Precise cloning and tandem integration of large polyketide biosynthetic gene cluster using Streptomyces artificial chromosome system
© Nah et al. 2015
Received: 14 July 2015
Accepted: 27 August 2015
Published: 16 September 2015
Direct cloning combined with heterologous expression of a secondary metabolite biosynthetic gene cluster has become a useful strategy for production improvement and pathway modification of potentially valuable natural products present at minute quantities in original isolates of actinomycetes. However, precise cloning and efficient overexpression of an entire biosynthetic gene cluster remains challenging due to the ineffectiveness of current genetic systems in manipulating large-sized gene clusters for heterologous as well as homologous expression.
A versatile Escherichia coli-Streptomyces shuttle bacterial artificial chromosomal (BAC) conjugation vector, pSBAC, was used along with a cluster tandem integration approach to carry out homologous and heterologous overexpression of a large 80-kb polyketide biosynthetic pathway gene cluster of tautomycetin (TMC), which is a protein phosphatase PP1/PP2A inhibitor and T cell-specific immunosuppressant. Unique XbaI restriction sites were precisely inserted at both border regions of the TMC biosynthetic gene cluster within the chromosome of TMC-producing Streptomyces sp. CK4412, followed by site-specific recombination of pSBAC into the flanking region of the TMC gene cluster. The entire TMC gene cluster was then rescued as a single giant recombinant pSBAC by XbaI digestion of the chromosomal DNA as well as subsequent self-ligation. Next, the recombinant pSBAC construct containing the entire TMC cluster in E. coli was directly conjugated into model Streptomyces strains, resulting in rapid and enhanced TMC production. Moreover, introduction of the TMC cluster-containing pSBAC into wild-type Streptomyces sp. CK4412 as well as a recombinant S. coelicolor strain resulted in a chromosomal tandem repeat of the entire TMC cluster with 14-fold and 5.4-fold enhanced TMC productivities, respectively.
The 80-kb TMC biosynthetic gene cluster was isolated in a single integration vector, pSBAC. Introduction of TMC biosynthetic gene cluster in TMC non-producing strains has resulted in similar amount of TMC production yield. Moreover, over-expression of TMC biosynthetic gene cluster in original producing strain and recombinant S. coelicolor dramatically increased TMC production. Thus, this strategy can be employed to develop a custom overexpression scheme of entire metabolite pathway clusters present in actinomycetes.
Microbial natural products are a major resource for drug discovery and development programs mainly due to their superior structural diversity and complexity . Isolation and characterization of the natural products of biosynthetic gene clusters have accelerated our understanding of the molecular mechanisms driving natural product biosynthesis and even guided the rational redesign of natural products through biosynthetic gene manipulation . Since most microbial biosynthetic genes are clustered within chromosomes, identification of the entire biosynthetic gene cluster is relatively straightforward. Unfortunately, some of these biosynthetic genes are derived from non-culturable or not amenable to genetic manipulation microorganisms and thus do not easily express the target compounds . To bypass such intrinsic limitations and achieve functional expression of uncharacterized potentially-valuable natural product biosynthetic pathways, a relatively well-characterized heterologous host should be utilized [4, 5]. Recent genome mining approaches have also identified a significant number of metabolite biosynthetic gene clusters, some of which must be expressed in a heterologous host. Synthetic biology techniques have also made it possible to produce novel and/or improved natural products through reconstitution of biosynthetic gene clusters in an appropriate host system [3, 6–8].
Since the gene clusters responsible for the biosynthesis of microbial secondary metabolites are typically as large as 100 kb, appropriate vector systems capable of cloning an entire gene cluster as well as transferring these genetic segments between different hosts are necessary. Capturing an entire biosynthetic gene cluster in a single E. coli clone can facilitate the genetic manipulation of secondary metabolite biosynthetic pathways using the PCR-targeted gene replacement method [9–11]. Recently, a new E. coli-Streptomyces shuttle bacterial artificial chromosomal (BAC) vector system, pSBAC, which conveniently switches single-copy to high-copy replication in E. coli as well as utilizes the phage ΦBT1 attP-int site-specific integration system, was successfully used for the heterologous expression of a meridamycin (mer) biosynthesis gene cluster in S. lividans . Specifically, to clone and express the entire mer cluster, a total genomic DNA library was first constructed through ligation of EcoRI-digested pSBAC and MfeI-digested total genomic DNA, followed by intergeneric conjugation of the mer cluster containing pSBAC into S. lividans . In this case, MfeI sites were present just outside of the mer cluster, and the large DNA fragment digested in the CHEF gel was also directly ligated with the MfeI-compatible EcoRI-digested pSBAC . Therefore, it is more desirable to develop a general cloning strategy to quickly capture an entire biosynthetic gene cassette without depending on endogenous restriction sites in the cluster or a large DNA fragment ligation process.
The plasmid rescue method is a technique for cloning and identifying the region of a locus adjacent to where an exogenous DNA fragment is inserted in the chromosome [13–15]. Based on the capability of pSBAC to accommodate large DNA fragments, the recombinant pSBAC rescue method was carried out to precisely clone a large (approximately 80 kb) polyketide biosynthetic gene cluster for tautomycetin (TMC), which is a protein phosphatase PP1/PP2A inhibitor and T-cell-specific immunosuppressant [16–18]. Unique XbaI restriction sites were first inserted at both border regions of the TMC biosynthetic gene cluster within the chromosome of TMC-producing Streptomyces sp. CK4412, followed by site-specific recombination of pSBAC into the flanking region of the TMC gene cluster. Moreover, introduction of the TMC cluster-containing pSBAC into wild-type Streptomyces sp. CK4412 as well as a recombinant S. coelicolor strain resulted in a chromosomal tandem repeat of the entire TMC cluster with 40-fold enhanced TMC productivities. This strategy consisting of site-specific restriction site insertion, recombinant pSBAC plasmid rescue, intergeneric conjugation, and cluster tandem repeat introduction can be employed to develop a custom overexpression scheme of entire metabolite pathway clusters present in actinomycetes (Additional file 1: Table S1).
PCR-targeting of unique restriction enzyme sites into borders of TMC gene cluster
Precise cloning of entire TMC biosynthetic gene cluster as a single giant recombinant pSBAC
The typical cloning method for large-sized DNA fragment isolation requires extra care so as to avoid unintended DNA fragmentation. Alternatively, the in vivo plasmid rescue method can be used to isolate a particular chromosomal locus through recovery of adjacent DNA sequences [13–15]. Here, we applied the plasmid rescue technique using pSBAC in order to clone a large DNA fragment containing the TMC biosynthetic gene cluster. A 3480-bp tmcI DNA fragment containing the gene at the left end of the cluster was first cloned into attP-int deleted pSBAC plasmid (named pSATNI), followed by conjugation into Streptomyces sp. CK4412. The presence of the tmcI fragment allowed pSATNI vector to integrate into the left site of the TMC biosynthetic gene cluster as a result of targeted homologous recombination (Fig. 2c). Genomic DNA from kanamycin-resistant conjugants was isolated and digested by XbaI restriction enzyme. XbaI-digested total chromosomal DNA fractions were self-ligated, followed by transformation into E. coli cells. DNA was then isolated from the transformants and analyzed by PCR, restriction enzyme digestion, and sequencing. Analysis revealed that the entire TMC biosynthetic gene cluster was successfully cloned as a single recombinant pSBAC vector (Additional file 3: Fig. S2). Finally, the DNA fragment containing ФBT1 attP-int was re-introduced into the rescued recombinant pSBAC vector and named pMMBL101 (Fig. 2d).
Heterologous expression of TMC biosynthetic gene cluster in Streptomyces strains
Homologous or heterologous tandem integration of entire TMC cluster
Isolation and characterization of a secondary metabolite pathway gene cluster in Streptomyces species can elucidate its molecular biosynthetic and regulatory mechanisms. Here, a secondary metabolite produced by Streptomyces sp. CK4412, originally isolated from Jeju Island, Korea, was identified as an activated T cell-specific immunosuppressive compound with novel pharmacological activities in both in vivo and in vitro studies, and its chemical structure was shown to be identical to that of tautomycetin (TMC), an antifungal compound with a structurally-unique ester bond linkage between its terminal cyclic anhydride moiety and linear polyketide chain . TMC is believed to specifically block tyrosine phosphorylation of intracellular signal mediators downstream of Src tyrosine kinases in a T cell-specific manner via selective inhibition of protein phosphatase 1 (PP1) and PP2A [18, 20, 21]. However, the Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) was also recently shown to be a putative target for the immunosuppressive activity of TMC . Moreover, TMC has been reported to inhibit growth of colorectal cancer cells, implying it is a potentially-valuable natural product with multiple medically relevant functions, including anti-fungal, immunosuppressive, and anti-cancer activities. Although the entire TMC biosynthetic and regulatory pathway gene clusters have been isolated from Streptomyces sp. CK4412 and characterized, pharmacokinetic application of TMC as a novel bioactive compound remains insufficient due to lack of knowledge of how its multiple biological activities correlate to specific moieties of its structure as well as its intrinsic low-level titer observed in wild-type.
Although heterologous expression of a metabolic pathway gene cluster is an attractive alternative to conventional cell culture, cloning and stable expression of large-sized DNA have become challenges to developing a heterologous expression system for Streptomyces. A few recent reports have examined the overexpression of a secondary metabolite gene cluster in Streptomyces species. Specifically, a site-specific recombination system was engineered to catalyze tandem amplification of a 35-kb actinorhodin biosynthetic gene cluster in S. coelicolor . In this case, the oriT-like recombination sites RsA and RsB as well as site-specific relaxase gene zouA were inserted into the S. coelicolor genome flanking an actinorhodin biosynthetic gene cluster. Recombination between RsA and RsB was followed by zouA-dependent DNA amplification, resulting in an average of nine tandem repeats of the actinorhodin gene cluster per genome as well as a 20-fold increase in actinorhodin production . Unfortunately, the actinorhodin cluster in a 110-kb AUD (amplifiable units of DNA) failed to overproduce actinorhodin, implying this approach may not be applicable to overexpression of a large-sized gene cluster in Streptomyces. Another study directly cloned a gene cluster via transformation-associated recombination (TAR) in yeast as an alternative approach . Further, a 67-kb cryptic non-ribosomal peptide synthase biosynthetic gene cluster identified by genome mining of the marine actinomycete Saccharomonospora sp. CNQ-490 was cloned and expressed in S. coelicolor, resulting in production of taromycin A as a new antibiotic related to daptomycin . Although a TAR-based system might be suitable for cloning and expression of a large cryptic gene cluster screened from actinomycetes genome mining, TAR-based cloning must be performed in yeast before intergeneric conjugation into Streptomyces. In addition, a tandem repeat of the target gene cluster in a heterologous host is not applicable in a TAR-based expression system.
pSBAC was previously applied for cloning a 95-kb Streptomyces meridamycin biosynthetic gene cluster into a heterologous S. coelicolor host . Although unique restriction enzyme MfeI sites were absent inside the mer cluster, they were present just adjacent to the mer cluster in the chromosome. However, most Streptomyces secondary metabolite gene clusters, including the tmc cluster, have no unique restriction enzyme sites in their border regions, which make the pSBAC system less attractive for heterologous Streptomyces expression. To overcome the limited applicability of the pSBAC-based heterologous expression system, we developed a general method for the site-specific introduction of unique restriction enzyme XbaI sites into border regions of the tmc biosynthetic gene cluster within the Streptomyces sp. CK4412 chromosome, as illustrated in Fig. 2. Using this approach, we demonstrated that any restriction enzyme site could be introduced at any specific location of the chromosome. Cloning of a large-sized DNA fragment is challenging due to its in vitro physical instability. An attractive alternative to conventional in vitro cloning is in vivo plasmid rescue. Instead of cloning the entire tmc gene cluster into the pSBAC vector in vitro, a small DNA fragment containing the flanking region of the tmc gene cluster, tmcI, was cloned into pSBAC and underwent in vivo homologous recombination. Simple XbaI digestion of total chromosomal DNA followed by self-ligation, E. coli transformation, and apramycin selection were performed to recover the giant recombinant pSBAC vector containing the entire tmc biosynthetic gene cluster. Finally, tmc-containing pSBAC in recombinant E. coli was successfully conjugated or transformed into Streptomyces species, implying that the described pSBAC approach could be generally applied to heterologous Streptomyces expression and synthetic biology. Further, genetic modification of the cluster, including target promoter optimization or specific gene manipulation, could be easily carried out in E. coli. Another advantage of the giant recombinant pSBAC system is the ability to re-introduce the entire gene cluster into both the original host chromosome as well as single cluster-containing recombinant host, resulting in a stable tandem repeat of the entire biosynthetic gene cluster. Two copies of the tmc gene cluster exhibited a 14-fold increase in TMC production, implying this pSBAC-driven tandem repeat approach is very effective for both homologous and heterologous overexpression [25, 26]. In conclusion, the Streptomyces pSBAC-driven precise cloning and tandem integration approach described here can be applied to the versatile overexpression of a large secondary metabolite gene clusters in actinomycetes, including novel cryptic clusters identified by genome mining .
Although actinomycetes continue to be a rich source of valuable secondary metabolites, their wild-type production levels are usually too low to be useful in the field of drug development. Here, we demonstrated an attractive genetic system for the efficient homologous and heterologous overexpression of a target cluster in Streptomyces species. Site-specific chromosomal integration of unique restriction sites as well as in vivo plasmid rescue of a Streptomyces bacterial artificial chromosome vector, pSBAC, containing a large biosynthetic gene cluster were performed for precise cloning and expression of the target cluster in a surrogate Streptomyces host. Re-introduction of a giant recombinant pSBAC vector into the chromosome of the Streptomyces host containing a single copy cluster resulted in a chromosomal tandem repeat of the entire TMC cluster with significantly enhanced TMC productivities. This approach demonstrates a platform technology for the precise cloning and functional overexpression of the entire biosynthetic gene cluster of any potentially-valuable low-titer metabolite in actinomycetes.
Bacterial strains and culture media
Bacterial strains and plasmids used in this study
aacIII(IV), oriT, attP-int, backbone of pCC1BAC
Modified pSBAC which deleted attP-int and inserted Kan R and tmcI fragment
pSATNI with 85 kb DNA insert containing whole tmc gene cluster
pTMC with attP-int
pMMBL101 which replaced Apr R into Spe R
F- mcrA-D(mrr-hsdRMS-mcrBC) trfA host for cloning and amplification of various BAC vectors and constructs derived from it
E. coli host for transferring various plasmids into Streptomyces via conjugation
E. coli host for transferring various plasmids into Streptomyces via conjugation
Original TMC-producing strain
CK4412 with pSATNI and XbaI recognition sequences in both flanking region of TMC biosynthetic gene cluster
Non TMC-producing strain
TK21 with pMMBL101
Non TMC-producing strain
M145 with pMMBL101
pMMBL102-containing S. coelicolor TMC003
Insertion of unique XbaI recognition sequences in both flanking regions of tautomycetin biosynthetic gene cluster
To isolate the TMC biosynthetic gene cluster, unique XbaI recognition sequences were inserted into both flanking regions of the TMC biosynthetic gene cluster using a PCR-targeted gene disruption system . Briefly, an apramycin resistance gene (aac(3)IV)/oriT cassette and spectinomycin resistance gene (aadA)/oriT cassette were used to insert XbaI recognition sequences into both flanking regions. These cassettes were amplified from pIJ773 and pIJ778 using XbaI recognition sequence-containing primers and then introduced into E. coli BW25113/pIJ790 containing pTMC2982 or pTMC2290, resulting in pTMC2982::aac(3)IV/oriT and pTMC2290::aad/oriT, respectively. Insertion of XbaI recognition sequences was confirmed by PCR applied to mutated pTMC2982 and pTMC2290. The mutated cosmids pTMC2982::aac(3)IV/oriT and pTMC2290::aad/oriT were then introduced into Streptomyce sp. CK4412 by conjugation with E. coli ET12567/pUZ8002. Conjugation experiments were performed as described previously . Conjugation was repeated using the isolated Streptomyces sp. CK4412::aac(3)IV strains in order to insert the XbaI recognition sequence into the opposite flanking region. The desired double cross-over mutants, selected by their apramycin-resistant (or spectinomycin-resistant) and kanamycin-sensitive phenotypes, were isolated. Their genotypes were verified using PCR.
Isolation of entire tautomycetin biosynthetic gene cluster into pSBAC
To isolate the entire TMC biosynthetic gene cluster from the chromosome by XbaI digestion and ligation, attP-int containing XbaI recognition sequences was removed from pSBAC by AvrII digestion and ligation. To select the right colony, a kanamycin resistance gene was ligated into BamHI-EcoRI-digested pSBAC. To integrate the modified pSBAC into the desired location by homologous recombination, a 3480-bp DNA fragment including a part of tmcI (tmcI′) was amplified by PCR using the pTMC2982 cosmid as a template. The amplified PCR products were then ligated into a RBC T&A cloning vector. The ligated vector was completely sequenced in order to ensure its integrity (Macrogen, Korea). The tmcI’ fragment, digested using BamHI and HindIII, was cloned into modified pSBAC to generate pSATNI. Conjugation was performed to integrate pSATNI into the chromosome by homologous recombination. The desired mutant (named CK4412-2XB) was selected on kanamycin-included MS agar medium, and its genotypes were verified using PCR.
CK4412-2XB strain was cultured at 28 °C in TSB media for 2 days, and preparation of genomic DNA of CK4412-2XB was carried out using a Wizard® genomic DNA purification kit (Promega). Genomic DNA was digested by restriction enzyme XbaI, purified, and concentrated by ethanol precipitation before self-ligation using T4 ligase (TaKaRa). After desalting, the ligation mixture was used for electroporation of E. coli EPI300. Recombinant colonies were selected onto apramycin- and kanamycin-containing LB medium. Plasmids were isolated by alkali denaturation and screened by PCR using randomly selected primers within the tmc cluster to identify pTMC.
A 2-kb DNA fragment containing the attP-int of ΦBT1 was amplified by PCR using pSBAC as a template and ligated into RBC T&A cloning vector. The ligated vector was completely sequenced to ensure integrity. The attP-int, digested using AvrII, was cloned into pTMC to generate pMMBL101.
HPLC quantification and antifungal bioassay for TMC
Extraction of TMC and HPLC analysis were carried out according to previously reported methods . Briefly, culture broth was extracted twice using an equal volume of ethyl acetate, followed by concentration using a rotary evaporator. The final extracts were dissolved in methanol. Analytical HPLC was carried out on a Grace C18 4-μm column at a flow rate of 1 ml/min with UV detection at 273 nm.
Antifungal bioassay was performed for qualitative analysis of TMC production yield of tmc-containing heterologous hosts. Evaluation of TMC production was carried out by the paper disc diffusion method. Aspergillus niger stock (1 ml) was inoculated onto ME agar medium, after which extract-soaked discs were placed onto the prepared medium. The plates were incubated at 30 °C for 2 days.
Isolation of total RNA and gene expression analysis by RT-PCR
For RNA preparation, CK4412, CK4412-TMC001, S. lividans TMC002, S. coelicolor TMC003, S. coelicolor TMC004 were grown for 5 days in R5 medium, after which samples were taken at 120 h. Mycelia were harvested by centrifugation and stored in a −40 °C deep freezer after washing twice with distilled water. RNA preparation and RT-PCR were carried out according to previously reported methods . Briefly, after the frozen mycelia were broken by shearing in a mortar, total RNA was isolated by using RNeasy mini kit [QIAGEN, Germany]. DNase-I treated RNA, AVM Reverse Transcriptase XL [TaKaRa, Japan] and random hexamers was used for cDNA synthesis. HrdB gene was used as internal control. Transcripts from three biosynthetic genes such as tmcB, tmcC, and tmcJ and two regulatory genes, tmcN and tmcT, were analyzed after 30 PCR cycles. Primers used for RT-PCR were previously reported .
Replacement of apramycin-resistant gene by spectinomycin-resistant gene and introduction into tmc-containing Strains
To introduce the tmc cluster into S. coelicolor TMC003, the apramycin-resistant gene of pMMBL101 was replaced by a spectinomycin-resistant gene (named pMMBL102) using a Quick & Easy BAC modification kit (GeneBridges). The Red/ET plasmid was introduced into pMMBL101-containing E. coli EPI300, after which BAC modification was performed according to the manufacturer’s guide using PCR to amplify the spectinomycin-resistant gene in the aprR-homologous region. Transformants were selected on spectinomycin-containing LB medium and confirmed by PCR. After pMMBL102 was transformed into E. coli S17-1, it was introduced into S. coelicolor TMC003 by conjugation.
Rapid genome sequencing of Streptomyces sp. CK4412-TMC001
Genomic DNA of Streptomyces sp. CK4412-TMC001 was prepared from 3-days culture with a Wizard® Genome DNA Purification Kit (Promega). The genomic DNA was fragmented by dsDNA fragmentase to make proper size for library construction. Resulting DNA fragments was processed to Illumine Nextera DNA sample preparation kit (Illumina, Inc., USA) following manufacturer’s instruction. Final library was quantified by Bioanalyzer 2100 (Agilent, USA) and average library size was 300 bp.
The genomic library was sequenced by Illumina MiSeq (Illumina, Inc., USA). Generated paired-end sequencing reads (23,891,700 method reads) were assembled using CLC genomics workbench 6.0 (CLC bio, Denmark) and resulted in 253 contigs. The contigs and PCR-based long reads were combined through manual curation by using CodonCode Aligner 3.7.1 (CodonCode Corp., Dedham, MA, USA). The final plasmid sequence was corrected by remapping with raw reads to check errors and dubious regions.
The coding sequences (CDS) were predicted by Glimmer 3.02 . tRNA were identified by tRNA-Scan-SE , and rRNA were searched using HMMER with EzTaxon-e rRNA profiles [33, 34]. The predicted CDSs were compared to catalytic families (catFam) and NCBI COG by rpsBLAST and NCBI reference sequences (RefSeq) and SEED databases by BLASTP for functional annotation [35–38].
H-J Nah carried out experiments, analyzed the primary data and drafted the manuscript. M-W Woo participated in the production analysis in heterologous hosts. S-S Choi participated in the data analysis. E-S Kim supervised the whole research work and revised the manuscript. All authors read and approved the final manuscript.
The pSBAC vector was kindly provided from Wyeth. The authors appreciate critical reading of the manuscript by Dr. Richard Baltz. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. NRF-2014R1A2A1A11052236).
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
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