Heterologous expression and antitumor activity analysis of syringolin from Pseudomonas syringae pv. syringae B728a

Background Syringolin, synthesized by a mixed non-ribosomal peptide synthetase/polyketide synthetase in Pseudomonas syringae pv. syringae (Pss) B728a, is a novel eukaryotic proteasome inhibitor. Meanwhile, directly modifying large fragments in the PKS/NRPS gene cluster through traditional DNA engineering techniques is very difficult. In this study, we directly cloned the syl gene cluster from Pss B301D-R via Red/ET recombineering to effectively express syringolin in heterologous hosts. Results A 22 kb genomic fragment containing the sylA–sylE gene cluster was cloned into the pASK vector, and the obtained recombinant plasmid was transferred into Streptomyces coelicolor and Streptomyces lividans for the heterologous expression of syringolin. Transcriptional levels of recombinant syl gene in S. coelicolor M145 and S. lividans TK24 were evaluated via RT-PCR and the production of syringolin compounds was detected via LC–MS analysis. The extracts of the engineered bacteria showed cytotoxic activity to B16, 4T1, Meth-A, and HeLa tumor cells. It is noteworthy that the syringolin displayed anticancer activity against C57BL/6 mice with B16 murine melanoma tumor cells. Together, our results herein demonstrate the potential of syrinolin as effective antitumor agent that can treat various cancers without apparent adverse effects. Conclusions This present study is the first to report the heterologous expression of the entire syl gene cluster in Streptomyces strains and the successful expression of syringolin in both S. coelicolor M145 and S. lividans TK24. Syringolin derivatives demonstrated high cytotoxicity in vitro and in vivo. Hence, this paper provided an important foundation for the discovery and production of new antitumor compounds.


Background
Pseudomonas syringae pv. syringae (Pss) is a foliar bacterial pathogen that causes brown spot disease in snap beans (Phaseolus vulgaris L.) [1,2] and produces a novel polyketide complex, known as syringolin [3,4]. Application of syringolin at micromolar concentrations onto rice plants can induce resistance against rice blast fungus. However, Pss mutants are incapable of syringolin biosynthesis and induce defense reactions and resistance, revealing its marginal therapeutic activity against systemic fungal infections [5,6]. Recently, syringolin has shown potent in vivo antitumor activity against neuroblastoma, ovarian, and leukemic cancer cells [7]. Such specificity of syringolin inhibits all three catalytic activities of eukaryotic proteasomes [8,9]. The proteasome acting during protein degradation has been known as a biological target for the clinic treatment lately [10,11]. Syringolin was confirmed as the notable peptide moiety in a 12-membered macrolactam ring structure, which also has two double bonds with (E)-configuration and an unusual urea moiety [12] (Fig. 1). The special chain reversal by ureido linkage was also present in natural products,
The syringolin biosynthetic gene cluster spanned 22 kb in length and included five open reading frames (sylA-sylE). The NRPS module sequence in the chromosome generally determines the amino acid sequence of the peptide product [21] (Fig. 2) where sylA is a putative transcription activator; sylB hypothetically encodes the lysine reductase; sylC encodes a module predicted for valine activation [22]; sylD codes two typical NRPS modules that activate lysine (or dehydrolysine) and 5-methyl-4-amino-2-hexenoic acid (or its precursor); and sylE possibly serves as an exporter [23].
The genetic manipulation for PKS/NRPS gene cluster is difficult to perform using conventional DNA engineering methods because of their large sizes (spanning 10-100 kb). Red/ET recombineering [17,18], which is Fig. 1 The structure of syringolin. The ring structure consists of two non-proteinogenic amino acids, namely, 3,4-dehydrolysine and 5-methyl-4-amino-2-hexenoic acid. The α-amino group of the latter attaches to valine through a peptide bond, which in turn, is linked to a second valine residue via an unusual ureido group Boxes labeled as C, A, and PCP represent condensation, activation, and peptide carrier protein domains of synthetase modules encoded by sylC and sylD, respectively. Circles denote putative β-ketoacyl synthetase (KS), acyl transferase (AT), dehydratase (DH), β-ketoreductase (KR), acyl carrier protein (ACP), and thioesterase (TE) domains of the polyketide-like module, which is also encoded by sylD (b) independent of restriction site location and DNA fragment size, has extraordinarily advanced the field of genetic manipulation by omitting many steps in standard restriction/ligation. Direct cloning was recently established based on full-length Rac prophage protein, RecE, and its partner RecT-mediated linear plus linear homologous recombination (LLHR) [24]. This efficient cloning approach, when applied to direct cloning of large gene clusters from genomic DNA, might greatly promote the course of genome mining and combinatorial biosynthesis of PKS/NRPS compounds.
In this study, we report the direct cloning of an intact syl gene cluster from the genomic DNA of Pss B301D-R and replaced its native promoter with P snpA , a strong native promoter in Streptomyces, to actively produce the syl gene in heterologous hosts. We analyze the bioactivity of the recombinant products by treatment of several cancer cell lines and tumor model in mice to provide a suitable protocol for syringolin production and clinical application in the future.

Recombineering
All engineering used Red/ET recombination techniques as described previously [27,28]. Red/ET-competent E. coli cells (50 μL) were electroporated with 0.3 μg of a linear fragment (either PCR product or fragment obtained from restriction). PCRs were performed with Phusion polymerase (New England Biolabs, GmbH, Frankfurt am Main, Germany). After electroporation, the selection of recombinants was carried out depending on antibioticresistant gene and examined by restriction analysis.

RNA extraction and RT-PCR analysis
Total RNA was extracted by the TRIzol ® (Invitrogen) method. The RNA quality was analyzed by absorbance measurement and formaldehyde-denatured agarose gel electrophoresis. RT-PCR of syl gene cluster was performed based on the previous protocol [29].

HPLC analysis and mass spectrometry of syringolin
Recombinant Streptomyces strains were grown on an M2 medium for 7 days at 30 °C. For the metabolite analyses, supernatant cultures were extracted with equal volumes of ethyl acetate after centrifugation and dried in a rotary evaporator. The extracts were then dissolved in methanol and filtered (0.22 μm pore size). LC-MS/MS experiments were performed on LTQ XL hybrid mass spectrometer (Thermo Fisher Scientific, USA) coupled to a Finnigan LC system (Thermo Fisher Scientific). The extracts were subjected and desalted online in a reverse-phase precolumn (C18 Pepmap column, LC Packings) and resolved on a nanoscale C18 Pepmap TM capillary column (LC Packings) at a flow rate of 0.4 mL/min (solvent A = 0.1% formic acid in H 2 O; solvent B = acetonitrile and 0.1% formic acid; 0-15 min 95% A and 5% B to 95% B [linear gradient], followed by 5 min 5% A and 95% B). Detection was carried out in positive ion model, auto MSn. Syringolins were identified by comparing the retention times and MS2 data identified from the original producer.
In vivo therapeutic assessment was carried out using 4T1 breast tumor model and B16 melanoma tumor model as described previously [1]. SPF female BALB/c and C57BL/6 mice, aged 6-8 weeks old, were purchased from the SLRC Laboratory Animal Company in Hunan, China. Animals were bred and maintained in SPF conditions and were kept for at least 3 days before use. Tumors in the fourth mammary pads of female BALB/c mice were established with 1 × 10 5 4T1 mouse breast tumor cells, and C57BL/6 mice were implanted with SC tumors by injecting with 1 × 10 5 B16 cells on the mid-right side. After the tumor volume reached ~ 0.2 cm 3 , breast tumor model BALB/c mice were randomly assigned to seven groups, and C57BL/6 mice bearing B16 tumor were randomly assigned to four groups. Syringolin extracts were injected for every 2 days within a span of 10 days. Tumor weights were estimated using two-dimensional caliper measurements conducted thrice per week using the formula: tumor weight (mg) = (a × b 2 )/2, where a and b are the tumor length and width in mm, respectively. At a defined time, mice were sacrificed by cervical dislocation. However, moribund animals characterized by irregular respiration, tremors, absence of voluntary response to external stimuli, and coma were killed for humane reasons and considered as animals that died during survival experiments. All animal experiments were repeated thrice in this study. All animal experiments followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and obtained the approval from the Animal Ethics Committee of Hunan Normal University.

Histology
Primary tumors, liver, kidney, and spleen from tumorbearing and control mice were harvested and fixed in 10% buffered formalin. Standard hematoxylin and eosin (HE) staining procedures were employed for morphological assessment [30]. The paraffin embedded samples were cut into 5 μm sections, and every twentieth section was stained and examined by microscopy.

Direct cloning of intact sly gene cluster
The syl gene cluster from Pss B301D-R was isolated through Red/ET direct cloning. Plasmid pAsk-amp-syl (Fig. 3a) was constructed after PCR amplification of the minimal replicon with 90-nucleotide homology arms at the start and end of the syl gene. Digestion with PstI and DNA sequencing revealed the presence of an intact syl gene. For successful integration into the chromosome and heterologous expression in Streptomyces strains, the resulting plasmid, p15A-Tpase-syl (Fig. 3b) was generated through triple recombination [31]. One fragment contained the BamH I/Xba I restriction enzyme sites of p15Adir while the second one employed from PCR introduces a new promoter, P snpA , a regulator snpR, and another IR sequence. BSD-and kanamycin-resistant colonies were cultured and verified by restriction analysis. This direct cloning method led to less mutations and much longer target DNA fragments than the approach achieved by PCR, because their cloning depends on the E. coli replication machinery and not on PCR, which is error-prone. Obviously, the technique is easier and faster than the standard method needed to establish and to screen the genomic DNA library.
The recombinant syl gene cluster was introduced and was integrated into Streptomyces strains and chromosome through a well-established E. coli: Streptomyces intergeneric conjugation protocol [32]. Syringolin compounds are synthesized by enzymatic actions of the sylB, sylC, and sylD gene products, and the generation and condensation of the ureido valine remained enigmatic. The modified P snpA promoter with native transcriptionactive sylA gene proved the efficient expression of the syl gene cluster in Streptomyces heterologous hosts.

Identification gene transcriptional levels of syl gene cluster in heterologous expression hosts
Transcriptional levels of recombinant syl gene in S. coelicolor M145 and S. lividans TK24 were evaluated via RT-PCR. Total RNA was extracted after fermentation for 24, 32, 38, 46, and 52 h. The syl gene cluster is well-expressed in both Streptomyces hosts (Fig. 4). sylC and sylD gene, which manages the extension of the core ring backbone, started the transcription process at 24 h. sylB catalyzed the reduction of the intermediate product. During the late fermentation period, sylE encoded thioesterase to release the syringolin compounds.

Biosynthesis of syringolin in heterologous Streptomyces host
HPLC analysis showed the presence of three metabolite peaks in the culture broth of both S. coelicolor M145/ P snpA -syl and S. lividans TK24/P snpA -syl mutants in   (Fig. 5a). In order to compare the yield of syringolin compounds in Streptomyces coelicolor M145 and Streptomyces lividans TK24, we measured and calculated the peak area of the major derivatives produced by the software Bruker Compass. The results showed that the production of syringolin derived compounds produced by heterologous host S. coelicolor M145 was about 1.3-fold that of S. lividans TK24. This 1.3-fold difference in the syringolin yield of S. lividans TK24/P snpA -syl might have resulted from the diverse regulatory system in heterologous hosts. Overexpression of novG CstrR-like positive regulatory protein confers a higher PKS/NRPS synthesis activity to S. coelicolor M145 [33]. UV spectra, retention time (R t ), and MS/MS2 results in HPLC/MS comparisons revealed that all known syringolins A, B, C, D, E, and F (SylA-SylF) were detected in the extract of the two heterologous production hosts (Fig. 5b-d). The three peaks represented C 24  . Low-resolution ESI-MS analyses further showed that the mass of 1 is lower by 14 and 28 amu compared with that of 3 and 2, respectively. These results indicated that their real molecular weights of 1, 3, and 2 might be 493, 507, and 521 Da, respectively, suggesting the single methylene group (-CH 2 -) difference, preferably at the R 1 or R 2 group among the compounds. The molecular formula of the syringolin derivative obtained through high-resolution EI-MS matched the predicted structure.

In vitro antitumor activity of recombinant syringolin compounds
We investigated the antitumor activity of recombinant syringolin on different tumor cell lines, and the results suggested that the extracts of the recombinant Streptomyces strains induced varying levels of cytotoxicity. All samples obtained from ethanol extraction methods were dried and resuspended in PBS, and the differences between the control and treatment groups were significantly and statistically different (P < 0.05), thus, confirming our results. Incubation of 4T1, B16, HeLa, and MthA cancer cells with 15,30,45, and 60 μM syringolin extracts for 24 h significantly reduced cell viability, as demonstrated by the reduction of MTT. Recombinant extracts showed dose-dependent cytotoxicity on all compounds. The effect of the extracts on cancer cells was evaluated (Fig. 6). Syringolin has IC 50 values of 22.5, 24.3, 26.4, and 35.2 μM towards 4T1, HeLa, B16, and MthA, respectively. MTT analysis showed that the syringolin compound has good cytotoxicity to the above four cell lines at low concentrations (Fig. 7).

In vivo antitumor activity of recombinant syringolin compounds
We constructed two tumor models to analyze the in vivo toxicity of syringolin compounds. All mice were given 30 or 60 μM dose of syringolin extract every 2 days for 10 days. BALB/c mice bearing 4T1 tumor were treated with syringolin compounds through gastric lavage (Fig. 8a), intratumoral injection (Fig. 8b), or intravenous injection (Fig. 8c), and syringolin compounds had significant effect on 4T1 from the latter two methods (P < 0.05), but had no in vivo activity from former treatment. Among the three methods, intravenous injection gave the greatest effect with 67% in vivo inhibition rate to the 4T1 tumor at 30 μM, and extended mice mortality, resulting in delayed growth and death. Syringolin compounds by intratumoral injection could inhibit 40% tumor growth at higher concentration, and the injected mice survived under good conditions. Tumor, liver, kidney, and spleen were harvested and subjected to HE staining. Syringolin did not only increase apoptosis of 4T1 cells but also protected the liver and kidney from injury (Fig. 9). Organs from control mouse models showed significant liver and kidney damage while cells from the drug-treated group were normal and did not change much. Syringolin treatment of C57BL/6 mice bearing B16 tumor by intratumoral injection also showed great in vivo antitumor activity in a dose-dependent manner at 30 μM concentration with about 75% inhibition rate to B16 (Fig. 8d). B16 tumor growth almost stopped after injection of syringolin compound. Together, our results herein demonstrate the potential of syrinolin as effective anitumor agent that can treat various cancers without apparent adverse effects.

Conclusions
This paper is first to express the whole syl gene cluster in heterologous Streptomyces strains. As sylA gene activates the expression of NRPS/PKS, acquisition of intact syl gene cluster uses the LLHR straightforward strategy mediated by Red/ET recombineering. The promoter underwent exchange after one round of LLHR. The results clearly indicate that the clusters of genes are capable of encoding proteins that synthesize syringolin. In comparison with the native promoter from Pseudomonas syringae pv. syringae, the general promoter, P snpA , successfully transcribed the whole gene cluster in heterologous strains.
Large natural product biosynthetic gene clusters traditionally require reconstruction from several cosmids, which is time-consuming given the required screening process from a genomic library and subsequent cloning steps. Our direct-cloning method furnishes a general tool of reconstituting large gene clusters. When coupled with suitable heterologous expression hosts, direct cloning is effective alternative in investigating or engineering known and unknown biosynthetic pathways, from slowgrowing bacteria and poorly established genetic systems.
Subsequently, the expression of the clone in both M145 and TK24 produce six syringolin family members, which show diverse transcriptions of the syl gene regulated by synthetic promoters. Syringolin yield is about 1.5 mg/ mL. Replacement of some strong promoters, like ermEp, fdmR1, or novG might regulate the gene transcription. Another possible measure in developing the production is to optimize the fermentation of heterologous stains.
Syringolin derivants demonstrated high cytotoxicity to B16, 4T1, Meth-A, and HeLa cells in vitro and to 4T1 model BALB/c mice and B16 melanotic C57BL/6 mice in vivo. SylA could preferentially target the β2 and β5 of the proteasome in vitro and in vivo. Structureactivity analysis revealed that the dipeptide tail of SylA contributed to β2 specificity and identified a nonreactive SylA derivative being essential for imaging experiments. The syringoline family members showed their activities of labeling nuclear and cytoplasmic proteasomes in our research.