1 Syngenta Biotechnology Inc., 3054 Cornwallis Road, Research Triangle Park, NC 27709, USA
2 Department of Microbiology, North Carolina State University, 4527 South Gardner Hall, Raleigh, NC 27695, USA
Correspondence
István Molnár
istvan.molnar{at}syngenta.com
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ABSTRACT |
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Present address: Martek Biosciences, 4909 Nautilus Court North, Boulder, CO 80301, USA.
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INTRODUCTION |
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Fermentation and industrial strain development of Sor. cellulosum strains is challenging. Sor. cellulosum strains grow slowly, with doubling times approaching 16 h (Reichenbach, 1999). Sorangium cultures do not grow readily on agar medium when plated under a certain cell density. This behaviour results from a quorum-sensing mechanism that prevents the futile growth of individual cells, reflecting the ecological need for Sorangium strains to form communities that can effectively degrade non-soluble polymeric substrates in the soil (Reichenbach, 1999
). Plated above the threshold density, the primary micro-colonies spread and aggregate to form macroscopic swarms. As a result of this process, distinct colonies derived from single cells do not form, making the isolation and propagation of discrete genetic events difficult.
The molecular genetic manipulation of Sor. cellulosum strains is also extremely challenging. Introduction of DNA into these strains relies on low-efficiency intergeneric conjugation from Escherichia coli. Phleomycin resistance has been the only effective selection marker described in Sor. cellulosum until recent reports on the use of a hygromycin resistance marker in strains So ce56 and So ce12 (Pradella et al., 2002; Kopp et al., 2004
). No plasmids have been found to replicate in Sor. cellulosum strains, thus the stable maintenance of transferred DNA requires integration into the chromosome via homologous recombination between a cloned DNA fragment and a homologous chromosomal locus (Jaoua et al., 1992
). Resolution of the cointegrate formed as a result of homologous recombination is extremely rare, preventing the isolation of gene replacement events.
Soraphen A is an 18-membered macrolide polyketide with an unsubstituted phenyl side ring, produced by Sor. cellulosum So ce26. Soraphen A has a unique mode of action in the inhibition of fungal acetyl-CoA carboxylase (Gerth et al., 1994; Vahlensieck et al., 1994
). Due to its potent activity against plant-pathogenic fungi, soraphen A was of considerable commercial interest until it was discovered that it is a weak teratogen. The gene cluster responsible for the biosynthesis of soraphen A in Sor. cellulosum So ce26 has been cloned and sequenced (Ligon et al., 2002
; Schupp et al., 1995
). The cluster encodes two type I polyketide synthases (PKSs) that together contain a starter module and seven extension modules for the biosynthesis of the soraphen polyketide core, several enzymes that take part in the biosynthesis of the putative polyketide chain extender unit methoxymalonyl-CoA, and at least two enzymes that tailor the nascent polyketide to produce soraphen A (Ligon et al., 2002
). Type I PKSs are multifunctional enzymes with domains for substrate recognition and loading (acyl transferase, AT), substrate anchoring (acyl carrier protein, ACP), condensation (ketoacyl synthase, KS) and
-keto processing (ketoreductase, KR; dehydratase, DH; enoyl reductase, ER). These domains are organized into modules and each module is responsible for one round of polyketide chain extension using acyl-CoA substrates (Hopwood, 1997
). Apart from the common substrates malonyl- and methylmalonyl-CoA, the soraphen PKS also uses benzoyl-CoA for chain initiation and a glycolate unit, probably methoxymalonyl-CoA, during chain extension.
Heterologous expression of secondary metabolic gene clusters in surrogate hosts is emerging as a viable alternative to both classical strain and fermentation process development, and molecular biological manipulation of the native producer strain (Pfeifer & Khosla, 2001). Heterologous expression of these clusters can provide strains with better fermentation characteristics that are more amenable to further optimization, or present the only fermentation-compatible alternative as with sponge- or symbiont-derived metabolites. Surrogate hosts with well developed genetic systems and higher production levels also open up the possibility of combinatorial genetic derivatization of secondary metabolites originating from genetically intractable strains for structureactivity relationship studies. Heterologous production of type I polyketides is, however, extremely challenging because of the large size (up to 10 kDa) of the synthases, the requirement for post-translational modification of the synthases by phosphopantheteinylation, the need for the availability of the activated substrates and our limited knowledge of the regulation of the interaction of primary and secondary metabolism. Despite these difficulties, the last few years have witnessed a growing number of reports on the successful production of these metabolites in surrogate hosts. Thus, Streptomyces coelicolor and its close relative Streptomyces lividans were used to express the polyketide cores for erythromycin, oleandomycin, picromycin, megalomicin, epothilone and their genetically engineered variants (Kao et al., 1994
; Shah et al., 2000
; Tang et al., 1999
, 2000
; Volchegursky et al., 2000
; Xue et al., 1999
). 6-Methylsalicylate and 6-deoxyerythronolide B (6-dEB, the macrolide core of erythromycin) were produced in yeast (Kealey et al., 1998
), 6-dEB and yersiniabactin were manufactured in E. coli (Pfeifer et al., 2001
, 2003
) and epothilone was biosynthesized in a Myxococcus xanthus heterologous host (Julien & Shah, 2002
). Although the initial titres with these engineered strains were usually low (0·250 mg l1), further improvements in the production processes by classical microbiological or metabolic engineering methodologies are becoming increasingly feasible (Desai et al., 2002
; Lau et al., 2002
; Murli et al., 2003
).
The formidable difficulties with the fermentation, classical strain improvement and molecular genetic manipulation of soraphen production in Sor. cellulosum So ce26 made it desirable, while the characterization of the gene cluster for soraphen A production made it feasible, to investigate a heterologous expression strategy of this metabolite in a more amenable host. The downstream border of the soraphen A biosynthetic gene cluster was reported previously by our group (Schupp et al., 1995). In this study, we have established the upstream border of the soraphen biosynthetic gene cluster by insertional inactivation of sorC and orf2 in Sor. cellulosum So ce26. Next, the soraphen PKS genes sorA and sorB, together with two genes downstream of sorB, the soraphen C methyltransferase sorM and orf4 of unknown function (Ligon et al., 2002
), were cloned into Streptomyces expression vectors and integrated into the Str. lividans ZX7 chromosome. The genes upstream of sorA that were implicated in soraphen A production (sorR, sorC, sorD, sorF and sorE), were also introduced into the engineered Str. lividans strain together with a gene from Rhodopseudomonas palustris that encodes a benzoate-coenzyme A ligase (Egland et al., 1995
). The resulting strains of Str. lividans produced soraphen A when sodium benzoate or trans-cinnamic acid was supplied to the fermentations. Further feeding experiments were used to shed light on the biosynthesis of the soraphen starter unit benzoyl-CoA in the engineered Str. lividans strain.
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METHODS |
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Media and growth conditions.
E. coli strains were grown at 37 °C in Luria broth or on Luria agar with the appropriate antibiotics. Sor. cellulosum So ce26 SJ3 was grown at 30 °C on S42 agar or in G51t broth (Jaoua et al., 1992). Str. lividans ZX7 and its derivatives were grown on ISP-2 agar for sporulation, R5 agar for protoplast regeneration and YEME broth for soraphen production (Kieser et al., 2000
), supplemented with antibiotics if necessary. C. albicans was grown at 30 °C in Bacto potato glucose broth (PDB) or agar (PDA; Becton Dickinson).
DNA manipulations.
Routine cloning and transformation procedures for E. coli were as described by Sambrook & Russell (2001). PCR for cloning was performed using Pfu polymerase and, for the analysis of strains, with Herculase polymerase (Stratagene) with 10 % (final concn) dimethyl sulfoxide supplementing each reaction. Oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA, USA). Genomic DNA was isolated from Sor. cellulosum So ce26 SJ3 with the Puregene Kit (Gentra) and from Str. lividans ZX7 with DNAzol (Invitrogen). The isolation of plasmid DNA from Str. lividans ZX7 was done with Tip-100 columns (Qiagen) as described by Kieser et al. (2000)
.
Gene disruptions.
Genomic DNA of Sor. cellulosum So ce26 SJ3 was used as template for PCR reactions to amplify internal fragments of the sorC and orf2 coding regions. The SC1 primers (Table 1) that contain NotI sites were used to amplify a 1506 bp fragment internal to the coding region of sorC and cloned into pCR-Blunt II-Topo to create pTsorCko. Plasmid pTsorCko was digested with NotI and the sorC fragment was cloned into NotI-digested pCIB132 to generate pCIB132sorCko. Similarly, the O2 primers (Table 1
) were used to generate a 1995 bp fragment internal to orf2, and the amplicon was cloned into pCR-Blunt-Topo to generate pTorf2ko. The orf2 fragment was then cloned into pCIB132 to create pCIB132orf2ko as described above for pCIB132sorCko. Introduction of plasmids pCIB132sorCko and pCIB132orf2ko into Sor. cellulosum So ce26 SJ3 by conjugation and selection of transconjugants were carried out as described by Jaoua et al. (1992)
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An integrative expression plasmid that contains the genes sorB, orf4 and sorM was constructed as follows. A 546 bp fragment containing the sorB start codon to a Bst1107I site within sorB was PCR-amplified from cosmid p98/1 and cloned into PacI/PmeI-digested pNEB193 as a PacIPmeI fragment using restriction sites from the amplification primers (PS3) to yield pSorBs. This plasmid was digested with Bst1107I and SanDI (present in PS3) and ligated to a 29·8 kb Bst1107ISanDI fragment from cosmid p98/1 containing the rest of sorB, the flanking genes orf4 and sorM, and the C-terminal part of orf5. The resulting plasmid, pNEBsorB was digested with PacI/PmeI and the 30·3 kb fragment was ligated into the PacI/PmeI-digested expression vector pTBBH (I. Molnár, unpublished), yielding pSorB4M.
Two expression plasmids containing the genes upstream of sorA were constructed. sorR was PCR-amplified from cosmid pM15-5 and cloned into XbaI/NdeI-digested pNEB193 as an NdeIXbaI fragment using sites introduced by the amplification primers (PS4) to yield pSorR. A DNA fragment containing the genes sorC, sorD, sorF and sorE was amplified by PCR from cosmid pM15-5 with primers (PS5) that introduced a ribosome-binding site (AGGAGG) upstream of sorC, and was cloned into the pCR-Blunt II-Topo vector, creating pTsorCDFE. This plasmid was digested with EcoRI and SpeI (sites introduced by PS5) and the 6 kb fragment was ligated into EcoRI/SpeI-digested pSorR to yield pNEBsorRCDFE. The badA gene was PCR-amplified (PS6) from plasmid pPE202 (Egland et al., 1995) with SpeI and XbaI restriction sites as well as the ribosome-binding site (AGGAGG) incorporated into the PCR product, and was cloned into pCR-Blunt II-Topo vector, creating pTbadA. This plasmid was digested with SpeI/XbaI and the fragment was cloned into SpeI/XbaI-digested pNEBsorRCDFE to create pNEBsorRCDFE+BL. The PacIPmeI fragments of plasmids pNEBsorRCDFE and pNEBsorRCDFE+BL were cloned into the Streptomyces expression vector pTUE (I. Molnár, unpublished), yielding pSorRCDFE and pSorRCDFE+BL, respectively.
Construction of Str. lividans ZX7 strains producing soraphen.
Plasmids pSorA and pSorB4M were co-transformed into protoplasts of Str. lividans ZX7 by polyethylene-glycol-mediated transformation (Kieser et al., 2000). The transformants were selected on R5 medium with hygromycin (100 µg ml1) and kanamycin (25 µg ml1) for the two integration events. Resistant strains were analysed by Southern hybridizations (data not shown) and strains with the expected integration events were labelled SorAB. Plasmids pSorRCDFE or pSorRCDFE+BL were transformed into protoplasts of SorAB and selected with hygromycin and kanamycin for the integration events and erythromycin (200 µg ml1) for the presence of the plasmid. These strains were analysed for the structural integrity of the two integrated expression cassettes by a series of PCR reactions covering the sorA (primer pairs SA14; Table 1
) and the sorB, orf4 and sorM genes (primer pairs SB16, Table 1
), and the ends of these overlapping 36 kb amplicons were sequenced and compared to the published sequence of the soraphen cluster (accession no. U24241). The plasmid pTUE was also transformed into Str. lividans ZX7 to create strain TUE. The presence of the plasmids pTUE, pSorRCDFE or pSorRCDFE+BL was ascertained by plasmid isolation and restriction analysis. Three isolates were selected from each transformed strain and named TUE, SorABRCDFE and SorABRCDFE+BL.
Fermentation of Str. lividans strains.
To test for the production of soraphen A, spores and vegetative mycelia from about 2 cm2 patches of Str. lividans strains TUE, SorAB, SorABRCDFE and SorABRCDFE+BL were inoculated into 25 ml YEME, supplemented with 20 µg erythromycin ml1 for the plasmid-containing strains, and incubated at 30 °C with shaking at 225 r.p.m. for 2 days. Six millilitres of these starter cultures was used to inoculate 600 ml YEME supplemented with 5 µg thiostrepton ml1, or 5 µg thiostrepton ml1 and 20 µg erythromycin ml1 as applicable. The cultures were grown at 30 °C with shaking at 225 r.p.m. Some of the cultures were also supplemented with one of the following (final concn): 2·5 mM trans-cinnamic acid, 5 mM L-phenylalanine, 5 mM sodium benzoate, 5 mM phenylpyruvic acid, 2·5 mM phenylacetate or 2·5 mM benzaldehyde (all from Sigma), where each stock solution had been adjusted to pH 7·5 with sodium hydroxide. After 3 days of growth, the cultures were freeze-dried and extracted with 500 ml methanol with shaking at 100 r.p.m. overnight at room temperature. The mixtures were centrifuged at 9000 g for 1 h, the methanol phases were concentrated in a SpeedVac and the extracts were redissolved in a final volume of 10 ml methanol. Fermentations were carried out with three independent isolates of each strain and repeated twice.
Sor. cellulosum fermentations.
To analyse strains of Sor. cellulosum for the production of soraphen, 250 ml G51t media were inoculated with 25 ml 5-day-old starter cultures and incubated at 30 °C with shaking at 225 r.p.m. After 12 days, the cultures were freeze-dried and extracted with 100 ml methanol by slow mixing overnight. The mixtures were centrifuged at 9000 g for 30 min, then the methanol phases were concentrated in a SpeedVac and redissolved in 5 ml methanol. Fermentations were carried out with four independent isolates of each strain and repeated twice.
Bioassay for soraphen A production.
Ten to 50 µl from the methanol extracts was spotted onto PDA plates and allowed to dry. An overnight culture of C. albicans was used to seed soft PDA (0·7 % agar) and used to overlay the PDA plates containing the dried extracts. Plates were incubated at 30 °C for a maximum of 3 days and observed every day for the inhibition of growth of C. albicans.
HPLC and LC-MS analysis.
Extracts from strains were analysed by reverse phase HPLC and LC-MS. HPLC was performed on an HP1100 series (Agilent Technologies) fitted with a Synergy 4 micron MAX-PR 80 Å 150x4·6 mm column (Phenomenex). Mobile phase A was 0·01 % trifluoroacetic acid (TFA) in water and mobile phase B was 0·0075 % TFA in acetonitrile. Elution was performed with a gradient of 2585 % B over 15 min at 40 °C with a flow rate of 0·8 ml min1 and monitored by a diode array detector. Soraphen A was detected by its absorption at 210 nm at a retention time of 14 min.
LC analysis of extracts was carried out on a HP1100 (Agilent Technologies) with the column described above. The gradient was 2585 % methanol in water in the presence of 0·1 % sodium formate and 0·1 % formic acid with a flow rate of 0·4 ml min1. Separation was monitored by a Finnigan LCQ Classic Mass Spectrometer (ThermoFinnigan) using positive mode ESI (capillary temp, 275 °C; capillary voltage, 3·22 V; spray needle voltage, 4·54 kV).
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RESULTS |
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The genes upstream of sorA were supplied to Str. lividans SorAB on a replicative expression vector that is compatible with the pSorA and the pSorB4M integrative vectors (Fig. 1). During the course of this work, it was brought to our attention that a small ORF encoding an ACP, not reported in our previous analysis, is located between the sorD and the sorE genes (Tin-Wein Yu, personal communication). This ORF, that we named sorF, extends from bp 8930 to 9181 (on the complementary strand) in the published sequence of the soraphen cluster (U24241) and would link the transcriptionally coupled sorCD genes to the sorE gene in an apparent operon.
Thus, the sorR gene and the sorCDFE operon were placed under the control of the thiostrepton-inducible PtipA promoter in an artificial operon setting. Since benzoate is the starter unit for soraphen biosynthesis (Hill et al., 2003), but no genes for benzoate biosynthesis or activation were found clustered with the soraphen biosynthetic genes, the benzoate-coenzyme A ligase gene badA from Rhodopseudomonas palustris (Egland et al., 1995
) was also fused to this artificial operon in construct pSorRCDFE+BL. This gene has previously been shown to be necessary to supply benzoyl-CoA for the biosynthesis of phenyl-substituted lactones in a Saccharopolyspora erythraea expression system utilizing the starter module of the soraphen PKS fused to a triketide lactone synthase derived from the erythromycin PKS (Wilkinson et al., 2001
). We have inserted copies of a Streptomyces ribosome-binding site (AGGAGG) into sorRCDFE, the sorRCDFE-badA artificial operons 12 bases upstream of the start codon of sorC and also upstream of the badA gene, as described in Methods. This ribosome-binding site occurs in the PtipA promoter and is widely distributed in genes that are highly expressed in Streptomyces (Strohl, 1992
). The expression plasmids pSorRCDFE and pSorRCDFE+BL were transformed into Str. lividans SorAB to produce Str. lividans SorABRCDFE and Str. lividans SorABRCDFE+BL. To ensure that no spurious recombination events disrupted the integrated PKS genes on the Str. lividans chromosome, the sorA and sorB-orf4-sorM expression cassettes were analysed in a series of overlapping PCR reactions as described in Methods. Due to the high level of homology amongst the repeated PKS domains, Southern hybridizations using probes derived from the soraphen PKS were of limited use for this purpose.
Soraphen production with the engineered Str. lividans strains fed with benzoate
Strains of Str. lividans ZX7 carrying the expression vector pTUE, and Str. lividans SorAB, SorABRCDFE and SorABRCDFE+BL were fermented in the presence of thiostrepton to induce the expression of the sorA, and the sorB4M, sorRCDFE or sorRCDFE-badA operons. The fermentations were analysed by Candida bioassays, HPLC and LC-MS, but no trace of soraphen A or soraphen-related compounds were detected in any of the cultures (Table 2). L-Phenylalanine was shown previously to be the precursor of benzoyl-CoA used for soraphen biosynthesis by Sor. cellulosum So ce26 (Gerth et al., 2003
; Hill & Thompson, 2003
). The Str. lividans fermentations were repeated, feeding L-phenylalanine to the cultures, but again no production of soraphen could be detected. Since benzoate feeding and the benzoate-coenzyme A ligase encoded by badA were both found to be necessary for the production of phenyl-substituted lactones in engineered Sac. erythraea systems (Wilkinson et al., 2001
), we have also tried feeding benzoate to the cultures. Str. lividans SorABRCDFE+BL, but none of the other strains, produced a compound that showed antifungal activity against C. albicans (Fig. 3
). HPLC analysis revealed a small peak co-migrating with authentic soraphen A standard in extracts of SorABRCDFE+BL only, and LC-MS assays showed the presence of the appropriate molecular ion (m/z 543 for the sodium adduct) in the peak co-eluting with soraphen A at 34·8 min. While the production of soraphen A under these initial conditions was less than 0·3 mg l1, these experiments indicated that the Str. lividans host is able to produce fully processed soraphen A. The results suggested that the host does not provide an adequate supply of benzoyl-CoA for the pathway, and also that externally supplied benzoate could be channelled into soraphen biosynthesis only in the presence of a suitable coenzyme A ligase.
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DISCUSSION |
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Heterologous production of polyketide natural products or their derivatives may circumvent the need for strain optimization and fermentation development for every native production strain, some of which cannot (sponges, symbionts, unculturable strains) or have not (environmental DNA libraries) been grown in standard laboratory conditions. Further, horizontal transfer of natural product biosynthetic gene clusters into host strains with well-developed genetics should facilitate the creation of unnatural natural products by combinatorial genetics (Pfeifer & Khosla, 2001), a task that has proven especially elusive with the soraphen A producer strain Sor. cellulosum So ce26.
Biosynthetic gene clusters with type I modular PKS have been expressed in several host strains. Thus, the synthase for 6-dEB has been expressed in E. coli together with the Sfp phosphopantheteinyl transferase to yield 10 mg of the macrolide product l1. E. coli does not produce methylmalonyl-CoA, so the biosynthesis of this substrate of the PKS had to be engineered into the host, requiring a substantial number of genetic modifications (Dayem et al., 2002; Pfeifer et al., 2001
). The Sorangium-derived natural product epothilone (Molnár et al., 2000
) has been expressed in the myxobacterium host Myxococcus xanthus with an initial yield of 0·2 mg l1 (Julien & Shah, 2002
). Industrial antibiotic-producer Actinomyces strains, optimized for high production levels during fermentation, seem to be especially well suited for heterologous production of polyketides (Martin et al., 2003
; Rodriguez et al., 2003
), but these strains are often proprietary and their molecular genetic manipulation is far from trivial. Str. coelicolor, and its close relative Str. lividans, have been used much more extensively for the production of complex polyketides, including the macrocyclic core of erythromycin (Kao et al., 1994
; Ziermann & Betlach, 1999
), megalomicin (Volchegursky et al., 2000
), picromycin (Tang et al., 1999
), oleandomycin (Shah et al., 2000
) or epothilone (Tang et al., 2000
). Engineered variants of these metabolites (Tang et al., 2000
; Xue et al., 1999
), or glycosylated derivatives (Tang & McDaniel, 2001
), were also produced in Str. lividans, with the initial titre of the heterologously produced metabolites ranging from 0·2 to 50 mg l1 in the two host strains (Pfeifer & Khosla, 2001
). Both Str. lividans and Str. coelicolor are able to synthesize malonyl- and methylmalonyl-CoA that serve as substrates for these PKS. Both strains also activate type I PKS by phosphopantheteinylation, with the Str. coelicolor PPTase reported to be promiscuous in recognizing diverse synthases as its substrate (Cox et al., 2002
). Str. coelicolor also has a wide range of transporters (Bentley et al., 2002
) that allowed the secretion of heterologously produced aromatic polyketides (Pfeifer & Khosla, 2001
). Based on its close taxonomic position, Str. lividans would also be likely to offer a broad-spectrum PPTase and diverse antibiotic transport systems, and has been described to support similar levels of production of type I polyketides as an expression host to those of Str. coelicolor (Ziermann & Betlach., 1999
). The absence of a requirement for methylation-free DNA for genetic transformation of Str. lividans, however, made this strain our preferred choice for the heterologous production of soraphen. Although single-plasmid expression systems have proved useful for the production of complex polyketides, the discovery that PKS complexes readily assemble from subunits expressed from multiple, chromosomally integrated or plasmid-based constructs (Kuhstoss et al., 1996
; Ziermann & Betlach, 2000
) has simplified the assembly, transfer and later modification of these large gene clusters that often cover 30100 kb. In the present work, we have used a three-construct approach, integrating the PKS genes (together with a post-PKS methyltransferase and an ORF of unknown function) into two loci in the Str. lividans chromosome for added stability, and supplying the sorR oxidase and the methoxymalonate biosynthetic operon sorCDFE on an autonomously replicating plasmid for added convenience. All these genes were placed under the control of the thiostrepton-inducible promoter PtipA (Murakami et al., 1989
) to provide for regulated expression of the cluster during fermentation (Ali et al., 2002
; aspects under investigation in our laboratory) that is also decoupled from the developmental regulation of secondary metabolic promoters (Kyung et al., 2001
).
The yield of heterologously produced soraphen A was less than 0·3 mg l1 in these experiments, six to ten times lower than the initial reported titres with the wild-type Sor. cellulosum So ce26 strain (Gerth et al., 1994), but within the range of other type I polyketides produced in Str. lividans or Str. coelicolor (Pfeifer & Khosla, 2001
). Although several years of industrial fermentation optimization and strain development had increased the titre of So ce26 fermentations to 150 mg l1 (Gerth et al., 1994
) and later to 1 g l1 (Gerth et al., 2003
), the strain is still largely refractory to molecular genetic manipulations, and challenging for classical genetic manipulations, while its slow growth (generation time 16 h; Reichenbach, 1999
) makes industrial-scale fermentations contamination-prone and the volumetric productivity is low. Despite the low initial titres, the Str. lividans soraphen production system might offer a more attractive starting point for fermentation optimization, and microbiological and genetic strain development. Indeed, both classical fermentation optimization and metabolic engineering has been used to increase low initial production levels in heterologous polyketide production systems. Introduction of an additional methylmalonyl-CoA supply pathway increased 6-dEB production in Str. coelicolor from 40 to 200 mg l1 (Lombo et al., 2001
), while media and fermentation regimen optimization boosted the production levels to 1·5 g l1 (Desai et al., 2002
). Fermentation optimization and utilization of an absorber resin to sequester the produced polyketide allowed epothilone titres to be increased from 0·2 to 23 mg l1 in a Myxococcus xanthus heterologous production system (Julien & Shah, 2002
; Lau et al., 2002
). A concerted approach of metabolic engineering, expression system optimization and fermentation development allowed the production of g l1 quantities of 6-dEB even in E. coli (Murli et al., 2003
; Pfeifer et al., 2002
). The availability of the Str. coelicolor genome sequence (Bentley et al., 2002
), together with our increasing understanding of its transcriptome and metabolome (Avignone-Rossa et al., 2002
; Huang et al., 2001
) also raises the possibility of a rational and rapid metabolic engineering of this host and, by extension, its close relative Str. lividans. Random mutagenesis coupled with whole-genome shuffling could provide an evolutionary approach aimed at rapid optimization of strains for polyketide metabolite production (Zhang et al., 2002
).
The biosynthesis of soraphen A is initiated by AT1a in the soraphen starter module loading a benzoate unit from benzoyl-CoA onto ACP1a (Ligon et al., 2002; Wilkinson et al., 2001
). The biosynthetic origin of the benzoyl-CoA substrate in Sor. cellulosum is not clear, however. Precursor feeding studies showed the incorporation of label into soraphen from cinnamate and phenylalanine, the latter often quoted as the ultimate source for the phenyl moiety of soraphen A (Gerth et al., 2003
; Hill & Thompson, 2003
). Phenylalanine might be converted to cinnamate via a phenylalanine ammonia lyase (PAL); although these enzymes are rare in bacteria in general, a PAL is present in the myxalamid biosynthetic gene cluster of the myxobacterium Stigmatella (Silakowski et al., 2001
) and a PAL was also detected in the Sor. cellulosum So ce56 genome project (Gerth et al., 2003
), indicating that this pathway might be more prevalent in myxobacteria. Interestingly, benzoate fed to So ce26 during fermentation does not seem to be utilized for soraphen biosynthesis (Gerth et al., 2003
; Hill et al., 2003
), probably due to the lack of a suitable coenzyme A ligase.
The substrate specificity of AT1a, at least in its original module context, seems to be rather strict for benzoyl-CoA, as was shown with an engineered polyketide synthetase in Sac. erythraea. This strain produced exclusively the expected phenyl-substituted lactone upon benzoate supplementation and co-expression of an additional benzoyl CoA ligase, but failed to produce any lactones without benzoate feeding and the additional ligase (Wilkinson et al., 2001). As with Sac. erythraea, there is no evidence that Str. lividans would biosynthesize benzoyl-CoA as part of its normal metabolism. Str. lividans was shown not to degrade benzoate (although it catabolizes p-hydroxybenzoate; An et al., 2000
; Grund & Kutzner, 1998
) and neither a benzoyl-CoA ligase, nor a PAL can be detected in the Str. coelicolor genome sequence (Bentley et al., 2002
). It was thus not surprising that Str. lividans SorABRCDFE did not produce soraphen, even in fermentations where benzoate was supplied to the strain (Table 2
). Co-expressing a benzoyl-CoA ligase, the badA gene from Rhodopseudomonas palustris with the soraphen gene cluster, however, allowed the Str. lividans strain SorABRCDFE+BL to produce soraphen A when benzoate was supplied to the medium. The strict requirement for both an external source of benzoate and for an activating enzyme highlights the importance of engineering an appropriate metabolic route for benzoyl-CoA into Str. lividans if this strain is to be utilized for soraphen production.
Benzoyl-CoA was shown to be biosynthesized via several routes in different organisms (Fig. 4). None of these pathways could be shown to supply benzoyl-CoA from phenylalanine for soraphen biosynthesis in Str. lividans, as exemplified by the absence of soraphen production with all the engineered strains in fermentations without feeding or with phenylalanine supplementation (Table 2
). Still, we were interested to map out whether parts of these pathways exist in our host and could be utilized to provide the soraphen starter unit by feeding a suitable intermediate. Anaerobic
-oxidation converts phenylalanine to benzoyl-CoA via phenylpyruvate and phenylacetate in Thauera aromatica (Schneider & Fuchs, 1998
; Schneider et al., 1997
; Route 1 on Fig. 4
). This catabolic pathway seems to be lacking in Str. lividans under the tested conditions, as shown by the absence of soraphen production in all the engineered strains fed with phenylpyruvate or phenylacetate. In plants and the marine actinomycete Str. maritimus, PAL converts phenylalanine to cinnamate that is activated to cinnamoyl-CoA by a CoA ligase, and this intermediate is converted to benzoyl-CoA via
-oxidation (Route 2; Moore et al., 2002
). While the Str. lividans host does not harbour a PAL, cinnamate could be fed to both SorABRCDFE and SorABRCDFE+BL to effect soraphen biosynthesis. A cinnamate/4-coumarate CoA ligase has recently been characterized in Str. coelicolor (Kaneko et al., 2003
), so its putative Str. lividans homologue should be able to activate cinnamate and commit it to
-oxidation. Generic
-oxidation enzymes were previously shown to complement for the conversion of cinnamoyl-CoA to benzoyl-CoA in Str. maritimus mutants inactivated in dedicated benzoyl-CoA biosynthetic genes located within the enterocin cluster (Xiang & Moore, 2003
). Since
-oxidation proceeds via CoA-activated intermediates, and its last step, the thiolase-catalysed conversion of
-ketophenylpropionyl-CoA yields benzoyl-CoA directly without the involvement of a benzoate-CoA ligase (Moore et al., 2002
), the badA gene incorporated into SorABRCDFE+BL was superfluous for soraphen biosynthesis upon cinnamate feeding as shown by the production of soraphen by SorABRCDFE. A non-oxidative, retro-aldol path from cinnamoyl-CoA through benzaldehyde was also reported in plants (Route 3; Moore & Hertweck, 2002
; and ibid.). Feeding benzaldehyde to the engineered Str. lividans strains did not provide for soraphen production, so the conversion of cinnamate to benzoyl-CoA in Str. lividans appears to proceed predominantly through the
-oxidative route. As the main focus of these experiments was to establish an effective and convenient supply route for the soraphen starter unit, the stability and bioavailability of the fed precursors during fermentation were not addressed separately from their accessibility to soraphen biosynthesis.
|
The heterologous production of soraphen thus required the augmentation of the Str. lividans host with both a methoxymalonate and a benzoyl-CoA biosynthetic pathway in addition to the expression of the soraphen polyketide synthetase. The success of this approach serves as a further example for the biosynthetic utility of this genetically well characterized strain and confirms the feasibility of engineering for the lateral transfer of secondary metabolic pathways from industrially and genetically intransigent strains into fermentation- and genetic-manipulation-friendly hosts. Increasing the titre of soraphen production by the engineered Str. lividans strains via fermentation optimization, strain improvement and metabolic engineering will allow us to utilize the flexibility offered by this expression system to decipher the exact role of each gene product encoded in the soraphen gene cluster and to shed more light on the more arcane processes of soraphen A biosynthesis like the introduction of the C9-C10 double bond. Such approaches would also provide strains with better prospects for industrial-scale fermentations and open up new possibilities for the production of unnatural natural products by combinatorial biosynthesis.
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ACKNOWLEDGEMENTS |
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Received 4 March 2004;
revised 20 April 2004;
accepted 18 May 2004.
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