Duplication of partial spinosyn biosynthetic gene cluster in Saccharopolyspora spinosa enhances spinosyn production

Abstract

Spinosyns, the secondary metabolites produced by Saccharopolyspora spinosa, are the active ingredients in a family of insect control agents. Most of the S. spinosa genes involved in spinosyn biosynthesis are found in a contiguous c. 74-kb cluster. To increase the spinosyn production through overexpression of their biosynthetic genes, part of its gene cluster (c. 18 kb) participating in the conversion of the cyclized polyketide to spinosyn was obtained by direct cloning via Red/ET recombination rather than by constructing and screening the genomic library. The resultant plasmid pUCAmT-spn was introduced into S. spinosa CCTCC M206084 from Escherichia coli S17-1 by conjugal transfer. The subsequent single-crossover homologous recombination caused a duplication of the partial gene cluster. Integration of this plasmid enhanced production of spinosyns with a total of 388 (± 25.0) mg L⁻¹ for spinosyns A and D in the exconjugant S. spinosa trans1 compared with 100 (± 7.7) mg L⁻¹ in the parental strain. Quantitative real time polymerase chain reaction analysis of three selected genes (spnH, spnI, and spnK) confirmed the positive effect of the overexpression of these genes on the spinosyn production. This study provides a simple avenue for enhancing spinosyn production. The strategies could also be used to improve the yield of other secondary metabolites.

secondary metabolite yield, Red/ET recombination, genetic engineering, polyketide, insecticide, actinomyces

Introduction

Saccharopolyspora spinosa was originally isolated in 1982 from a soil sample collected in a Caribbean island (Mertz & Yao, 1990). Fermentation broth extracts from this strain contain a series of spinosyn factors that are highly efficient against a broad range of pests, and appear to have little or no effect on non-target insects and mammals (Sparks et al., 1998). Previous studies showed that spinosyns are derived from nine acetate and two propionate units, which produce a cyclized polyketide molecule; three carbon–carbon bonds are soon formed to obtain the tetracyclic aglycone (AGL). The rhamnose is subsequently attached and is tri-O-methylated to yield the intermediate pseudoaglycone (PSA), followed by the incorporation of forosamine sugar, giving the final spinosyns product. The most active and abundant spinosyns from S. spinosa fermentation broth are spinosyn A and spinosyn D. They differ from each other by a single methyl substituent at position 6 of the polyketide. Other factors of the spinosyn family, produced as minor components, exhibit different methylation patterns and are significantly less active (Crouse et al., 2001). A naturally occurring mixture of spinosyn A (c. 85% of spinosad) and spinosyn D (c. 15% of spinosad) is called spinosad (Waldron et al., 2001). The c. 74-kb spinosyn biosynthetic gene cluster contains 23 open reading frames (ORF) including five genes encoding a type I polyketide synthase (PKS) (spnA, B, C, D, and E); four genes involved in intramolecular C–C bond formation (spnF, J, L, and M); four genes responsible for rhamnose attachment and methylation (spnG, I, K, and H); six genes participating in forosamine biosynthesis (spnP, O, N, Q, R, and S) and four genes (ORF-L15, ORF-L16, ORF-R1, and ORF-R2) with no proven role in spinosyn biosynthesis (Waldron et al., 2001). The genes involved in rhamnose biosynthesis (gtt, gdh, epi, and kre) are not linked to this cluster (Madduri et al., 2001b).

Traditionally, improvement of secondary metabolite-producing strains is achieved by random mutagenesis and selection techniques (Parekh et al., 2000). Although these techniques have succeeded in generating many industrial strains, they are time-consuming and costly. Rational strain improvement strategies overlap with classical approaches in generating a mutant population (Adrio & Demain, 2006). Unfortunately, the regulatory mechanism for the spinosyn biosynthesis still remains unclear, so strain improvement through genetic manipulation of the regulatory network is currently impractical. Nevertheless, some reports revealed that massive amplification of antibiotic biosynthesis gene cluster is often one of the outcomes of empirical strain improvement programs. The kanamycin-overproducing strain, Streptomyces kanamyceticus 12-6 generated by classical mutagenesis possesses tandem amplification of the entire kanamycin (Km) biosynthetic gene cluster and the level of Km production is linearly co-related with the copy number of the Km biosynthetic gene cluster (Yanai et al., 2006). A penicillin-overproducing strain of Penicillium chrysogenum contains a large number of copies of penicillin biosynthetic genes (pcbAB, pcbC, and penDE) in tandem on a c. 57.9-kb DNA fragment (Fierro et al., 1995). In the industrial strain Streptomyces lincolnensis 78-11; the non-adjacent gene clusters for the production of lincomycin and melanin are duplicated (Peschke et al., 1995). These examples imply that introduction of extra copies of biosynthetic gene clusters into a wild-type strain might be an effective approach to improve the yield of the corresponding product.

However, most of the antibiotic biosynthetic genes often cluster in a contiguous region containing tens of thousands of nucleotide base pairs in the chromosome. They are consequently almost impossible to manipulate via restriction endonucleases and DNA ligases due to the frequent occurrence of cleavage sites. Reports focusing on overexpressing these biosynthetic gene clusters in parental strain through directed genetic approaches are few. The Red/ET recombination technology provides a convenient and simple method for engineering large DNA fragments in Escherichia coli. The recombineering is mediated by homologous recombination, which occurs between two DNA molecules and requires only short homology regions (c. 40–50 bp) for efficient recombination (Zhang et al., 2000). The myxochromide S (mchS, c. 30 kb) and myxothiazol (mta, c. 60 kb) gene clusters from the myxobacteria Stigmatella aurantiaca, the epothilone (epo, c. 60 kb) gene cluster from myxobacteria Sorangium cellulosum have all been successfully engineered for heterologous expression by different strategies based on Red/ET technology (Wenzel et al., 2005; Perlova et al., 2006).

The S. spinosa CCTCC M206084 isolated by our laboratory has a low capability for spinosyn production. We thus attempted to improve its spinosyn productivity through duplication of the spinosyn biosynthetic genes. It is difficult to obtain the c. 74-kb gene cluster on one single vector by one step and therefore we first directly cloned part of the spinosyn biosynthetic gene cluster (c. 18 kb) which encoded the enzymes for cross-bridging of the cyclized polyketide, for deoxysugar biosynthesis, attachment and methylation from the genomic DNA of S. spinosa CCTCC M206084 with the assistance of Red/ET recombination instead of constructing a genomic library. The resultant plasmid pUCAmT-spn was then introduced into S. spinosa CCTCC M206084 through conjugal transfer. The dosage effect of these genes on spinosyn production was investigated. Quantitative real time PCR was also performed to validate the corresponding rise in the transcript levels of these genes.

Materials and methods

Bacterial strains, plasmids, and culture conditions

Escherichia coli YZ2005 for Red/ET homologous recombination was kindly provided by Dr Youming Zhang (Genebridges GmbH, Germany). Escherichia coli S17-1 was used as the donor strain in intergeneric conjugation. The spinosad-producing strain S. spinosa CCTCC M206084 was isolated by our laboratory from the south of China. For routine use, all strains of E. coli were grown in Luria–Bertani medium at 37 °C supplemented with antibiotics as required (apramycin, Am, 50 μg mL⁻¹). Saccharopolyspora spinosa was grown in tryptic soy broth (TSB; Difco) at 30 °C. For fermentation, S. spinosa and its exconjugants were first grown for 2 days at 30 °C in the seed medium containing 1% glucose, 0.9% yeast extract, 0.2% MgSO₄·7H₂O, and 0.05% KH₂PO₄, followed by 10 days in production medium PM1 containing 0.1% KNO₃, 0.05% K₂HPO₃·3H₂O, 0.001% FeSO₄, 0.05% MgSO₄·7H₂O, 0.4% yeast, and 0.4% tryptone. To improve yield further, fermentation was performed in a modified production medium PM2 containing 6% glucose, 2% starch, 2% soybean meal, 1% fish meal, 1% corn syrup, 0.3% glutamine, 1% soybean oil, and 0.4% CaCO₃. Plasmid pSET152 was obtained from Dr Meifeng Tao (Central China Agricultural University, China) and was used as template for PCR amplifying the linear cloning vector.

Cloning by Red/ET recombination

The Red/ET recombination was performed as described previously (Zhang et al., 2000). To clone the partial spinosyn biosynthetic gene cluster (c. 18 kb) directly, a 50-μL aliquot of Red/ET-competent (ET⁺) E. coli YZ2005 cells was co-electroporated with 0.3 μg of linear cloning vector and 5 μg genomic DNA of S. spinosa CCTCC M206084 in a Bio-Rad Gene Pulser Apparatus (Bio-Rad Ltd, Richmond, CA). The linear cloning vector was amplified with primer pair P1/P2 (Supporting Information, Table S1) using pSET152 as template. Each primer P1/P2 contains a 50-bp homologous arm for the cloning of the spinosyn gene cluster. To guarantee the correction of the sequence of the homologous arms, two c. 800-bp fragments covering the homologous arms from S. spinosa CCTCC M206084 were amplified and sequenced using primer pairs P3/P4, P5/P6 designed according to the published spinosyn biosynthetic gene cluster sequence of S. spinosa NRRL 18538 (GenBank accession number: AY007564, Waldron et al., 2001). The sequencing results had 99% identities with the corresponding sequences of S. spinosa NRRL 18538. Two 50-bp regions were chosen as homologous arms. The genomic DNA was isolated according to Kieser et al. (2000) and was completely digested by Xho I (Takara, Japan) which occurs outside the c. 18-kb target genes to expose the homologous arms. After electroporation, colonies that grew under selection for the apramycin resistance were identified for the intended Red/ET recombination product by restriction analysis, PCR, and sequencing (Invitrogen, Shanghai, China).

PCR amplifications were carried out in a thermal cycler (Tgradient; Biometra, Germany) with LA Taq DNA polymerase (Takara) according to the manufacturer's protocol. The conditions were 95 °C for 10 min, followed by 30 cycles at 95 °C (30 s), 58 °C (30 s), 72 °C (150 s) and finally 72 °C 10 min. The PCR product was purified with Agarose Gel DNA Fragment Recovery Kit Ver.2.0 (Takara). PCR for verifying the resultant plasmid was carried out with primer pairs P7/P8, P9/P10, P11/P12, and P13/P14 targeted at spnK, spnG, spnF, and spnS, respectively (Table S1). The PCR conditions were similar to the above one except for the annealing temperature (63 °C) and the extension time (90s for spnK, spnG; 45s for spnF, spnS). Primer synthesis was performed at Shanghai Sangon Biological Engineering Technology & Service Co., Ltd.

Intergeneric conjugation

Intergeneric conjugation from E. coli S17-1 to S. spinosa CCTCC M206084 was carried out according to a standard procedure (Kieser et al., 2000). Escherichia coli S17-1 harbored pUCAmT-spn was served as donor strain. Genotypes of the exconjugants were confirmed by PCR amplification using apramycin resistance gene specific primer pair P15/P16 (Table S1).

Fermentation and metabolite analysis

Fermentation experiments were conducted in triplicate. Saccharopolyspora spinosa CCTCC M206084 and the exconjugants were grown in 20 mL seed medium for 2 days at 30 °C. From this seed culture, 2 mL was inoculated into 20 mL fermentation medium in a 250-mL flask, and was grown for another 10 days in a humidified shaker (Innova 4900; NBS, Edison, NJ) at 30 °C and 80% relative humidity. Final culture (0.5 mL) was mixed with 0.5 mL methanol for 12 h, followed by centrifugation at 5900 g for 10 min (Centrifuge 5415R; Eppendorf, Germany). The liquid phase (10 μL) was analyzed by high-performance liquid chromatography using a C₁₈ reverse-phase column (AQ12S05-1546WT, 150 × 4.6 mm; Waters). The column was developed at a flow rate of 1.5 mL min⁻¹ with acetonitrile–methanol–2% ammonium acetate (45 : 45 : 10, by vol.), and metabolites were monitored at a wavelength of 250 nm. According to the standard curve of spinosad, the spinosad content was calculated using the following formula: Y = 1.1956476 + 0.22728109 × X, where the X is the content of spinosad (mg L⁻¹) and Y peak area (mAU mL⁻¹). The coefficient correlation, namely R is 0.992. The identity of spinosyns was confirmed by HPLC-MS using LTQ XL mass spectrometer (ThermoFisher). The samples were eluted by methanol containing 0.1% formic acid (by vol.) with a gradient procedure starting from 50 and reaching 80% in 18 min. Data were collected over the range (m/z): 300.00–2000.00. Analysis of variance (anova) was performed on spss 16.0 (SPSS Inc., Chicago, IL) statistical software to compare the spinosad production between the exconjugants and the wild-type strain. The significance level was set at 0.05.

RNA extraction and transcription analysis

Total RNA of S. spinosa CCTCC M206084 and its exconjugant S. spinosa trans1 were separately isolated from the fifth day fermentation in broth cultured in medium PM1 using TRIzol Reagent (Invitrogen). RNA concentration and purity were determined by measuring the ratio of OD_{260 nm} to OD_{280 nm}.

The transcript levels of spnK, spnH, and spnI were assayed by two-step quantitative real-time PCR analysis with a 7500 Real-Time PCR System (Applied Biosystems). DNase treatment and cDNA synthesis were carried out using RNase-free DNase 1 (Invitrogen) and a High-capacity cDNA Archive kit (Applied Biosystems) according to each manufacturer's instructions. The real time PCR amplification was performed on the 25-μL mixture [consisting of 1 μg mL⁻¹ template cDNA, 2× Power SYBR^® Green PCR Master Mix (Applied Biosystems), and 0.4 μM forward and reverse primers] under the following conditions: 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles of 30 s at 95 °C and 1 min at 60 °C. A control (RT-minus) reaction which included all components for real time PCR except for the reverse transcriptase was always performed. Specification of PCR amplification was checked with a melting curve using an additional stage of dissociation after the final cycle, beginning at 60 °C for 30 s and then incrementally increasing the temperature until 95 °C. The data was normalized with the transcript level of principal sigma factor (sigA) (Tanaka et al., 2009) and analyzed according to 2^−ΔΔCT method (Livak & Schmittgen, 2001). Results were shown as the means of three replicate experiments. Primer pairs P17/P18, P19/P20, P21/P22, and P23/P24 were used to amplify fragments of spnH, spnK, spnI, and sigA (Table S1).

Results

Direct cloning of the partial spinosyn biosynthetic gene cluster by Red/ET recombination

As illustrated in Fig. 1, the strategy of direct cloning based on Red/ET recombination was used. The minimum linear cloning vector containing pUC replication origin, apramycin resistance gene, and oriT of RK2 and flanked by 50-bp homology arms each to the targeting molecule was directed to clone c. 18-kb spinosyn biosynthetic genes from the purified total genomic DNA of S. spinosa CCTCC M206084 in a precise, specific and faithful manner. PvuII digestion of the final constructs (designated as pUCAmT-spn) from five different transformants all matched well with the theoretical pattern via agarose gel electrophoresis (Fig. S1a, lanes 1–5). PCR products of spnG (c. 1188 bp), spnK (c. 1173 bp), the c. 524-bp fragment of spnF, and c. 576-bp fragment of spnS were successfully achieved using pUCAmT-spn as template (Fig. S1b).

1

Cloning the partial spinosyn biosynthetic gene cluster by Red/ET recombination. The linear cloning vector carrying the pUC replication origin, oriT, and an antibiotic selectable marker (Am) was PCR amplified using primers containing the homology arms at their 5′end. Total genomic DNA was precleaved using Xho I. The arrows represented ORFs and point in the direction of transcription. Am, apramycin resistance selection marker; pUC, origin of replication; oriT, oriT of RK2; h.a, homology arms.

Exconjugant identification

The resultant pUCAmT-spn was transferred into S. spinosa CCTCC M206084 through conjugation, yielding three exconjugants (designated S. spinosa trans1, trans2 and trans3). All the c. 18-kb spinosyn biosynthetic genes were integrated into the chromosome by a single-crossover homologous recombination because plasmid pUCAmT-spn lacked the integrase gene, attP site, and an origin of replication in S. spinosa. The integration was checked by PCR using vector-specific primers. PCR amplification for the apramycin resistance gene yielded a band of c. 0.7 kb, the same size as the theoretical value by agarose gel electrophoresis in the exconjugants but not in the parental strain (Fig. S2).

Qualitative and quantitative analysis of spinosyns in fermentation

Fermentation broths of S. spinosa CCTCC M206084 and three exconjugants were detected by HPLC and HPLC-MS. All samples revealed two compounds with the same retention times as those of the standard spinosyn A and spinosyn D (Fig. 2). Their identities were further confirmed by HPLC-MS, showing a measured m/z of 732.6 and 747.0 (M + H)⁺ which were consistent with the molecular formula C₄₁H₆₅NO₁₀ for spinosyn A and C₄₂H₆₇NO₁₀ for spinosyn D, respectively (Fig. S3). Three exconjugants enhanced their production of spinosad ranging from 1.90- to 2.24-fold when fermented in the production medium PM1. Fermentation in the modified production medium PM2 showed a similar trend of increased spinosad production, with S. spinosa trans1 showing the highest increase. According to the standard curve, the total concentration of spinosyns A and D of S. spinosa trans1 in production medium PM2 was 388.0 (± 25.0) mg L⁻¹, which overproduced 3.88-fold spinosad compared with 100.0 (± 7.7) mg L⁻¹ in the parental strain. Analysis of variance by spss 16.0 showed that the increases of spinosad production in the three exconjugants when compared with that of the wild-type strain were statistically significant. Furthermore, three extra peaks were observed in the chromatogram of the mutant strain but not of the wild-type strain (peaks marked with an asterisk, Fig. 2). The HPLC-MS result indicated that these peaks might have a m/z of 718.0 (M + H)⁺. As the spinosyns B, E, F, H, J, and K all had a relative molecular mass of 718.0 (Sparks et al., 2008), we speculated that they could be minor spinosyn derivatives. The exconjugants and the wild-type strain shared a comparable final biomass (data not shown), implying that higher biomass was not an overproduction mechanism.

2

HPLC analysis of the spinosyn production in Saccharopolyspora spinosa CCTCC M206084 and its exconjugant S. spinosa trans1. (a) Fermentation broth of wild-type strain S.spinosa CCTCC M206084. (b) Fermentation broth of the exconjugant S. spinosa trans1. (c) Standard spinosyn A. (d) Standard spinosyn D. Fermentation experiments were conducted in triplicate in production medium PM2. Peaks marked with an asterisk are assumed to be three minor spinosyn derivatives.

Quantitative real time PCR analysis of selected genes

Saccharopolyspora spinosa trans1, which had the highest increase in spinosad production among the three exconjugants, was chosen to further assess gene dose effect on increasing the enzyme production. According to the time course for spinosad production of the parental strain and S. spinosa trans1 in production medium PM1 (Fig. S4), the total RNA was extracted from the fifth day fermentation culture for RT-PCR analysis. spnK, spnI and spnH were selected from three different transcript units. The transcript level of the gene fragment of sigA served as a control in this study. The transcript levels of spnK, spnH, and spnI in recombinant strain trans1 were 3.203-, 3.524- and 3.495-fold higher than those in the parental strain, respectively (Fig. 3). The increase in transcript levels for spnK, spnI, and spnH agreed with the high yield of spinosad in the exconjugants.

3

Quantitative real time PCR analysis of the transcript levels of spnK, spnH, and spnI in Saccharopolyspora spinosa CCTCC M206084 and exconjugant S.spinosa trans1. The total RNA was extracted from the fifth day culture in production medium PM1. spnK, spnH, and spnI from three different transcript units were used as the representative genes of the c. 18 kb spinosyn biosynthetic genes. sigA was used as a control. Results are shown as the means with error bars from three replicate experiments. The red columns represent the transcript levels of the genes in the exconjugants S. spinosa trans1; the green columns represent those in the wild-type strain S. spinosa CCTCC M206084.

Genetic stability of the exconjugants

Exconjugants of S. spinosa CCTCC M206084 were passaged in the absence of selection in TSB for 16 culture doublings (Matsushima et al., 1994), and then plated on brain heart infusion broth (BHI; Difco) with Am (50 μg mL⁻¹) and without Am. The exconjugants formed colonies on BHI agar plus Am (50 μg mL⁻¹) at 90% efficiencies compared to the plating efficiencies on BHI agar lacking Am, which implied that the insertion of the biosynthetic genes could yield relatively stable recombinants that do not require antibiotic selection for maintaining their inserts.

Discussion

Duplication of biosynthetic genes to increase the yield of corresponding secondary metabolite is a practicable and successful approach. The introduction of cosmid pML48 containing partial compactin gene cluster into Penicillium citrinum 41520 enhanced compactin production (Abe et al., 2002). A large increase in nikkomycin production was obtained when an extra nikkomycin biosynthetic gene cluster was integrated into the genomic of Saccharopolyspora ansochromogenes (Liao et al., 2010). Partial duplication of the moenomycin cluster in Saccharopolyspora ghanaensis also increased average moenomycin production (Makitrynskyy et al., 2010). In these cases, constructing and screening the BAC or cosmid library was the routine method for obtaining the biosynthetic gene cluster, which is time- and labor-consuming. In our study, the strategy of direct cloning based on Red/ET technology was applied to obtain the spinosyn biosynthetic gene cluster from the genomic DNA of S. spinosa, which is simple and convenient. This straightforward technique is particularly suitable for large DNA molecules and is therefore ideal for engineering PKS and non-ribosomal peptide synthetase pathways.

The spinosyn-producing microorganism, S. spinosa, has been shown to be recalcitrant to genetic manipulation and gene transfer processes (Matsushima et al., 1994). A plasmid containing a large fragment of S. spinosa DNA can integrate at high frequencies into the S. spinosa chromosome apparently by homologous recombination, whereas a plasmid containing a small sequence (c. 2 kb) of S. spinosa DNA integrated at low frequencies into the S. spinosa chromosome at one of two bacteriophage φC31 attB sites (Matsushima et al., 1994). Our previous experiments also showed low frequencies when the integrative vector pSET152 was used for conjugation from E. coli S17-1 to S. spinosa. Therefore, we only amplified the pUC replication origin, apramycin resistance gene, and oriT of RK2 from this plasmid as the linear cloning vector. The c. 18-kb spinosyn genes in plasmid pUCAmT-spn served as the homologous sequence and guided a single-crossover homologous recombination to generate stable, apramycin-resistant exconjugants with all the genes duplicated.

HPLC results showed that the yield of spinosyns A and D was significantly greater in the exconjugants than in the parental strain. The exconjugants also produced three more substances which might be the minor spinosyn components. As previously described, during the early part of a spinosyn fermentation, S. spinosa accumulates PSAs, the intermediates that lack forosamine, which indicates that the biosynthesis or attachment of forosamine can be limiting under certain conditions (Madduri et al., 2001a). Furthermore, it was formerly shown that a two- to fourfold increase in spinosad yield was attained through duplication of the rhamnose biosynthetic genes gtt and gdh simultaneously, which implied that an extra copy of the genes involved in the conversion of the cyclized polyketide to spinosyn could enhance the spinosyn yield (Madduri et al., 2001b). Thus, we hypothesized that the overexpression of the genes for cross-bridging of the polyketide, for deoxysugar biosynthesis, attachment, and methylation could increase the flux through the pathway and accelerate the conversion of cyclized polyketide to spinosyns. The quicken conversion could lead to a faster deprivation of the cyclized polyketide and thus might stimulate the strain to synthesize more cyclized polyketide for conversion into fully glycosylated spinosyns. Interestingly, the introduction of the cosmid pRHB9A6 containing all the genes on the spinosyn gene cluster but not the PKS genes in S. spinosa A83543.3 accelerated the conversion of the PSAs, but did not enhance the spinosyn production (Madduri et al., 2001a). This may be due to the different genetic background between the two parental strains, or the negative effect of ORF-L15 and ORF-L16. The culture conditions could also be a cause.

In a more recently published report (Pan et al., 2011), the gtt and gdh genes were further overexpressed by PermE* promoter in S. spinosa SIPI-A2090, leading to a 3.81-fold increase in spinosad production. In our study, a 3.88-fold increase was achieved merely by duplicating the 14 genes involved in the conversion of the cyclized polyketide to spinosyns, which indicated that an overall rise in the expression level of the spinosyn biosynthetic genes could be more effective in enhancing the spinosyn production, and our exconjugants have the potential for further improvements in production. As the Red/ET recombination is more convenient in genetic manipulating, and the integration frequency augments with the length of the homologous sequence, our strategy may be simpler and possess higher frequencies.

The corresponding antibiotic might be indispensable during the fermentation of genetically engineered high-producing strains which contain a self-replicating plasmid or cosmid. In this work, the pUCAmT-spn was integrated into the chromosome by single-crossover homologous recombination, and the insertion of cloned DNA into the chromosome did not cause observable instability or loss of product yield in most cases, which provided an alternative to obtain a stable engineered strain.

In conclusion, the Red/ET approach was applied to clone part of the spinosyn biosynthetic gene cluster from the genomic DNA of S. spinosa, allowing the generation of a stable spinosyn overproducer. Our investigation indicated that introduction of an extra copy of the biosynthetic gene cluster into the parental strain could be a simple and widely applicable approach for enhancing the corresponding product. The method in this study could also provide a feasible strategy for duplicating the five large spinosyn genes encoding the type I PKS and the four rhamnose biosynthetic genes in S. spinosa for increasing spinosyn production.

Acknowledgements

We wish to thank Prof. Mark Goettel (Lethbridge Research Centre of Agriculture & Agri-Food Canada) for revising the manuscript. This investigation was supported by National Natural Science Foundation of China (30870064, 30970066), National High Technology Research and Development Project (863) of China (NC2010GA0091), and Key Project of Hunan Provincial Science & Technology Department (2010FJ2002).

Supporting Information

Fig. S1. Restriction digestion (a) and PCR analysis (b) of plasmid pUCAmT-spn.

Fig. S2. PCR analysis of the integration of pUCAmT-spn into the chromosome of S. spinosa CCTCC M206084.

Fig. S3. Mass spectra of spinosyns from S. spinosa CCTCC M206084 and exconjugant S. spinosa trans1.

Fig. S4. Time course for spinosad accumulation by S. spinosa CCTCC M206084 and the exconjugant S. spinosa trans1 in production medium PM1.

Table S1. Sequences of the primers used in this article.

Author notes

Editor: Andre Klier