- Open Access
Heterologous expression and antitumor activity analysis of syringolin from Pseudomonas syringae pv. syringae B728a
- Fan Huang†1,
- Jianli Tang†1,
- Lian He1,
- Xuezhi Ding1,
- Shaoya Huang1,
- Youming Zhang1,
- Yunjun Sun1 and
- Liqiu Xia1Email authorView ORCID ID profile
© The Author(s) 2018
- Received: 30 June 2017
- Accepted: 15 January 2018
- Published: 26 February 2018
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.
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.
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.
- Heterologous expression
- Red/ET recombineering
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 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) . 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 PsnpA, 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.
Bacterial strains and culturing conditions
Various E. coli strains were cultured at 37 °C in Luria–Bertani (LB) medium supplemented with antibiotics. Kanamycin (30 μg/mL; Sigma Chemical Co., St. Louis, Mo.), blasticidin S (50 μg/mL), tetracycline (5 μg/mL), and ampicillin (100 μg/mL) were added to the growth media as required. Heterologous hosts, S. lividans TK24 and S. coelicolor M145 strains, were grown at 30 °C on M2 (0.4% glucose, 1% malt extract, 0.4% yeast extract, and 0.1% CaCO3) or in TSB medium (tryptic soy broth (Oxoid), 30 g/L) for metabolite analyses as previously reported . MS-agar medium  was used to transfer the cosmids from E. coli into Streptomyces in accordance with the standard protocol . Apramycin (50 μg/mL) and nalidixic acid (25 μg/mL) were supplemented in the medium whenever necessary.
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 antibiotic-resistant gene and examined by restriction analysis.
Direct cloning of the syl gene cluster
PCR product and linear genomic DNA were co-transformed into recombineering proficient competent GBdir cells to obtain p15A-syl-IR-Tpaes-BSD-oriT-IR. Sequencing the syl gene used primers sylseq-up (5′-ccggcctacacgcattc sylA end) and sylseq-down (5′-agcaacctggatgtacgg sylE end).
Engineering the syl gene cluster
To obtain highly heterologous expression in Streptomyces strain, a strong promoter PsnpA was inserted in front of the syl gene in p15A-syl-IR-Tpaes-BSD-oriT-IR to form the p15A-syl-IR-Tpaes-BSD-oriT-IR construct. The PsnpA-apra cassette (apra: apramycin-resistant gene) was prepared with Psnpsyl5 (5′-TTAATGATGTCTCGTTTAGATAAAAGTAAAGTGATTAACAGCGCATTAGCGCGCCTATCCTCCATGGTATAAATCG-3′) and Psnpsyl3 (5′-GGAATTAATCATCTGGCCATTCGATGGTGTCGGGTCATGTGAGCAAAAGGGAAGCCGCGGGAGTAATCCT-3′).
Conjugation into streptomyces
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 . Control (RT-minus) reaction including all components for RT-PCR except the reverse transcriptase enzyme excluded the presence of genomic DNA. The expression of 16S rRNA gene from heterologous host served as a positive internal control. Reverse transcription reactions were conducted with the primers: sylB5 (5′-TGGCGCATGACCGATTGCGT-3′), sylB3 (5′-TCGGCATGCACGGGGACAAC-3′), sylC5 (5′-ACTGCCAATGGGAGCGCGAC-3′), sylC3 (5′-CAACTTACCCG GCAGCGGCA-3′), sylD5 (5′-ACTATCGCGCTCGTGTCCAA-3′), sylD3 (5′-CAGCCCGATACCGTCAGAAA-3′), sylE5 (5′-AAAGCCTTGCGGCCGAGCAT-3′), sylE3 (5′-AACCAGGAGCACGTCGCAGC-3′), 16SRNA-F (5′-CTACCTCAAGCAGATCGGCAAG-3′), and 16SRNA-R (5′-GATCAGGTC CAGGAACGCCATG-3′).
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 pre-column (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 H2O; 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.
Cell viability and death was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay for adherent B16, HeLa, 4T1, and MethA cells in 96-well plates as described [12, 25]. Cells were incubated with 10 or 20 μL syringolin extracts from Streptomyces strains for 48–72 h, and the optical density (OD) of each well was determined in an ELISA reader at 560 nm.
In vivo therapeutic assessment was carried out using 4T1 breast tumor model and B16 melanoma tumor model as described previously . 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 × 105 4T1 mouse breast tumor cells, and C57BL/6 mice were implanted with SC tumors by injecting with 1 × 105 B16 cells on the mid-right side. After the tumor volume reached ~ 0.2 cm3, 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 × b2)/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.
Primary tumors, liver, kidney, and spleen from tumor-bearing and control mice were harvested and fixed in 10% buffered formalin. Standard hematoxylin and eosin (HE) staining procedures were employed for morphological assessment . 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 recombinant syl gene cluster was introduced and was integrated into Streptomyces strains and chromosome through a well-established E. coli: Streptomyces intergeneric conjugation protocol . 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 PsnpA promoter with native transcription-active 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
Biosynthesis of syringolin in heterologous Streptomyces host
In vitro antitumor activity of recombinant syringolin compounds
In vivo antitumor activity of recombinant syringolin compounds
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, PsnpA, 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 slow-growing 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. Structure–activity 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.
This research provided new avenues and ideas for the discovery and production of new antitumor compounds. The antitumor effects of syringolin may be attributable to the inhibition of proteinase and cancer cell invasion, and its concrete mechanism of inducing apoptosis in cancer cells still needs further studies.
FH and LH designed and carried out the experiments, JLT analysed the data and prepared the manuscript. XZD and SYH carried out the HPLC analysis and LC/MS analysis of the syringolin. YMZ and YJS gave valuable suggestions in the experiments and manuscript editing. LQX supervised the research. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article.
Consent for publication
The authors are consent for publication.
Ethics approval and consent to participate
All animal experiments followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Hunan Normal University.
This work was supported by the National Basic Research Program (973) of China (2012CB722301), the National High Technology Research and Development Program (863) of China (2011AA10A203), the International Cooperation Project (0102011DFA32610), and the Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (20134486).
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