted again with 1 vol of chloroform, and concentrated with methanol. The extract was analyzed for polyketide production on a reverse-phase C18 column (4.6×250 mm Watchers 120 ODS-BP, 5 μm; Daiso Watchers) with a binary mobile phase (solvent A: acetonitrile/methanol=4:1 with 5 mM ammonium acetate; solvent B: 5 mM ammonium acetate in H2O; A: 20–75%, 30 min; 75%, 10 min; 75–20%, 10 min) at a flow rate of 1 ml/min (Rodriguez et al. 2004) by the combination of liquid chromatography/mass spectrometry (LC/MS) and mass spectrometry/mass spectrometry (MS/MS) using a Finnigan LCQ-DecaXP with an electrospray source. A purified tylactone standard (kindly provided by Professor Eric Cundliffe at University of Leicester) was used to generate a calibration curve for titer determination. The production level of tylactone was averaged from five separate cultivations and extractions.

Results

Generation of Pik PKS deletion mutant of S. venezuelae DHS2001

A strain for the expression of heterologous PKS (DHS2001) was constructed by deleting the native Pik PKS from the chromosome of S. venezuelae and by replacing with the hygromycin resistance gene through a plasmid-mediated homologous recombination. A pikAV gene encoding a type II thioesterase desosamine (des) biosynthetic gene cluster, a pikC gene encoding a cytochrome P450 hydroxylase, and a pikD gene encoding a pathway-specific activator (Xue et al. 1998a,b; Wilson et al. 2001) downstream of Pik PKS (Fig. 1b) remain intact in DHS2001. The genomic DNA of the resulting mutant strain was analyzed by Southern blot hybridization to confirm double crossover (data not shown). The culture extract of strain DHS2001 was analyzed, and no antibiotic production was observed. Feeding the pikromycin aglycone, narbonolide, to the growing medium of DHS2001 restored the production of pikromycin, which is synthesized through the attachment of desosamine to narbonolide at C-5 oxygen by DesVII glycosyltransferase and by the addition of a hydroxyl group at C-12 position by PikC hydroxylase (Fig. 1b). This result indicates that the remaining des cluster, the monooxygenase pikC, and the pathway-specific activator pikD genes are functional in DHS2001.

Heterologous expression of the entire Tyl PKS in S. venezuelae DHS2001

A previous study revealed that two SCP2*-derived plasmids carrying different antibiotic markers were compatible and stable during growth (Xue et al. 1999). For the expression of the entire Tyl PKS, two plasmids containing SCP2* replication origin and two different resistance genes were used. The expression plasmids pBB155 (carrying tylGI–III; Fig. 2a) and pDHS3003 (carrying tylGIV–V; Fig. 2b) were constructed and transformed into the pikA deletion mutant DHS2001. Transformants resistant to apramycin and thiostrepton were isolated and designated YJ005. The organic extract of YJ005 was analyzed by LC/MS and MS/MS for identification of tylactone. A major peak was observed at a retention time of 30.7 min with m/z=395 and 377, consistent with the calculated molecular weight for tylactone and its dehydrated form, respectively, from LC/MS analysis (Fig. 3a). MS/MS fragmentation of this compound with m/z=395 produced a pattern identical to the characteristic fragmentation pattern of a tylactone standard (data not shown), thus confirming the presence of tylactone in the growing medium of YJ005. In addition, a peak with m/z=552 was observed at a retention time of 43.0 min from LC/MS analysis (Fig. 3a), corresponding to desosaminyl tylactone, which is known to be a biologically active compound against the majority of Gram-positive bacteria (Furumai et al. 1977). The compound with m/z=552 fragmented into peaks at m/z=534, 395, 377, and 158, corresponding to the dehydrated form of desosaminyl tylactone, tylactone, dehydrated form of tylactone, and oxonium ion fragment derived from desosamine (Butler et al. 2002), respectively (Fig. 3b). This fragmentation pattern is consistent with the previous report in which 5-O-desosaminyl tylactone was synthesized by feeding tylactone to a mutant of Sa. erythraea that is deficient in eryA-encoding erythromycin PKS and both eryCIII and eryBV glycosyltransferases but containing the tylosin tylMII glycosyltransferase gene (Gaisser et al. 2000). The yield of tylactone in S. venezuelae YJ005 was approximately 0.5 mg/l, and the percentage conversion of tylactone into desosaminyl tylactone was approximately 8.3% based on the peak intensity from LC/MS analysis. The maximum level of tylactone production was reached after a 4-day culture (Fig. 4).

Fig. 3
figure 3

LC/MS and MS/MS analyses of compounds from the extract of YJ005. a A major peak was observed at a retention time of 30.7 min with m/z=395 and 377, consistent with the calculated molecular weight for tylactone and its dehydrated form, respectively. A minor peak was detected at 43.0 min with m/z=552, which corresponds to desosaminyl tylactone. b The compound with m/z=552 fragmented into peaks at m/z=158, 377, 395, and 534, corresponding to characteristic desosamine moiety ion (oxonium ion), a dehydrated form of tylactone, tylactone, and a dehydrated form of desosaminyl tylactone, respectively

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Fig. 4
figure 4

Production level of tylactone on R2YE solid medium from YJ005. S. venezuelae YJ005 was cultured on R2YE solid medium up to 5 days at 30°C

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Feeding several precursors for ethylmalonyl-CoAto the culture medium of YJ005

Besides malonyl-CoA and methylmalonyl-CoA, the biosynthesis of tylactone requires ethylmalonyl-CoA as an extender unit, which is incorporated into the tylosin polyketide backbone by an ethylmalonyl-specific acyltransferase in module 5 of Tyl PKS. However, Pik PKS does not require ethylmalonyl-CoA as an extender unit for the biosynthesis of pikromycin. Successful production of tylactone in S. venezuelae suggests that this strain certainly contains the biosynthetic pathway for ethylmalonyl-CoA. However, it is uncertain whether S. venezuelae contains a sufficient pool of intracellular ethylmalonyl-CoA to support the efficient biosynthesis of tylactone. To address this question, S. venezuelae YJ005 was grown on R2YE medium containing precursors for ethylmalonyl-CoA, such as butanol, butyrate, and diethylmalonate (Stassi et al. 1998). Consequently, supplementation with the precursors for ethylmalonyl-CoA into the growing medium resulted in enhancement of tylactone productivity. When 50 mM butanol, 50 mM butyrate, and 10 mM diethylmalonate were added to the growing medium during inoculation, the production of tylactone increased by about 1.4-fold (0.7 mg/l), 1.6-fold (0.8 mg/l), and 2.8-fold (1.4 mg/l), respectively.

Tyl PKS expression under the control of actI promoter

The actI promoter and its cognate activator gene actII–ORF4 from the actinorhodin biosynthetic gene cluster of S. coelicolor (McDaniel et al. 1993) have been widely used to express several large PKS genes in genetically engineered “clean” host strains of S. coelicolor and S. lividans (Pfeifer and Khosla 2001). Erythromycin PKS was expressed under the control of the actI promoter after integration into the chromosome by homologous recombination in a mutant strain of Sa. erythraea that is deficient in native PKS, and the resulting strain produced a high quantity of erythromycin when compared with the wild-type Sa. erythraea (Rowe et al. 1998). In contrast, use of this promoter–activator in a Sa. erythraea overproducer resulted in a >20-fold decrease in erythromycin titers compared to the strain using a native eryAI promoter. (Rodriguez et al. 2003). To investigate the effect of promoters on the productivity of heterologous polyketide in S. venezuelae, the actI promoter and the actII–ORF4 activator gene were tested to express Tyl PKS in the S. venezuelae mutant strain. The plasmids containing Tyl PKS under the control of the actI promoter were constructed and introduced into the pikA deletion mutant DHS2001 to generate YJ048. The organic extract of YJ048 was analyzed by LC/MS and MS/MS. The calculated molecular weights corresponding to tylactone and desosaminyl tylactone were detected. The amount of tylactone produced from YJ048 was similar to levels produced from the YJ005 strain, demonstrating that substitution of the pikAI promoter with the actI promoter did not affect the production level of heterologous polyketides in this host–vector system.

Discussion

S. venezuelae is a suitable heterologous host for the expression of polyketide biosynthetic genes due to its a fast growth, which allows large quantities of cell mass and metabolites to be harvested relatively quickly compared to other Streptomyces species (Xue and Sherman 2001). Development of the genetically engineered strain of S. venezuelae DHS2001 and rapid production of tylactone in the mutant strain validated the unique advantage of S. venezuelae as an attractive host for the production of heterologous polyketides. In addition, biosynthetic pathways for the three most common precursors found in modular polyketide biosynthesis (malonyl-CoA, methylmalonyl-CoA, and ethylmalonyl-CoA) seem to be present in S. venezuelae as proven by the production of tylactone from the YJ005 strain, which requires all of the three common precursors. The presence of a range of precursors for the biosynthesis of polyketides in S. venezuelae would facilitate the heterologous expression of a diverse class of PKS.

The flexibility of DesVII glycosyltransferase in accepting new sugar donors as substrate has been demonstrated from previous reports (Zhao et al. 1998a,b, 2001; Yamase et al. 2000; Borisova et al. 1999; Hong et al. 2004). DesVII has also been shown to be flexible toward hybrid aglycones, to which the sugar is attached (Yoon et al. 2002). The unique property of DesVII has allowed researchers to explore the possibility of generating novel macrolide derivatives attached with new sugars. The current study takes this a step further by showing that DesVII, which normally accepts both 12- and 14-membered ring macrolactones, is capable of accepting a 16-membered ring, tylactone, as an aglycone substrate. The successful generation of desosaminyl tylactone has further expanded the usefulness of glycosyltransferases as a tool for the generation of new hybrid polyketide structures. In another study, a pikromycin producer (Streptomyces sp. no. 4,900, in which the activity of the KS domain of PKS was inhibited by cerulenin) also successfully converted tylactone (200 mg) into 5-O-desosaminyl tylactone (30 mg) with a 15% conversion yield (Omura et al. 1980), which is approximately 2-fold higher than that of S. venezuelae (DesVII). The glycosyltransferase of another pikromycin producer Streptomyces sp. no. 4,900 seems to have a higher efficiency in converting tylactone to desosaminyl tylactone than DesVII, and its catalytic efficiency toward unnatural substrates would be worth investigating further.

Although the glycosylation site of tylactone was not determined due to this low conversion yield, it is likely that desosamine was introduced to the oxygen at position C-5 of tylactone, according to the intrinsic catalytic specificity of DesVII. Further analysis, such as by nuclear magnetic resonance spectroscopy, is required to confirm the glycosylation site of tylactone. No hydroxylated forms of tylactone and desosaminyl tylactone were detected, although the monooxygenase PikC of S. venezuelae was known to have considerably relaxed substrate specificity (Yoon et al. 2002; Lee et al. 2005) toward alternative substrates, indicating that tylactone and desosaminyl tylactone may be poor substrates for PikC.

The level of production of tylactone in S. venezuelae is relatively low and requires further improvement. In this study, two simple strategies were employed to enhance the production level of tylactone. First, the limiting precursor was added to the growing medium. Ethylmalonyl-CoA, which is required for the biosynthesis of tylactone, was expected to be limiting in the S. venezuelae system since pikromycin production in wild type does not require ethylmalonyl-CoA as a precursor. Supplementation with several precursors for ethylmalonyl-CoA, such as butanol, butyrate, or diethylmalonate, into the growing medium led to improvement in tylactone production, suggesting that ethylmalonyl-CoA may be a limiting factor for the biosynthesis of tylactone in S. venezuelae. Second, Tyl PKS was expressed under the control of the actI promoter. Similar levels of tylactone production were observed when Tyl PKS was expressed under the control of either an actI promoter or a pikAI promoter. Switching promoters had no observable effect, although expression levels of Tyl PKS may have been different in using the actI promoter or the pikAI promoter. Taken together, the major factor limiting the production level of tylactone seems to be deficiency in the intracellular pool of ethylmalonyl-CoA rather than the expression level of PKS in this system. Previous work revealed that Tyl PKS genes have an independent expression pattern in which tylGI and tylGIII–V are under differential regulation in S. fradiae (Stratigopoulos and Cundliffe 2002). According to this observation, tylGIII expression in pBB155 and pYJ153 could be expressed independently under the control of its intrinsic promoter in S. venezuelae, which would be another explanation for the fact that switching the pikA promoter by actI/actII–ORF4 had no observable effect on tylactone production.

The newly developed S. venezuelae system described in this study has both promising and limiting factors as a heterologous host. The limiting factor is the relatively low production yield of heterologous polyketides compared to other well-developed Streptomyces host. Features of S. venezuelae, including rapid growth rate and quick production of metabolites, are favorable characteristics for heterologous polyketide production that cannot be easily endowed by genetic engineering. Reducing the time for bacteria culture has important implications for the production of metabolites through the fermentation process; thus, this unique advantage of S. venezuelae as a fast-growing bacteria (doubling time, approximately 1 h) is an attractive feature in developing a heterologous host. Further works on enhancing the production level of heterologous polyketides through metabolite engineering strategies will facilitate the development of S. venezuelae as a competent heterologous host.