Reconstitution of Kinamycin Biosynthesis within the Heterologous

Jan 17, 2018 - The three BACs were then introduced to a second heterologous host, S. albus J1074, and the resulting transformants similarly analyzed b...
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Reconstitution of Kinamycin Biosynthesis within the Heterologous Host Streptomyces albus J1074 Xiangyang Liu,† Dongxu Liu,† Min Xu,† Meifeng Tao,† Linquan Bai,† Zixin Deng,† Blaine A. Pfeifer,‡ and Ming Jiang*,† †

State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, People’s Republic of China ‡ Department of Chemical and Biological Engineering, University at Buffalo, The State University of New York, Buffalo, New York 14260, United States S Supporting Information *

ABSTRACT: Diazofluorene compounds such as kinamycin and lomaiviticin feature unique molecular structures and compelling medicinal bioactivities. However, a complete understanding of the biosynthetic details for this family of natural products has yet to be fully elucidated. In addition, a lack of genetically and technically amenable production hosts has limited access to the full medicinal potential of these compounds. Here, we report the capture of the complete kinamycin gene cluster from Streptomyces galtieri Sgt26 by bacterial artificial chromosome cloning, confirmed by successful production of kinamycin in the heterologous host Streptomyces albus J1074. Sequence analysis and a series of gene deletion experiments revealed the boundary of the cluster, which spans 75 kb DNA. To probe the last step in biosynthesis, acetylation of kinamcyin F to kinamycin D, gene knockout, and complementation experiments identified a single gene product involved with final acetylation conversions. This study provides full genetic information for the kinamycin gene cluster from S. galtieri Sgt26 and establishes heterologous biosynthesis as a production platform for continued mechanistic assessment of compound formation and utilization.

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be fully elucidated. Previous biosynthetic studies in S. murayamaensis revealed kinamycin’s polyketide origin and identified several intermediates such as dehydrorabelomycin, kinobscurinone, and stealthin C (Scheme 1).14−16 Early biosynthetic studies were hindered by incomplete genetic information regarding the compound’s biosynthetic cluster and relied mainly upon isotope feeding experiments.17 The partial biosynthetic gene cluster of kinamycin was cloned from S. murayamaensis, and production of related compounds, but not the final product, was achieved through heterologous transfer to Streptomyces lividans ZX7.18 Recently, a new Streptomyces ambofaciens kinamycin production strain was discovered, and its biosynthetic gene cluster (the alp cluster) was identified, providing a new opportunity to study and reconstitute kinamycin biosynthesis.19,20 Specifically, two enzymes, AlpJ and AlpK, were identified to be involved in the oxidative cleavage and the following C−C bond formation of the compound’s B-ring.21,22 Further, the structure of the unique C−C bond cleaving enzyme AlpJ was determined.23 The majority of enzymes involved in the proposed kinamycin biosynthetic pathway could be identified in the S. ambofaciens cluster, among which an

romatic polyketides, derived from type II polyketide synthase (PKS) pathways, represent a group of natural products with important pharmacological uses; the anticancer and antibiotic compounds doxorubicin and oxytetracycline serve as prime examples.1−3 Natural products of the kinamycin family are aromatic polyketide antibiotics first isolated from Streptomyces murayamaensis by Omura and co-workers.4 Specifically, kinamycins A, B, C, and D were isolated and confirmed to differ by the position and degree of polyketide acetylation.5 Later, several other kinamycin family metabolites were also isolated.6,7 The kinamycin compounds are wellknown for displaying cytotoxic activity with additional potential as antitumor agents.8 More recently, lomaiviticin, which features a polyketide backbone similar to kinamycin but in a dimeric configuration, has been identified and shows similarly promising biological activity.9−11 The interesting bioactivity associated with kinamycin-based compounds and the unique benzo[b]fluorene skeleton featuring a diazo functional group has also attracted numerous synthetic or semisynthetic efforts to generate additional kinamycin or lomaiviticin compounds in order to leverage their medicinal properties and better understand the chemical underpinnings of compound construction.12,13 Despite successful efforts in the total synthesis of kinamycin,12,13 its complete biosynthetic pathway has yet to © 2018 American Chemical Society and American Society of Pharmacognosy

Received: July 28, 2017 Published: January 17, 2018 72

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Scheme 1. Proposed Biosynthetic Pathway of Kinamycin D

Figure 1. Kinamycin biosynthetic cluster. (A) Overlapping maps of three positive bacterial artificial chromosomes (BACs; 3C2, 3A4, 2E9) containing the full or nearly full kinamycin gene cluster from S. galtieri Sgt26. Clone 3C2 lacks alpZ and 3A4 contains a partial alp2I reading frame. Base pair numbering is based upon the homologous kinamycin sequence from S. ambofaciens. (B) Genetic organization of the kinamycin biosynthetic cluster in S. galtieri Sgt26 (the genes labeled as red were not included in the previously published cluster).

enzymes involved in the kinamycin pathway and provide insight into the final acetylation steps, we heterologously expressed the biosynthetic cluster in S. albus J1074. A series of gene deletion experiments confirmed the boundaries of the cluster and revealed eight additional genes related to the cluster from S. ambofaciens. We further confirmed that the alp2F and 2G genes are involved in compound formation and the acetyltransferase alp2D gene is involved in the conversion of kinamycin F to D.

epoxy hydrolase was found to catalyze the hydrolysis of an epoxy intermediate.24 However, due to the complexity of kinamycin biosynthesis and the lack of successful complete heterologous production, confirmation of the biosynthetic boundaries of the S. ambofaciens cluster has not been firmly established. For example, there remain questions regarding the final kinamycin acetylation steps. In previous studies, a membrane-associated enzyme or enzyme complex with a large apparent molecular weight was partially purified from S. murayamaensis with the ability to convert kinamycin F to D.25 However, the gene(s) encoding this large enzyme could not be identified, opening the possibility of genetically profiling the S. ambofaciens or alternative kinamycin gene clusters via knockout mutants or heterologous transfer to identify the gene(s) responsible for this conversion.25 In this study, we present a kinamycin gene cluster newly identified within S. galtieri Sgt26. In order to identify all the

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RESULTS AND DISCUSSION Heterologous Production and Identification of the Entire Biosynthetic Gene Cluster of Kinamycin. Previous studies of kinamycin production from S. ambofaciens revealed a 63 kb DNA region as the gene cluster.24,19 However, the complete boundaries of the cluster had not been confirmed, and efforts were unsuccessful at establishing heterologous kinamycin biosynthesis. Future efforts at both mechanistic understanding and compound utilization would benefit from 73

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heterologous kinamycin production from S. albus J1074 (Figure 3). In contrast, deletion of alp2H dramatically decreased

accomplishing these tasks. In a separate study devoted to screening for bioactive compounds from microbial sources, we identified a kinamycin gene cluster in S. galtieri Sgt26. Due to the large size of the cluster (≥63 kb DNA), bacterial artificial chromosome (BAC) cloning was selected to capture the entire kinamycin gene cluster. A genomic BAC library of S. galtieri Sgt26 was prepared and screened using two pairs of primers for alpZ and alp1X, which represent the currently accepted ends of the known kinamcyin cluster. Several BACs were identified including three (3C2, 3A4, and 2E9) containing the expected full kinamycin biosynthetic gene cluster region (Figure 1). By bioinformatics assessment, alpW was determined to encode a key repressor of kinamycin biosynthesis19 and, as such, was removed from BACs 3C2, 3A4, and 2E9 via in-frame deletion. The three BACs were then separately introduced to Streptomyces coelicolor M115226 by conjugation. However, when the resulting transformants were cultured on agar plates and analyzed by HPLC and LC-MS, no production of kinamycin compounds could be detected. In contrast, several associated intermediates were identified including dehydrorabelomycin and seongomycin, a shunt product from kinamycin biosynthesis,18 indicating polyketide biosynthesis derived from the introduction of the BAC clones. The three BACs were then introduced to a second heterologous host, S. albus J1074, and the resulting transformants similarly analyzed by LC-MS. A new compound with the same molecular weight (454.2 Da) and UV spectrum as kinamycin D was observed. The identity of kinamycin D was further confirmed by comparison of the LC profile with authentic kinamycin D (Figure 2). Thus, we concluded that all three BACs contained the necessary set of genes for complete kinamycin biosynthesis.

Figure 3. Cluster boundary determination for kinamycin D heterologous biosynthesis via HPLC analysis. (A) ΔalpW/3C2; (B) ΔalpWΔorf1-2/3C2; (C) ΔalpWΔalp2I/3C2; (D) ΔalpWΔalp2H/ 3C2; (E) ΔalpWΔalp2F-2G/3C2.

production and resulted in no accumulation of other compounds. Bioinformatic analysis suggests alp2H encodes an alkylhydroperoxidase and may function as a resistance gene. Thus, alp2H was determined to be the 3′ boundary of the cluster. The kinamycin biosynthetic cluster in S. galtieri Sgt26 is almost identical in gene organization to the published cluster from S. ambofaciens. Among the 62 genes in the cluster, eight (alp2A, alp2B, alp2C, alp2D, alp2E, alp2F, alp2G, and alp2H) are not reported in the original cluster.24 Among the eight genes, alp2A encodes a NAD(P)-dependent oxidoreductase that shows high similarity with Lom18. Alp1T and Lom19 were proposed for the reduction of the two ketone groups of the Aring in kinamycin and lomaiviticin biosynthesis, respectively.24 Since there are two ketone groups in the A-ring, Alp2A and Lom18 may also be involved in these reduction steps. BLAST searches revealed three (alp2B, alp2C, and alp2H) among the eight genes encode peroxidase-like enzymes that may be involved in self-resistance. The putative gene product of alp2D is an O-acyltransferase and may be responsible for one of the acetylation steps in kinamycin biosynthesis. The gene product of alp2E is an ECF-subfamily sigma factor and may not be related to kinamycin biosynthesis. Interestingly, we found two genes, alp2F and alp2G, to be homologous to creE and creD in Streptomyces cremeus.27 These two genes were proposed to function in diazo incorporation in cremeomycin biosynthesis27 with the possibility of serving the same role in kinamycin biosynthesis. However, we could not find homologous genes in the lomaiviticin biosynthetic cluster or throughout the genomes of its native producers. To determine whether alp2F and alp2G are indeed involved in kinamycin biosynthesis, the two genes were deleted. Only a trace amount of kinamycin D could be detected in the alp2F and 2G deletion mutant (Figure 3), confirming the important role of these genes in kinamycin biosynthesis. Kinamycin Biosynthesis O-Acetylation. Previous studies showed an apparent multifunctional protein or protein complex with a molecular weight greater than 600 kDa that could catalyze the consecutive acetylations of the tetraol kinamycin F at C-4 and C-2 to kinamycin D; however, the enzyme was only partially purified and no genetic information was reported.25 Successful heterologous production of kinamycin D prompted us to genetically probe for acetyltransferase activity. The

Figure 2. Heterologous production of kinamycin D from S. albus J1074 assessed by HPLC. (A) ΔalpW/3C2; (B) ΔalpW/3A4; (C) ΔalpW/2E9; (D) pHL931 (empty BAC control); (E) standard kinamycin D.

Boundary Determination of the Kinamycin Biosynthetic Gene Cluster. The individual inserts within the three BAC plasmids were each more than 120 kb DNA. When comparing each insert sequence with the kinamycin biosynthetic cluster in S. ambofaciens (alp gene cluster),19 a 73kb homologous DNA region was identified (Figure 1). BAC 3C2 lacks alpZ while still retaining kinamycin production from S. albus J1074. Hence, AlpZ (a putative negative regulator) was considered the 5′ terminal of the kinamycin biosynthetic cluster. BAC 3A4 lacks the full sequence of alp2I yet retained heterologous kinamycin D production capability. In addition, the deletion of orf1-2 and alp2I in BAC 3C2 still yielded 74

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considered a strong candidate as the missing bifunctional acetyltransferase catalyzing the acetylation of kinamycin F to D (Figure 5).

kinamycin gene cluster, however, revealed no encoded genes predicted to approach the size of the previously reported acetyltransferase protein product. Hence, by bioinformatic analysis, we identified two genes, alp1N and alp2D, predicted to encode associated acyltransferase activity. Sequence analysis showed that alp1N encoded an N-acyltransferase. Furthermore, the product of its homologous gene ( fzmQ) in the fosfazinomycin biosynthetic cluster was recently determined to catalyze the N-acetylation of hydrazinosuccinate and is also proposed to be involved in the incorporation of the N−N bond.28 There is no homologous gene of alp2D in the lomaiviticin cluster, which is consistent with the lack of similar acetylation steps in its biosynthesis. As such, we focused on alp2D as the possible gene involved in the O-acetylation of kinamycin F. To validate the functionality of Alp2D, the corresponding gene was deleted from the heterologously transferred cluster. The Δalp2D mutant abolished the production of kinamycin D while accumulating a more hydrophilic compound with a similar UV spectrum (Figure 4). LC-MS analysis suggested the compound was kinamycin F,

Figure 5. Kinamycin D conversion through the exogenous addition of kinamycin F fed to S. albus J1074 strains either with or without alp2D. (A) S. albus J1074 with empty vector (pMS82); (B) S. albus J1074::alp2D; (C) standard kinamycin F.

In summary, the entire kinamycin biosynthetic gene cluster was identified by heterologous expression of BAC clones generated from an S. galtieri Sgt26 genomic library. From this new cluster, eight additional genes were identified. In particular, an acetyltransferase Alp2D was discovered to be involved in the conversion of kinamycin F to D through a combination of gene deletion, complementation, and in vivo feeding experiments. As a result, a strain was generated capable of overproducing kinamycin F for future studies and applications. Homologous genes of creE and D, alp2F and 2G, were found in the kinamycin gene cluster and confirmed to improve the biosynthesis of kinamycin. In the future, the detailed mechanism of these and other gene products of kinamycin biosynthesis will be investigated.

Figure 4. HPLC profiles of the crude extracts from mutant strains. (A) ΔalpW/3C2, producing kinamycin D; (B) ΔalpWΔalp2D/3C2, producing kinamycin F; (C) ΔalpWΔalp2D/3C2::alp2D, restoring the production of kinamycin D; (D) standard kinamycin F; (E) standard kinamycin D.

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EXPERIMENTAL SECTION

General Experimental Procedures. Bacterial strains, plasmids, and BACs used in this study are listed in Table S1, and PCR primers are listed in Table S2. Streptomyces galtieri Sgt26 (China Center for Type Culture Collection No. AA97007) was isolated from forest soil of Shengnongjia, Eastern Hubei Province, China. All E. coli strains were cultured in lysogeny broth at 37 °C supplemented with appropriate antibiotics as needed. E. coli DH10B was used for plasmid propagation, and E. coli BW25113/pIJ790 was used for PCR-targeted mutagenesis. E. coli ET12567 bearing the RK2-derived helper plasmid pUB307 30 was used to facilitate the intergeneric triparental conjugation from E. coli ET12567/BACs to Streptomyces spp. S. coelicolor M1152 (Δact Δred Δcpk Δcda rpoB[C1298T])26 and S. albus J1074,31 which were the hosts tested for heterologous expression of the S. galtieri Sgt26 kinamycin biosynthetic gene cluster. S. coelicolor M1152, S. albus J1074, and their derivatives were grown on soya flour medium agar for sporulation and conjugation, in R2 liquid medium containing 5% HP-20 resin, or on R3 solid medium plates for metabolite production at 30 °C. Genomic Library Construction and Screening. Plasmid pHL931, containing the origin of transfer from RK2 (oriTRK2) and int and attP from the Streptomyces phage ΦC31, was used as a vector to construct the genomic BAC library of S. galtieri Sgt26 following published procedures.32 The clones containing the kinamycin gene cluster were screened by PCR with specific primers alpZ-partial-FR and alp1X-partial-FR. Heterologous Expression of BAC Kinamycin Gene Clusters. The transfer of 3C2 and its derivatives from E. coli to S. albus J1074 and S. coelicolor M1152 was accomplished using E. coli ET12567/

a known metabolite isolated from the kinamycin-producing strain S. murayamaensis.29 The identity of kinamycin F was supported by chromatographic comparison to the standard compound. To exclude a genetic polarity effect as a result of the Δalp2D mutant upon subsequent heterologous biosynthesis of kinamycin D, one copy of alp2D was introduced to the Δalp2D mutant strain by intergeneric conjugation, and the production of kinamycin D was restored. These results support Alp2D as the acetyltransferase in the conversion of kinamycin F to E with the possibility of also catalyzing kinamycin E to D. Enzymatic Activity of Alp2D via in Vivo Biotransformation. The in vivo gene knockout and complementation experiment unambiguously implicated the involvement of alp2D in the final acetylation steps. However, the molecular weight of Alp2D is only ∼45 kDa and is not consistent with the large molecular weight of the partially purified acetyltransferase previously reported.25 It was also unknown whether Alp2D alone or additional enzymes were involved in the conversion of kinamycin F to D. To further explore the activity of Alp2D and investigate acetylation across the C-4 and C-2 functionalities of kinamycin F, Alp2D was expressed in S. albus J1074 under the ermE promoter. Kinamycin F was added to the culture after 24 h, and metabolite profiles were analyzed. Complete conversion of kinamycin F to D was found after 1 h. Thus, Alp2D is 75

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pUB307-mediated triparental conjugation.30 Integration of the BACs was confirmed by apramycin resistance and diagnostic PCR. Preparation of Kinamycin F. Kinamycin F was prepared from kinamycin D through LiOH hydrolysis using a previous reported procedure.24 Kinamycin D was a kind gift from Prof. Keqian Yang.24 Production and Analysis of Kinamycin and Related Metabolites. For kinamycin production, spore suspensions were inoculated in liquid trypticase soy broth (TSB) and incubated at 30 °C for 24 h as a seed culture. A total of 1 mL of seed culture was then transferred into 30 mL of fermentation medium and incubated with shaking (220 rpm) for an additional 1−2 days at 30 °C. For solid fermentation, S. coelicolor M1152 (3C2ΔalpW3) was cultured on R3 agar-solidified medium (35 mL medium/9 cm Petri dish) for 1−2 days at 30 °C. For metabolite analysis, solid medium from each dish was sliced individually and extracted with 35 mL of ethyl acetate/acetic acid (99:1 v/v) three times. For liquid cultures, 30 mL of broth was extracted with an equal volume of ethyl acetate after the pH was adjusted to 3−4. The ethyl acetate was concentrated by rotary evaporation, and the final extract was dissolved in methanol. Samples were then analyzed with an Agilent 1200 HPLC system using a ZORBAX XDB-C18 column (5 mm, 4.6 × 250 mm) under the following conditions: 75% solvent A (water with 0.1% trifluoroacetic acid [TFA]) to 100% solvent B (acetonitrile with 0.1% TFA) over 20 min and then held for 10 min at a flow rate of 1 mL/min. The absorbance was monitored at 424 nm. Construction of Gene Disruption Mutants and Their Complementation. All genetic manipulations of 3C2 and other BACs were conducted using λ-Red recombination-mediated PCRtargeted gene deletion, as described by Gust et al.33 Individual gene disruption cassettes (for alpW, alp2D, and alp2F-2G) were generated via PCR amplification of the ermE erythromycin resistance gene cassette with flanking FRT sites from pSJTU6722. Primers were designed with 39 nucleotides matching the adjacent sequences of the targeted gene. Gene replacement by λ-Red recombination was achieved in E. coli BW25113/pIJ790 carrying the 3C2 or other BACs. Mutated constructs were confirmed by PCR and then transferred to E. coli BT340 for flippase (FLP) recombinase-mediated excision of the erythromycin resistance gene. The corresponding final knockouts, with FLP scar, were verified by PCR prior to transfer into S. albus J1074. For knockouts alp2I, alp2H, and orf1-2, the procedure was repeated but using a kanamycin resistance cassette without FRT sites from SK-kana (this study) such that final mutants retained the kanamycin resistance gene. For complementation experiments, the integrative plasmid pHXZ215 (pMS82 derivative constructed containing the ermE promoter) was used. The alp2D PCR product was amplified from BAC 3C2 using primers alp2D-NdeI-F and alp2DEcoRV-R prior to digestion with NdeI and EcoRV and ligation to the predigested pHXZ215 to generate gene complementation plasmid pMS82-alp2D. Plasmid construction was confirmed by sequencing at Majorbio. The resulting pMS82-alp2D was introduced into the S. albus J1074 (3C2ΔalpWΔalp2D) mutant to obtain the complementation strain S. albus J1074 (3C2ΔalpWΔalp2D)/pMS82-alp2D. Exconjugants were selected on MS agar plates with apramycin and trimethoprim. Expression of Enzymes in S. albus J1074 and Feeding Experiments. The complementation plasmid pMS82-alp2D and control plasmid pMS82 were transformed to S. albus J1074 to generate S. albus J1074 (pMS82-alp2D) and S. albus J1074 (pMS82), respectively, via conjugation from ET12567/pUZ8002. Transformants were cultured at 30 °C to isolate single colonies of corresponding strains. For feeding experiments, spore suspensions of the S. albus J1074 (pMS82-alp2D) or S. albus J1074 (pMS82) were inoculated in liquid TSB and incubated for 24 h as a seed culture. A total of 1 mL of seed culture was then transferred into 30 mL of R2 liquid medium at 30 °C and 220 rpm before the substrate kinamycin F was added at 24 h. The culture broths were harvested after 1 h and extracted with an equal volume of ethyl acetate after pH adjustment to 3−4 prior to metabolite analysis.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00652. Bacterial strains, plasmids, and primers used in this study (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Blaine A. Pfeifer: 0000-0002-9338-2794 Ming Jiang: 0000-0001-9437-0632 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Science Foundation (31300033 and 21661140002) of China. We thank Prof. K. Yang of the Institute of Microbiology, Chinese Academy of Sciences, for the kind gift of kinamycin D.

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REFERENCES

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