Construction of over-expression shuttle vectors in Streptomyces

Abstract

Two high-copy number vectors, pL97 and pL98, that contain the strong constitutive ermEp* from Saccharopolyspora erythraea and the ssrA promoter (ssrAp) from Streptomyces coelicolor, respectively, and an inducible high-copy vector, pL99, with the PnitA-NitR system from Rhodococcus rhodochrous, were constructed based on the pIJ101 replicon. These vectors have pUC18 and pIJ101 replication origins for high-copy plasmid number in Escherichia coli and Streptomyces, respectively, and the oriT (RK2) allows the efficient and convenient plasmid transfer from E. coli to Streptomyces. The transformants can be easily selected with apramycin. There is also a multiple cloning site (MCS) between promoter and terminator convenient for heterologous gene cloning. The enhanced green fluorescent protein (EGFP) gene and redD, a pathway-specific regulatory gene for the production of undecylprodigiosin in S. coelicolor, were inserted into these plasmids and could be over-expressed in S. coelicolor. Western blotting revealed that the expression of EGFP was dramatically increased compared to the cell with a single copy of EGFP. Furthermore, the production of undecylprodigiosin was also greatly enhanced in the cells constitutively over-expressing redD. Hence, the high-copy plasmids could be readily used to express foreign proteins and for production of secondary metabolites, especially the antibiotics, potentially for industrial applications.

Introduction

Streptomyces are Gram-positive, soil-dwelling microorganisms, which can produce many kinds of antibiotics, enzymes, immuno-suppressives, etc., with commercial and academic value. Moreover, Streptomyces have also been developed as the hosts for the expression of heterogonous proteins, which might aggregate in the inclusion bodies of E. coli.

In order to maximize the productivity of Streptomyces, an easy and routine strategy is to over-express some pivotal genes in multiple-copy plasmids under strong promoters (Ann and van Mellaert 1993). pIJ101, a wide host range and multiple-copy plasmid from Streptomyces lividans ISP5434, can reach up to 100–200 copies per chromosome or 30% of the total DNA of a cell (Kieser et al. 1982, 2000). Several pIJ101 replicon-based over-expression vectors have previously been constructed, such as pIJ6021, pIJ4123 (Takano et al. 1995), and pHZ1272 (Yang et al. 1998), which all contain thiostrepton-inducible tipAp but do not have oriT. Recently, pTONA5 was reported as another E. coli–Streptomyces shuttle plasmid with a metalloendopeptidase promoter for hyperexpression of foreign proteins (Hatanaka et al. 2008).

ermEp*, a mutant promoter originating from erythromycin resistant gene of Saccharopolyspora erythraea, is a strong and constitutive promoter widely used in Streptomyces (Bibb et al. 1985; Janssen et al. 1989; Schmitt-John and Engels 1992). The ssrA gene, encoding the transfer-messenger RNA (tmRNA) for protein turn-over (Withey and Friedman 2003; Yang and Glover 2009), is also strongly expressed in Streptomyces, though a condition-dependent expression profile was observed (Paleckova et al. 2006, 2007; Yang and Glover 2009). Though this is a convenient method, there is the disadvantage that constitutively expressed proteins or secondary metabolites might be nocuous to cell growth, and have a severe effect on the production of the favored products. Accordingly, the introduction of an inducible expression system may be a preferable way. PnitA-NitR system has been demonstrated to be a powerful inducible expression system used in mycobacteria based on the positive feedback regulation of nitAp activity (Pandey et al. 2009). The expression of nitA, encoding nitrilase in Rhodococcus rhodochrous J1, is regulated by a positive regulator nitR. It has been reported that NitR coupling with inducer (ε-caprolactam) could bind to the specific site of the nitA promoter region to activate the expression of nitA (Komeda et al. 1996a, b).

In this paper, we present three multiple-copy plasmids in Streptomyces based on pIJ8668 backbone and pIJ101 replicon, two with strong and constitutive promoters and one with inducible system. Moreover, the egfp and redD were highly expressed in S. coelicolor to demonstrate the feasibility of these three over-expression vectors.

Materials and methods

Bacterial strains, plasmids and media

Escherichia coli TG1 was used as the host for plasmid construction and E. coli ET12567 (MacNeil et al. 1992) with the helper plasmid pUZ8002 was used for plasmid introduction into S. coelicolor by conjugation according to procedures previously described (Kieser et al. 2000). Escherichia coli strains were cultured in liquid or solid Luria–Bertani (LB) medium containing appropriate antibiotics at 37°C for plasmid propagation (Sambrook et al. 2000). Streptomyces coelicolor strains in this study are listed in Table 1. Wild-type strain M145 and its derivatives were maintained at 30°C on MS or R5 medium or in yeast extract–malt extract (YEME) liquid medium according to the manual (Kieser et al. 2000).

Table 1 Streptomyces strains used in this study

Plasmid construction

All primers and plasmids used in this study are listed in Tables 2 and 3, respectively. Promoter ermEp* was amplified by PCR using vector pLM1 (Mao et al. 2009a) as a template with primers 1 and 2, and then subcloned into pTA2 (Toyobo). Then, the ermEp* (0.2 kb) was obtained by BamHI digestion and ligated into the BglII site of pIJ8668 (5.0 kb) (Sun et al. 1999) resulting in the 5.2 kb of pIJ8668-ermEp. Subsequently, two synthetic oligonucleotides (primer 7 and 8) containing the multiple cloning sites (MCS) were heat denatured in buffer A (50 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA) for 5 min and cooled down slowly to room temperature for renaturation. Then, the annealed double helix replaced the EGFP gene in pIJ8668-ermEp after digestion with NdeI-NotI to generate pIJ8668-ermEp-MCS. In parallel, both pIJ8600 (Sun et al. 1999) and pHZ1272 (Yang et al. 1998) were digested with XbaI-ClaI to get 6.3 kb of vector from pIJ8600 and 3.2 kb of pIJ101 replicon from pHZ1272, respectively, which were ligated for plasmid pIJ8600-101 ori (9.5 kb). Then, the resultant vector was digested with BamHI-BglII and self-ligated to create pIJ8600-101 ori-B/B. Finally, the 4.0-kb EcoRI-HindIII pIJ101 replicon-tfd from pIJ8600-101 ori-B/B was inserted into EcoRI-HindIII site of pIJ8668-ernEp-MCS to generate plasmid pL97. The construction of pL98 (pIJ8668-101 ori-ssrAp-MCS) is the same as described above except that S. coelicolor M145 genomic DNA served as a template for ssrAp amplification with primers 3 and 4.

Table 2 Primers used in this study

Table 3 Plasmids used in this study

The synthetic PnitA-NitR system (Shanghai Qinglan Biotech) cloned in pUC19 was digested with SphI-XbaI and then inserted into SphI-XbaI site of pIJ8600-101 ori to create pL99 (pIJ8600-101 ori-nit). The sequence of PnitA-nitR system was synthesized according to pNIT-1 (Genbank accession number FJ173069) (Pandey et al. 2009) with codon optimization for nitR according to Streptomyces codon usage (Kieser et al. 2000). The optimized sequence has been deposited in GenBank (accession number JN636796).

The KpnI-EcoRI egfp gene fragment from pIJ8668 was inserted into the KpnI-EcoRI site of pTA2 (Toyobo) generating pTA2-egfp. Then, the NdeI–BamHI egfp from pTA2-egfp was inserted into the three high-copy plasmids digested with NdeI-BglII, generating pL100, pL101, and pL102, respectively.

redD was amplified using primers 5 and 6, and cloned into pTA2 (Toyobo) after addition of dA. Then, the redD fragment digested by NdeI-BglII from pTA2-redD was inserted into pL97 and pL98 to generate pL103 and pL104, respectively.

SDS-PAGE and western blot analysis

Streptomyces coelicolor mycelia were collected after inoculation of spores in YEME liquid medium for the indicated time and resuspended in lysis buffer as described previously (Mao et al. 2009b). About 20 μg of total protein were separated in 12% SDS-PAGE followed by western blot with rabbit polyclonal anti-EGFP antibody (Proteintech, USA).

Antibiotic (undecylprodigiosin) production assay

For phenotype analysis, about 10¹⁰ spores were streaked on the R5 plate, incubated at 30°C for 30 h, and photographed at the bottom. Quantitative analysis of Red production has been previously described (Kieser et al. 2000; Mao et al. 2009b). Briefly, about 10 mg of mycelia collected from R5 medium overlaid with cellophanes at different developmental stages was extracted with acidified methanol overnight before vacuum freeze-drying, and the A₅₃₀ of the supernatants was determined after centrifugation at 5,000  g for 5 min.

Results and discussion

Construction of novel multiple-copy vectors pL97, pL98 and pL99

Fragments harboring three different promoters, transcriptional terminators, selection markers, and the replicons for plasmid propagation were combined for a series of novel high-expression vectors (Figs. 1 and 2). The pIJ101 replicon required for the high copy plasmid number maintenance in S. lividans ISP5434 served as a replication element in Streptomyces (Kieser et al. 1982) and pUC18 ori was the replication origin in E. coli. The three plasmids also contained oriT origin for inter-species DNA transfer, which allowed convenient mobilization of shuttle–plasmid from E. coli to Streptomyces. The difference between the three vectors was mainly based on the strong promoters, among which ermEp* and ssrAp were constitutive while nitAp in PnitA-NitR system was inducible with the inducer ε-caprolactam. Additionally, the multiple cloning site (MCS) flanked by the strong promoter and transcriptional terminators tfd, which represented a major transcription terminator of bacteriophage fd, also allowed the convenient cloning of foreign genes.

Fig. 1

Maps of high-copy number vectors pL97 (a) and pL98 (b). Unique restriction endonuclease sites of the plasmids are in bold

Fig. 2

Map of inducible high-copy number plasmid pL99. Unique restriction sites are indicated in bold

Over-expression of EGFP in S. coelicolor

To evaluate the applicability of these vectors, the endogenous egfp gene from pIJ8668 (Sun et al. 1999) was cloned in these plasmids and expressed in S. coelicolor. The cell-free extract analysis by western blot revealed that the EGFP protein was expressed much more abundant in high-copy plasmid under ermEp* in comparison with the integrative single-copy plasmid (Fig. 3, lane 2 and 3). However, though much higher than that in the single copy plasmid (Fig. 3, lane 2 and 4), EGFP production under ssrAp was found to be slightly lower than that under ermEp* (Fig. 3, lane 3 and 4), suggesting that, though ssrAp was comparably a strong promoter, the foreign gene was transcribed more efficiently under ermEp*. Moreover, dramatically increased production of EGFP protein was observed when the inducer (ε-caprolactam) was added in the PnitA-NitR inducible system in multiple-copy plasmid, and 0.1% ε-caprolactam was sufficient for the full induction of PnitA-NitR system after 1 day (Fig. 3, lane 7–9). In addition, the PnitA-NitR system presented the highest expression level among the three promoters (Fig. 3, lane 3 and 4 and 8 and 9). However, the basal leaky expression was also observed without inducer (Fig. 3, lane 7), which might be caused by transcriptional read-through resulting from the inefficient synthetic short terminator.

Fig. 3

Over-expression of EGFP. Strain L48, L49, and L50 were cultured in a 250-ml baffled flask containing 50 ml of YEME medium supplemented with 50 μg/ml apramycin and shaked at 210 rpm at 30°C to exponential phase. As for the PnitA-NitR inducible system, ε-caprolactam was added to the medium (final concentrations 0.1 and 1%) when cells grew to pre-exponential phase, and then further cultivation was carried out at 30°C for 24 h. Streptomyces coelicolor wide-type strain M145 without plasmid or with integrated plasmid pLM1 was cultured at the same condition. The cell extract was prepared and subjected to western blot with anti-EGFP antibody

Over-production of undecylprodigiosin by over-expression of redD in S. coelicolor

Streptomyces are the major producer of secondary metabolites including antibiotics and immunosuppressives, etc., especially for clinical applications. Then, the antibiotic undecylprodigiosin (Red) production from S. coelicolor was tested as a model for the production capacity of secondary metabolites after the positive regulator was over-expressed in these high-copy plasmids.

redD, the gene encoding the pathway specific transcription activator for Red production in S. coelicolor (Takano et al. 1992), was over-expressed in the multiple-copy vectors under ermEp* and ssrAp in strains L51 and L52, respectively, and L49 with ssrAp-egfp served as a control (Fig. 4). It was observed that after 30 h on the R5 plate, the lawn of cells over-expressing redD was much redder than the negative control, which only had the endogenously expressed redD (Fig. 4a). Consistent with the phenotypical result, quantitative measurement also showed that over-expression of redD in our constitutive multiple-copy plasmids could remarkably enhance the Red production at all developmental stages. Especially after 72 h, both strain L51 (redD under ermEp*) and L52 (redD under ssrAp) showed about 40 times higher than the control strain L49 (egfp under ssrAp) (Fig. 4b).

Fig. 4

Undecylprodigiosin production assay. a L49, L51, and L52 were streaked on R5 medium, incubated at 30°C for 30 h and photographed from the bottom of the plate. b Quantitative measurement of Red production. The ratios of absorbance at A₅₃₀ to mycelium dry weight of strains L51 (●), L52 (■), and L49 (▲) were calculated, respectively, and numbers in the graphs are the mean values of three independent experiments; bars standard deviations

One of the most effective strategies to improve the production of a particular protein or secondary metabolite in Streptomyces is to over-express the gene, the synthesis gene cluster, or the positive regulatory gene for this gene/gene cluster. In this paper, we constructed three high-copy expression vectors derived from pIJ8668 and pIJ101 to enlarge options for the choice of expression plasmids. The plasmids in our report were all E. coli–Streptomyces shuttle vectors, and the oriT in these vectors made the plasmid introduction into Streptomyces via conjugation very efficient and convenient, which was quite different from the classical pIJ101-derived vectors pIJ6021 and pIJ4123 (Takano et al. 1995). Moreover, the strong constitutive promoters ermEp* and ssrAp made it possible to permanently highly express foreign genes. However, especially for the expression of toxic compounds that would greatly affect the physiological processes of Streptomyces, the inducible system could be one of the best choices. After reaching the desired cell growth stage, the Streptomyces cells could be induced to express a large amount of toxic components in a comparably short time. The extensively used high-copy plasmids pIJ6021, pIJ4123, and pHZ1272 all contain the strong inducible promoter tipAp, whose activity could be induced by thiostrepton (Takano et al. 1995). However, thiostrepton is expensive, which limited it to the research area, and it could not be applied in the industrial field. In comparison, the inducer ε-caprolactam in PnitA-NitR system is quite cheap, and also the high expression level makes it possible for industrial production.

In this study, we presented two constitutive and one inducible high-expression plasmids based on pIJ101 replicon. These vectors could therefore be used to express heterogeneous genes in a high expression level, for the expression of biologically active foreign proteins that would be in the inclusion bodies of E. coli in a denatured form, and also some positive regulators that control the synthesis of secondary metabolites, such as antibiotics.