Heterologous production of epothilones B and D in ... - Springer Link

Appl Microbiol Biotechnol (2008) 81:109–117 DOI 10.1007/s00253-008-1674-0

APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY

Heterologous production of epothilones B and D in Streptomyces venezuelae Sung Ryeol Park & Je Won Park & Won Seok Jung & Ah Reum Han & Yeon-Hee Ban & Eun Ji Kim & Jae Kyung Sohng & Sang Jun Sim & Yeo Joon Yoon

Received: 3 July 2008 / Revised: 9 August 2008 / Accepted: 19 August 2008 / Published online: 4 September 2008 # Springer-Verlag 2008

Abstract Epothilones, produced from the myxobacterium Sorangium cellulosum, are potential anticancer agents that stabilize microtubules in a similar manner to paclitaxel. The entire epothilone biosynthetic gene cluster was heterologously expressed in an engineered strain of Streptomyces venezuelae bearing a deletion of pikromycin polyketide synthase gene cluster. The resulting strains produced approximately 0.1 μg/l of epothilone B as a sole product after 4 days cultivation. Deletion of an epoF encoding the cytochrome P450 epoxidase gave rise to a mutant that selectively produces 0.4 μg/l of epothilone D. To increase the production level of epothilones B and D, an additional copy of the positive regulatory gene pikD was introduced into the chromosome of both S. venezuleae mutant strains. The resulting strains showed enhanced production of corresponding compounds (approximately 2-fold). However, deletion of putative transport genes, orf3 and orf14 in the epothilone D producing S. venezuelae mutant strain, led to an approximately 3-fold reduction in epothilone D production. These results introduce S. venezuelae as an alternative heterologous host for the production of these S. R. Park : J. W. Park : W. S. Jung : A. R. Han : Y.-H. Ban : E. J. Kim : Y. J. Yoon (*) Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, Republic of Korea e-mail: [email protected] J. K. Sohng Department of Pharmaceutical Engineering, Institute of Biomolecule Reconstruction, Sun Moon University, Asan 336-708, Republic of Korea S. J. Sim Department of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea

valuable anticancer agents and demonstrate the possibility of engineering this strain as a generic heterologous host for the production of polyketides and hybrid polyketide-nonribosomal peptides. Keywords Epothilone . Heterologous production . Streptomyces venezuelae

Introduction Many of the natural products with useful biological activities belong to the polyketide and nonribosomal peptide families (O’Hagan 1991; Kleinkauf and Döhren 1996; Finking and Marahiel 2004). Recently, characterization of genes responsible for the polyketide and hybrid polyketide-nonribosomal peptide production has increased rapidly, and the newly developed techniques and increased genetic knowledge have enabled the manipulation of their biosynthetic pathways to yield novel compounds. However, this approach is often limited by the poor growth and yield titer associated with the original polyketide producer. The transfer of natural product biosynthetic pathways from the original microorganisms into more amenable heterologous hosts represents an attractive alternative to producing high levels of desired natural products or to generating novel bioactive compounds (Wenzel and Müller 2005). Among well-developed heterologous hosts, Streptomyces coelicolor and Streptomyces lividans are most commonly used for polyketide production (Rude and Khosla 2004). The pikromycin-producing Streptomyces venezuelae has recently been developed as a favorable heterologous host for the polyketide production due to its fast growth characteristics, relative ease of genetic manipulation, and the abundance of substrate pools available for the polyketide biosynthesis (Yoon et al. 2002; Hong et al.

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Appl Microbiol Biotechnol (2008) 81:109–117

2004; Jung et al. 2006; Park et al. 2007; Thapa et al. 2007; Jung et al. 2008). In our previous study, the heterologous expression of tylosin polyketide synthase (PKS) encoded by tylGI-V of Streptomyces fradiae in the pikromycin PKSdeleted strain of S. venezuelae DHS2001 produced tylacone (Jung et al. 2006). In addition, improved production of hetereologous polyketide was achieved by the overexpression of a pikD regulatory gene of the pikromycin biosynthetic gene cluster (Jung et al. 2008). In order to probe the possibility to develop the S. venezuelae-based heterologous system for the production of hybrid polyketide-nonribosomal peptide compound such as epothilone, we expressed the epothilone biosynthetic gene cluster containing hybrid PKSnonribosomal peptide synthetase (NRPS) genes in the S. venezuelae mutant DHS2001. The epothilones are a class of 16-membered polyketide macrolactones with a methylthiazole group attached by an olefinic bond. They are potent anticancer agents that bind to tubulin and inhibit the disassembly of microtubules in a manner similar to paclitaxel (Taxol; Bollag et al. 1995). Earlier studies in human cancer patients indicate that these agents are effective against tumors resistant to conventional chemotherapy and have an advantage over Taxol in terms of water solubility (Bollag et al. 1995; Su et al. 1997a). These valuable features make epothilones the next generation of chemotherapeutic compounds for the treatment of cancer (Chou et al. 1998). Epothilones were originally identified as secondary metabolites produced by the slowgrowing myxobacterium Sorangium cellulosum (Gerth et al. 1996). The major metabolites of this bacterium are epothilones A and B, whereas epothilones C and D are the early biosynthetic intermediates of epothilones A and B, respectively (Fig. 1). Of these four congeners, epothilone D has the most promising therapeutic index, but is produced only in small amounts (320.1; epothilone D, 491.9>303.9. Epothilones A and B as authentic standards were purchased from Calbiochem, whereas epothilone D was obtained from the culture of S. cellulosum So ce90 as previously described (Park et al. 2006). The production levels of epothilones were averaged from three separate cultivations and extractions.

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Results Heterologous production of epothilone B Epothilone biosynthetic genes and a putative transporterencoding gene set were individually cloned into two replication vectors, pDHS702 and pDHS618 (Xue and Sherman 2001), which are E. coli–Streptomyces shuttle vectors containing a pikAI promoter, SCP2* origin, and different antibiotic resistance genes. The epoA–B–C PKS genes and epoP NRPS gene were cloned into pDHS702, whereas the remaining epoD–E PKS genes, the epoF cytochrome P450 gene plus orf6–orf3–orf14 genes were cloned into pDHS618 (Fig. 2). The orf6 gene, immediately downstream of epoF, encodes an unknown protein with membrane-spanning regions (Tang et al. 2000). The orf3 and orf14 genes encode putative transport proteins (Molnár et al. 2000). These constructs (pDHS702-APBC and pDHS618-DEF) were introduced into S. venezuelae DHS2001 and the transformants produced only epothilone B as verified by LC–ESI-MS/MS (Fig. 3c), while no epothilones were detected in the S. venezuelae strain harboring empty plasmids (Fig. 3a). Neither epothilone A nor epothilone D, the biosynthetic intermediate to epothilone B, was detected. The metabolite eluted at 28.6 min shows a molecular ion [M + H]+ at m/z 508.1, with typical fragments at m/z 420.2 and 320.1, which can be interpreted as epothilone B (Fig. 3g). An epothilone B standard showed identical retention time and fragmentation pattern (data not shown). The initial yield of epothilone B was approximately 0.1 μg/l. Because there was no gene encoding ferredoxin and ferredoxin reductase in the epothilone biosynthetic gene cluster, the functional expression of cytochrome P450 EpoF, which requires ferredoxin and ferredoxin reductase, in S. venezuelae indicates that the one of the native ferredoxin and ferredoxin reductase in S. venezuelae could support the function of the heterologously expressed EpoF, as in the case for S. coelicolor (Tang et al. 2000) and Myxococcus xanthus (Julien and Shah 2002). Heterologous production of epothilone D At previous study, deletion of epoF and the downstream gene (orf6) produced epothilones C and D from S. coelicolor (Tang et al. 2000). Removal of both epoF, which is involved in the formation of the epoxide at C-12 and C-13 position (Julien et al. 2000; Molnár et al. 2000), and orf6 from pDHS618-DEF (by expressing pDHS702-APBC and pDHS618-DE-2 in DHS2001), gave rise to S. venezuelae mutant that produces 0.4 μg/l of epothilone D as a sole product (Fig. 3e). The metabolite eluted at 38.3 min shows a molecular ion [M + H]+ at m/z 491.9, with typical fragments at m/z 404.0 and 303.9, which can be identified as epothilone

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Fig. 3 LC–ESI-MS/MS analyses of extracts from a Streptomyces venezuelae strain harboring empty plasmid, b wild-type S. cellulosum So ce90, c S. venezuelae mutant DHS2001 harboring pDHS702APBC and pDHS618-DEF, d pikD overexpressing mutant of S. venezuelae YJ112 harboring pDHS702-APBC and pDHS618-DEF, e S. venezuelae mutant DHS2001 harboring pDHS702-APBC and pDHS618-DE-2, and f pikD overexpressing mutant of S. venezuelae YJ112 harboring pDHS702-APBC and pDHS618-DE-2. ESI-MS/MS spectra of g epothilone B shown in (c), and h epothilone D shown in (e)

D (Fig. 3h). By comparison of the retention time and MS/ MS spectral data with those of epothilone D produced from wild-type S. cellulosum (Fig. 3b), the compound produced from S. venezuelae strain harboring pDHS702-APBC and pDHS618-DE-2 was identified as epothilone D. Consistent with the previous report, orf6 does not appear to be a prerequisite for the biosynthesis of epothilones (Tang et al. 2000). The reason for the 4-fold increase in epothilone D production compared to its final product epothilone B is currently unknown. Enhanced production by pikD and the effect of ORF3 and ORF14 The PikD regulatory factor that originates from S. venezuelae was the sole transcriptional regulator of the pikromycin biosynthetic cluster and required for the macrolide antibiotic biosynthesis (Wilson et al. 2001; Jung et al. 2008). A recent study showed that the overexpression of PikD regulator in S. venezuelae DHS2001 resulted in upregulated expression of heterologous PKS by binding to pikAI promoters on the two plasmids (pDHS702 and

pDHS618) used to express heterologous PKS genes, thus improved production of heterologous polyketides could be achieved (Jung et al. 2008). To increase the production of epothilones in S. venezuelae, the different sets of replication vectors carrying the epothilone B and D biosynthetic genes were individually introduced into the pikD-overexpressed S. venezuelae mutant YJ112 (Jung et al. 2008). The resulting mutant strains showed approximately 2-fold enhanced production of both epothilones B (0.2 μg/l) and D (0.8 μg/l; Fig. 3d, f). There was an unknown peak at 43.0 min in chromatograms of epothilone D-producing recombinant strains with or without an additional copy of pikD (Fig. 3e, f). Based on its detection with epothilone D in MRM (491.9>303.9) mode, this relatively hydrophobic peak appears to be an analogue of epothilone D; however, further analyses would be required to confirm this. It has been suggested that the heterologously produced epothilones may have a cytotoxic effect on S. coelicolor (Julien and Shah 2002). A set of putative transport genes orf3 and orf14 was co-expressed in S. venezuelae with the intention of stimulating the secretion of epothilones from this heterologous host, thereby reducing the cytotoxicity and


improving the production level. Removal of this putative transport gene set from pDHS618-DE-2 (by expressing pDHS702-APBC, pDHS618-DE-1 in YJ112) led to an approximately 3-fold reduction in epothilone D production (0.27 μg/l, data not shown). As the production levels of epothilones were determined by extracting the total culture containing mycelia and agar medium, it is unclear whether ORF3 and ORF14 are involved in the export of epothilones; however, their co-expression increased the total production level of epothilones in S. venezuelae.

Discussion In this study, heterologous production of epothilones in S. venezuelae mutant strain demonstrated some advantages of S. venezuelae as an attractive host for the heterologous production of epothilones. S. venezuelae grows faster (doubling time, approximately 1 h) and produces epothilones more rapidly than other epothilone heterologous hosts, S. coelicolor, M. xanthus, and E. coli (Tang et al. 2000; Julien and Shah 2002; Mutka et al. 2006). S. coelicolor has a moderate doubling time of 2 h. Doubling time of M. xanthus (approximately 5 h) is significantly longer than S. coelicolor or E. coli (Tang et al. 2000; Mutka et al. 2006). A long cultivation period (6 days) at low temperature (15°C) was required in E. coli (Mutka et al. 2006). In addition, epothilone B or D could be generated in S. venezuelae system as a sole product probably due to the higher intracellular concentration of methylmalonyl-CoA pool compared with malonyl-CoA. The production ratio of epothilone B to A (or epothilone D to C) seems to be influenced by the intracellular concentrations of malonylCoA and methylmalonyl-CoA pools (Gerth et al. 2000; Han et al. 2008). In our previous report, the intracellular concentration of methylmalonyl-CoA in wild-type S. venezuelae and DHS2001 was 170 and 430 pmol/g cell wet weight, respectively, which is approximately 2-fold higher than that of malonyl-CoA (Park et al. 2007). This offers an advantage in recovery and purification of the desired product. The productivity of epothilone D from S. venezuelae system is approximately 0.2 μg l−1 day−1 (0.8 μg/l per 4 days) which is similar to the calculated productivity of the sum of epothilones C and D from E. coli (less than 0.17 μg l−1 day−1 or 1.0 μg/l per 6 days) (Mutka et al. 2006). These results show the possible economic advantage of S. venezuelae as a rapid and selective heterologous host for epothilone D production although its current productivity is low. There might be a possibility that epothilones A and C could be produced at a trace level if the total yields could be improved. However, because the LC–ESI-MS/MS analysis employed in this study is able to detect epothilones at sub-nanogram levels (0.1 ng of

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epothilone detection limit at the signal to noise ratio of 10:1, corresponding to 0.003 μg/l in S. venezuelae culture), the relative ratio of epothilones B (0.2 μg/l) and D (0.8 μg/l) to A and C produced from S. venezuelae would be at least 60:1 or higher if the productions of maximum undetectable levels epothilone A or C were assumed. In another study, heterologous expression of propionyl-CoA synthetase gene (prpE) from Ralstonia solanacearum in S. cellulosum increased the yield of epothilone B with an epothilone B to A ratio of 127:1, demonstrating its potential use for the selective production of epothilone B (Han et al. 2008). The expression of various genes to increase the concentration of methylmalonyl-CoA or propionyl-CoA would be worth investigating further to enhance the ratios of epothilone B and D to epothilone A and C in S. venezuelae. One of the key challenges of S. venezuelae-based heterologous expression system described in this study is relatively low production yield of epothilones. To improve the production level of epothilones in S. venezuelae, we introduced the positive regulatory gene pikD into the epothilone producing strain, leading to a 2-fold enhanced production of epothilones. However, these increased yields of epothilones in S. venezuelae were still significantly low compared to those of other heterologous hosts, S. coelicolor and M. xanthus. The reported yields of epothilones in S. coelicolor were 50 to 100 μg/l (Tang et al. 2000). M. xanthus showed the highest yields (200 to 400 μg/l) among those heterologous hosts (Julien and Shah 2002). While E. coli has been developed as an efficient host for polyketide production (Murli et al. 2003; Pfeifer et al. 2001), the titers of epothilones in E. coli were less than 1.0 μg/l (Mutka et al. 2006). Although the genome sequence of S. coelicolor (Bentley et al. 2002) and the draft genome sequence of S. venezuelae (our unpublished data) are available, it is hard to pinpoint the reason for low production in S. venezuelae by comparing the genome sequences of both strains without further detailed investigation such as transcriptome or proteome analysis of both strains expressing epothilone biosynthetic genes. One possible explanation is that the expression plasmid system used in this study was not as efficient as the system used in the previous report (Tang et al. 2000). Further detailed investigation is needed to find out the exact reason for the differences of epothilone production between the S. coelicolor and S. venezuelae systems. The S. venezuelae system described in this study has advantages as well as challenges as a heterologous host for the production of epothilones. Although the current productivity of epothilones in S. venezuelae is low, S. venezuelae has rapid growth rate and quick production of metabolites are possible. These are favorable characteristics of S. venezuelae for heterologous polyketide production that cannot be easily endowed by genetic engineering. And

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the availability of the draft genome sequence of S. venezuelae (our unpublished data) and ease of genetic manipulation will facilitate the development of S. venezuelae as a competent heterologous host for epothilone production and combinatorial biosynthesis of novel epothilone derivatives. Finally, the heterologous production of epothilones in S. venezuelae opened the possibility to develop this strain as a generic heterologous host for the production of polyketide-nonribosomal peptide hybrid compounds as well as polyketides. Acknowledgments This work was supported by the Korea Science and Engineering Foundation (KOSEF) funded by the Korea government (MEST) (R11-2005-008-00000-0), the National Research Laboratory (NRL) program grant (R0A-2008-000-20030-0), the Marine and Extreme Genome Research Center Program of the Ministry of Land, Transportation and Maritime Affairs, Republic of Korea, and grants from the National R&D Program for Cancer Control (0620300-1).

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