The screening and development of Streptomyces natural products (NPs) as antibiotics, antifungals, antivirals, immunosuppressants, and anti-cancer drugs have played essential roles in human medicine for the past several decades [1-5]. Recent post-genomic approaches have stimulated the development of microbial genome mining to identify previously unsuspected and low-titer NP biosynthetic gene clusters (BGCs) [6-9]. The heterologous expression of Streptomyces NP BGC has become an attractive method for the titer improvement and pathway engineering of a range of potentially valuable Streptomyces NPs. Because the typical size of Streptomyces NP BGC is usually larger than 20 kb (sometimes over 100 kb), a range of strategies, such as a transformation-associated recombination (TAR) system, integrase-mediated recombination (IR) system, and plasmid Streptomyces bacterial artificial chromosome (pSBAC) system, have been developed to isolate large-sized Streptomyces NP BGCs [10-13].

The pSBAC system is an efficient tool for Streptomyces heterologous expression with site-specific restriction site insertion, recombinant pSBAC in vivo rescue, and E. coli-Streptomyces intergeneric conjugation [13]. Previously, meridamycin (MER, ~95 kb), tautomycetin (TMC, ~80 kb), and pikromycin (PIK, ~60 kb) BGCs were isolated success-fully as a single giant recombinant plasmid using the pSBAC system and those BGCs were expressed functionally in several Streptomyces cell factories, such as S. lividans and S. coelicolor [12-14]. These results showed that the pSBAC system can be an effective platform technology for the precise cloning and functional overexpression of large-sized BGC of any potentially valuable low-titer metabolite in Streptomyces and its physiologically related actinomycetes. Because all three BGCs previously cloned in the pSBAC system belong to the polyketide synthase (PKS) family, it could be argued whether a different structural family BGCs, such as non-ribosomal peptide synthase (NRPS), are also feasible for pSBAC-driven heterologous expression.

Daptomycin is an FDA-approved highly-valuable antibiotic exhibiting strong bioactivity against Gram-positive pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), vancomycin-resistant enterococci (VRE), and other antibiotic resistant strains [15-17]. Daptomycin belongs to A21978C family of lipopeptides containing thirteen amino acids produced by S. roseosporus [18]. The three major components, A21989C_1-3, have 11-, 12- or 13-carbon branched-chain fatty acids, respectively, attached to the terminal amino group of L-Trp [18]. After the fatty acid side chains of A21978C are removed by incubation with Actinoplanes utahensis or with the A. utahensis deacylase produced by recombinant S. lividans, daptomycin can be produced by reacylation of the cyclic peptide with n-decanoyl fatty acid [19]. Alternatively, for daptomycin production biosynthetically, decanoic acid can be supplied to cultures of S. roseosporus to allow the direct incorporation of the straight chain supplement [20].

Previously, the heterologous expression of daptomycin BGC was performed to improve the production and generate novel analogues. A 128-kb fragment and a 157-kb fragment, including 65-kb daptomycin BGC, were isolated by the construction of a BAC library and IR system, respectively [21]. Because these clones contained huge extra sequences, which are not related to daptomycin production, it might impart some genetic burden to the heterologous host. Here, the exact daptomycin BGC was isolated precisely using the pSBAC system and the recombinant pSBAC was introduced into a model surrogate host, S. coelicolor M511 [22]. This was the first example of NRPS BGC to be cloned into the pSBAC-driven heterologous expression system. The daptomycin and its derivatives were produced successfully in the heterologous host, and their levels of production varied according to decanoic acid feeding into the culture media.

Bacterial Strains and Culture Media

Table 1 lists the bacterial strains and plasmids used in this study. E. coli strains were cultured in Luria-Bertani (LB) broth or agar supplemented with the appropriate antibiotics at 37°C. Spores and hyphal fragments of S. roseosporus ATCC31568 were grown on DA1 agar medium (0.4% glucose, 1% malt extract, 0.4%yeast extract, 0.2% CaCO₃, and 1.5% agar) for five days at 30°C [23]. The MS agar medium was used for the sporulation of S. coelicolor. For the production of daptomycin and its derivatives, S. coelicolor were grown in TSB media for two days and cultured for five days in R5 media at 30°C. Conjugation was carried out on a modified ISP4 medium.

Table 1 . Bacterial strains and plasmids used in this study..

Strain/plasmid	Relevant characteristics	Source/reference
Plasmid
pATTP	attP-intΦC31-containing pGEM-T easy vector	This work
pSBAC	accIII(IV), oriT, attP-int, backbone of pCC1BAC	[10]
pSA	Modified pSBAC which deleted attP-int	[11]
pSAMD	Modified pSBAC with deleted attP-int and inserted dptPfragment and NheI site	This work
pDPT	pSAMD with 70kb DNA insert containing whole dpt gene cluster	This work
pDPT001	pDPT with attP-intΦC31	This work
E. coli
EPI300	F-mrc-A-D (mrr-hsdRMS-mcrBC) trfA host for cloning and amplification of various BAC vectors and constructs derived from it	Epicenter
S17-1	E. colihost for transferring various plasmids into Streptomycesvia conjugation
ET12567/pUZ8002	E. colihost for transferring various plasmids into Streptomycesvia conjugation
Streptomyces roseosporus
ATCC31568	Original daptomycin-producing strain	[18]
Streptomyces coelicolor
M511	ΔactII-orf4-deleted M145, Non daptomycin-producing strain	[20]
DPT101	M511 with pDPT001	This work

Insertion of Recombinant pSBAC in Vicinity of Daptomycin Biosynthetic Gene Cluster

attP-int_ΦBT1 was removed from pSBAC by AvrII digestion and ligation (named pSA) to isolate the daptomycin biosynthetic gene cluster from the chromosome by NheI digestion and ligation. To integrate the pSA into the vicinity of dptP by homologous recombination, a 1,720-bp DNA fragment (HR fragment) was amplified by PCR using the genomic DNA of the S. roseosporus ATCC31568 wild-type strain as a template. The amplified PCR products were analyzed by electrophoresis in 1% (w/v) agarose gel and purified using a DNA extraction kit. The amplified PCR products were then ligated into RBC T&A cloning vector (Real Biotech Co., Taiwan). The ligated vector was sequenced completely to confirm its integrity (Macrogen, Korea). The HR fragment, which was digested using EcoRI and HindIII, was cloned into pSA to generate pSAMD. The pSAMD was then introduced into E. coli S17-1 and conjugated directly with S. roseosporus ATCC31568 by homologous recombination. The desired mutant was selected on apramycin-included DA1 agar medium, and its genotypes were verified by PCR.

Isolation of the Entire Daptomycin Biosynthetic Gene Cluster into pSBAC

To isolate the entire daptomycin biosynthetic gene cluster, the pSAMD containing the S. roseosporus strain was cultured in TSB media for 1 day at 30°C, and the genomic DNA of the mutant was prepared using a Wizard genomic DNA purification kit (Promega, USA). The genomic DNA was digested using the restriction enzyme, NheI, purified, and concentrated by ethanol precipitation before self-ligation using T4 ligase (TaKaRa, Japan). After desalting, the ligation mixture was used for the electroporation of E. coli EPI300. Recombinant colonies were selected on an apramycin-containing LB medium. The plasmids were isolated using a TIANprep Mini Plasmid Kit (Tiangen, China), and screened by PCR using DptM, DptA, DptA-B-C, DptD, DptI, and DptRI check primers in daptomycin cluster to identify pDPT.

A 1,989-bp DNA fragment including the attP-int_ΦC31 and AvrII was amplified by PCR using the pSET152 vector as a template. The amplified PCR products were analyzed by electrophoresis in 1% (w/v) agarose gel, purified using a DNA extraction kit, and then ligated into a pGEM-T Easy vector (named pATTP). The pATTP vector was completely sequenced to ensure integrity (Macrogen, Korea). The attP-int_ΦC31 was digested by AvrII and cloned into pDPT to generate pDPT001.

Production and Analysis of Daptomycin and Its Derivatives

Spores of S. coelicolor M511 and its mutants prepared from MS medium were inoculated into 250 ml baffled flasks containing 50 ml TSB and shaken at 230 rpm for two days. A 1 ml volume of seed culture was inoculated into the flasks containing 50 ml of R5 medium and then fermented at 30°C on a rotary shaker at 220 rpm for five days. Decanoic acid and methyl oleate were used as the feeding medium. After 48 h of fermentation, the feeding medium (final concentration, 1 mM) was added to the fermentation media once after 48 h or two, four, and six times (every 12, 18, 36 h) after 48h. The culture broth was mixed with an equal volume of methanol to disrupt the cells and harvested by centrifugation. The supernatant was concentrated using a vacuum concentrator and dissolved in methanol. The final extracts were analyzed by HPLC (high pressure liquid chromatography) on a reverse phase column (Agilent ZORBAX SB-C18, USA) at a flow rate of 1 ml/min with UV detection at 222 nm. Solutions A (H₂O containing 0.01% TFA) and B (acetonitrile containing 0.01% TFA) were used to isolate daptomycin and the compounds of the A21978C family.

Mass spectrometric (MS) data were obtained by LC-MS analysis on Triple TOF 5600+ (AB Sciex, USA) system using electrospray ionization in positive ion mode, with a scan range of 50 ~ 2000 m/z. The LC method was run at 40°C on a Phenomenex Kinetex 1.7u C18 (2.1 mm × 150 mm, 1.7 um). The initial composition of 95%water, 10% acetonitrile and 0.01% formic acid were maintained for 3 min, followed by a linear gradient to 95% acetonitrile and 0.01%formic acid over 21 min, and this composition was held 25 min before re-equilibration; the flow rate was 0.4 ml/min. The electrospray capillary voltage was 5.5 kV and capillary temperature was maintained at 500°C.

Antibacterial Assays against Staphylococcus aureus

The antimicrobial activities of the daptomycin and its derivatives were assessed using the paper disc diffusion method. The bioassay was performed using Staphylococcus aureus as the indicator organism. A 1 ml sample of overnight-cultured St. aureus was mixed with 10 ml of a sterile solution of 1% agarose in H₂O. A 5 ml volume of the agarose/growth media mixture was added to a prewarmed NA medium and solidified for 30 min. The discs (6 mm diameter) were saturated with 10 µl of the extracts (dissolved in methanol) and placed onto an NA medium overlaid with St. aureus. After incubation for 20 h at 37°C, the diameter of the inhibition zone surrounding the discs, resulting from the diffusion of bacterial compounds, was then measured.

Isolation of the Daptomycin Biosynthetic Gene Cluster Using the pSBAC System

Unique restriction enzyme site in both border regions of NP BGC is necessary to isolate the BGC using pSBAC system. While the MER BGC was isolated using the unique restriction enzyme MfeI sites present in the border region of MER BGC, artificial XbaI or HindIII sites were inserted precisely near the borders of the TMC BGC and PIK BGC to capture the BGCs. No unique restriction enzyme sites were available at the border regions of TMC BGC and PIK BGC for pSBAC cloning. Fortunately, the genome sequence analysis of S. roseosporus ATCC31568 revealed two NheI restriction enzyme sites at the both border regions of the daptomycin BGC (Fig. 1). To integrate a pSBAC vector into the vicinity of the dptP, a 1,720-bp gene fragment was cloned into a pSA using In-Fusion cloning kit. The resulting construct was called pSAMD, which was then introduced directly into the daptomycin-producing S. roseosporus. The integration of pSAMD was confirmed by PCR analysis (data not shown). The NheI-digested total chromosomal DNA fractions were then self-ligated and transformed directly into E. coli EPI300 (Fig. 2). The transformants were selected using apramycin antibiotics and confirmed by PCR (Fig. S1). As a final step of pSBAC cloning, attP-int_ΦC31 was introduced into the daptomycin BGC-captured vector for the stability of daptomycin BGC in a heterologous host. The isolated TMC BGC or PIK BGC were expressed stably in Stretpomyces cell factories via integration using ΦBT1 integrase. On the other hand, the integration efficiency of the ΦBT1 attP-int system was quite low. Therefore, to increase the integration efficiency, the ΦC31 attP-int system, which is an advantage for high integration efficiency and a broad host range, was utilized for the integration of daptomycin BGC into S. coelicolor. The AvrII-digested attP-int_ΦC31 from pATTP was ligated with pDPT, and the entire daptomycin BGC-containing modified pSBAC, called pDPT001, was constructed successfully for heterologous expression.

Figure 1. Daptomycin biosynthetic pathway and its biosynthetic gene cluster.

Figure 2. Schematic description of pDPT001 construction. Modified pSBAC called pSAMD was constructed by removing attP-int_ΦBT1 and inserting 1,720-bp sequences in the vicinity of dptP. pSAMD was then introduced into the chromosomal DNA via homologous recombination. The daptomycin biosynthetic gene cluster was isolated by NheI digestion of the modified chromosomal DNA and its self-ligation, called pDPT. A DNA fragment containing attP-int_ΦC31 was inserted into the AvrII recognition site of pDPT to generate pDPT001.

Using the pSBAC system, the precise cloning of the target cluster can be achieved through site-specific chromosomal integration of the vector and unique restriction sites as well as in vivo plasmid rescue. Previously, the daptomycin biosynthetic gene cluster was cloned by the construction of the BAC library, but approximately the 60 kb region of the BAC clone was not associated with the daptomycin biosynthetic gene cluster. Here, the daptomycin biosynthetic gene cluster (~65 kb) was isolated precisely using pSBAC and endogenous restriction enzyme, NheI, sites. Therefore, isolation of the daptomycin biosynthetic gene cluster can be another example of the isolation of natural product biosynthetic gene cluster belonging to not only type I polyketide, but also non-ribosomal polypeptide using the pSBAC system.

Heterologous Expression of Daptomycin Biosynthetic Gene Cluster in Streptomyces coelicolor

To confirm the heterologous and functional expression of the daptomycin BGC, the newly formed pDPT001 was introduced into S. coelicolor M511 through conjugation (named S. coelicolor DPT101). S. coelicolor M511 is one of the most widely used Streptomyces surrogate strains for heterologous expression, which is variant of S. coelicolor M145 with deletion of actII-orf4 for repression the actinorhodin cluster that might be a competitive pathway for heterologous BGC. The constructed strain was cultured along with the parental strain, S. coelicolor M511. HPLC analysis revealed highly noticeable peaks showing identical retention times to the authentic daptomycin only in the S. coelicolor DPT101 (Fig. 3A). LC-MS analyses with the collected fraction of the peak between 26 min and 28 min showed that the m/zof 1 was 810.8630, and its UV max was approximately 222 nm, which is characteristic of the daptomycin. This suggests that daptomycin was produced heterologously in S. coelicolor M511 (Fig. S2). Compounds 2, 3, and 4 were also identified as daptomycin derivatives, A21798C_1-3 (Fig. S2). A21978C_1-3 containing a cyclic anionic 13-amino acid lipopeptide can be distinguished by the 11 to 13-carbon branched-chain fatty acyl moiety attached to the N-terminal L-Trp. This fatty acid comprises n-decanoic acid for daptomycin, anteiso-undecanoic acid, iso-dodecanoic acid, and anteiso-tridecanoic acid for A21798C_1-3, respectively. In contrast, the production of daptomycin and its derivatives were inconsistent in S. roseosporus (data not shown); the production yields of daptomycin were 3.24 mg/l and the total yields of daptomycin and its derivatives were 24.22 mg/l in the heterologous host (Fig. 3B). This shows that the pSBAC-driven heterologous expression of an entire daptomycin BGC resulted in consistent production of daptomycin in a surrogate host S. coelicolor. As expected, the antimicrobial activity against Staphylococcus aureus was detected only in the extracts of DPT101 (Fig. 3C).

Figure 3. (A) HPLC analysis of DPT101. (B) Production yield of daptomycin and its derivatives. (C) Antimicrobial assay against Staphylococcus aureus. a, authentic daptomycin 5 mg; b, culture media with decanoic acid; c, S. coelicolor M511 wild type; d, S. coelicolor DPT101.

Previously, the attP-int_ΦBT1 system was used for the stable expression of TMC BGC and PIK BGC in heterologous hosts, despite its integration efficiency being very low. In this study, the phage ΦC31 attP-int system, which has an advantage for high integration efficiency, was utilized for the integration of daptomycin BGC into S. coelicolor. After conjugation, many colonies (> 30 colonies) were shown in the ΦC31 system while only a few colonies were observed in the ΦBT1 system (data not shown). Integration into the ΦC31 attB site in S. coelicolor did not affect the production of daptomycin and its derivatives. These results suggest that the pSBAC system combined with the ΦC31 system can be used for the development of various NRPS-driven NP BGCs.

Heterologous Production of Daptomycin Via Decanoic Acid Feeding

The biosynthesis of daptomycin is initiated by the condensation of decanoic acid (a 10-carbon branched chain fatty acid) and L-Trp [24]. Therefore, the addition of decanoic acid to the fermentation medium is essential for daptomycin production [20]. However, decanoic acid feeding should be optimized because high concentration of decanoic acid could affect the surrogate host cell growth. To improve daptomycin production in a S. coelicolor DPT101 strain, 1 mM decanoic acid was added to the shake flask culture once after 48 h or two, four and six times after 48 h [25, 26]. Compared to repeated feeding of decanoic acid, two, four and six mM of feeding medium is added among the cultures tested. Although precursor was fed repeatedly or once in higher concentration, it resulted in similar level of daptomycin and its derivatives production (Fig. 4). These results suggested that adding higher amount of decanoic acid has no effect on improvement of daptomycin production yield for S. coelicolor DPT101. Increasing daptomycin production yield in a heterologous strain still needs further improvement with several strategies such as BGC refactoring, regulatory network optimization, and heterologous expression in more diverse Streptomyces cell factories. In summary, the 65-kb daptomycin BGC was cloned successfully in a single pSBAC vector system and expressed functionally in a heterologous host, suggesting that a pSBAC-driven heterologous expression strategy is an efficient approach for the production of Streptomyces NRPS-family NP BGC.

Figure 4. Comparison of the production of daptomycin and its derivatives by optimization of the number of times decanoic acid was fed in DPT101. Quantitative production yield of daptomycin and its derivatives.

JMB-29-1931_Supple.pdf

This study was supported by “National Research Foundation of Korea (NRF) (Project No. NRF-2017R1A2A2A05069859). This work was also funded by Agricultural Microbiome R&D Program, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea (as part of the (multi-ministerial) Genome Technology to Business Translation Program). No. 918008-04.

The authors have no financial conflicts of interest to declare.