Letters Cite This: ACS Chem. Biol. 2019, 14, 1135−1140
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Comprehensive Derivatization of Thioviridamides by Heterologous Expression Kei Kudo,†,¶ Hanae Koiwai,‡,¶ Noritaka Kagaya,† Makoto Nishiyama,§,∥ Tomohisa Kuzuyama,∥,⊥ Kazuo Shin-ya,*,†,§,∥ and Haruo Ikeda*,‡ †
National Institute of Advanced Industrial Science and Technology, 2-4-7 Aomi, Koto-ku, Tokyo, 135-0064, Japan Kitasato Institute for Life Sciences, Kitasato University, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0373, Japan § Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan ∥ Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan ⊥ Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan
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‡
S Supporting Information *
ABSTRACT: New technology for the derivatization of peptide natural products is required for drug development. Despite the recent advances in the genome sequencing technique enabling us to search for the biosynthetic genes for wide variety of natural products, the technical methods to get access to them are limited. A class of RiPPs, a recently emerged natural product family such as thioviridamide, is one of those possessing such unexplored chemical space. In this paper, we report a streamlined method to generate new thioviridamide derivatives and to assess their biological activities. Heterologous expression of 42 constructs in an engineered Streptomyces avermitilis host gave 35 designed thioviridamide derivatives, along with several unprecedented analogues. Moreover, cytotoxicity assay revealed that several derivatives showed more potent activities than those of prethioviridamide. These results indicate that this strategy can become one of the potential ways to produce supreme unnatural products.
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cluster for TVA in Streptomyces olivoviridis was identified, and heterologous expression in S. lividans TK23 demonstrated that TVA is a member of ribosomally synthesized and posttranslationally modified peptides (RiPPs).4 Later, thioviridamide was determined to be an acetone adduct of prethioviridamide, which is the true product of the biosynthetic gene cluster.5 Since only two types of secondary metabolites that possess polythioamide functional groups are known (TVA and closthioamide),6 three research groups independently investigated novel TVA analogues using the genome mining approach and seven TVA analogues were reported.7−10 From these reports, as well as our independent genome database search, more than 15 thioviridamide-like biosynthetic gene clusters were found. All 10 essential genes encoding tailoring enzymes are conserved between the gene clusters, except for the one from Mastigocladus laminosus UU774, while the slight variety of primary sequence of the precursor peptide plausibly reflects the differences among final
atural products have given great benefits to humankind over more than 70 years, although the rate of discovery of skeletally novel compounds from microorganisms has significantly decreased over time.1 During the development of drugs, delicate fine tuning such as the improvement of pharmacokinetics, drug metabolism, etc., is indispensable; derivatization of these developments is required. Structure− activity−relationship (SAR) study is also important to select pipeline candidates. Despite necessity, difficulty in the derivatization of middle- and large-molecular-weight natural products is considered to be one of the severe bottlenecks of natural product drug development. Thus, new technology for the derivatization of middle- and large-molecular-weight natural products has been explored to expand the capacities of natural products. Recent advances in genome sequencing enable genome mining approaches to determine the cryptic state of biosynthetic gene cluster for natural products. Thioviridamide (TVA) is an N-acylated undecapeptide antibiotic that induces apoptosis selectively in EIA-transformed cells.2 The most unique structural feature of TVA is the poly thioamide backbone, instead of amide bonds.3 The biosynthetic gene © 2019 American Chemical Society
Received: April 25, 2019 Accepted: June 7, 2019 Published: June 7, 2019 1135
DOI: 10.1021/acschembio.9b00330 ACS Chem. Biol. 2019, 14, 1135−1140
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Figure 1. Genome mining of a thioviridamide-like compound. (A) Alignment of 15 core peptides found in public database. Accession No. (in order from the top); BAN83916, BBC15198, ATJ00792, WP_107048385, OMI33604, OIJ91358, KIE26472, WP_018962002, KPC82025, WP_017595621, KIY13273, OXM55618, SCL51081, WP_027655658, and PRX68470. (B) Structures of thioviridamide and related derivatives. The planar structures of neothioviridamide and thioholgamide A are identical.
Figure 2. Design, construction, and production of mutated biosynthetic gene clusters: (A) schematic chart for the construction of the mutated biosynthetic gene cluster; (B) sequence comparison between the core peptide regions of tvaA (wild type) and tvaAsyn; and (C) an example of an oligonucleotide pair. The case of V1I substitution is shown as a representative. Blue represents the installed restriction sites; red represents the mutated position for amino acid substitution. 1136
DOI: 10.1021/acschembio.9b00330 ACS Chem. Biol. 2019, 14, 1135−1140
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to verify the productivity. Fermentations were performed under the same conditions that were optimized for TVA and NTV production.7,10 The culture extracts were subjected to liquid chromatography−mass spectrometry (UPLC-TOF-MS) analysis. Since the molecular formula of designed derivatives can be anticipated from the core peptide sequences, the detection of each product was based on the calculated m/z value, as well as UV absorption spectra, with maximum absorption at 271 nm, which shows the presence of thioamide functional groups.11 Every product was further confirmed to have a TVA-like structure by comparing the mass fragmentation pattern obtained from MS/MS experiment. As the result, 35 out of 42 designed derivatives were successfully produced (see Figure 3A, as well as Figures S1 and S2 and Tables S2 and S3 in the Supporting Information). The 27 designed constructs, which are classified into 19 single
structures. Thus, despite the success of some genome-based approaches, many TVA derivatives presumed from genome mining are still inaccessible (Figure 1). As the conventional approach to utilize these cryptic genes, improvement of fermentation conditions has been employed; however, it is not a realistic and universal way to express all candidate gene clusters one by one via the fermentation of wild-type strains or the heterologous expressions of the gene clusters. To fully evaluate this unexplored chemical space of TVA for the contribution to biological activities, we employed systematic approaches involving gene editing. Since TVA is the RiPPs compound, derivatization can be performed by modifying the core peptide region of precursor peptide. Here, we report a newly established methodology for introducing the amino acid substitutions into the core peptide region of the precursor peptide gene. By using this method, we designed and generated a series of TVA derivatives with a sufficient amount of each derivative for the evaluation of the biological activity in the genome engineered host strain, S. avermitilis SUKA. As the targets of RiPPs compounds, we selected the biosynthetic machineries for TVA from S. olivoviridis and neothioviridamide (NTV) from Streptomyces sp. MSB090213SC12, because the heterologous expression systems have already been established, as previously reported.7,10 We considered that a cassette exchanging system could be an efficient strategy for the diversifying amino acid sequences. To introduce the amino acid substitution(s) without changing the allele of precursor peptide gene within the entire gene cluster, we first replaced the precursor peptide gene with the selection marker by using λRED recombinase system to yield the pKU592A::tva cluster ΔtvaA::aph(3′) or pKU592A::ntv cluster ΔntvA::aph(3′) (see Figure 2A). Next, silent mutation was designed to introduce the BamHI and NheI sites at the 5′ and 3′ regions of the core peptide coding sequence, respectively (see Figure 2B). The designed sequence then was synthesized and cloned to pRED, yielding the pRED::tvaAsyn (228-bp) or pRED::ntvAsyn (264-bp). An artificial core peptide-coding region was synthesized as a pair of complemental oligonucleotides (40-mer each; see Figure 2C, as well as Table S1 in the Supporting Information). The pair was designed where the annealing product gives short dsDNA flanking with the sticky ends complementing the BamHI−NheI site in order to ligate to the same site of pRED::tvaAsyn or pRED::ntvAsyn. The ligation products then were designated to the pRED::tvaA_X#Y or pRED::ntvA_X#Y, where the reside X# was substituted with residue Y. Each of artificial precursor peptide genes was then introduced into the original allele in the biosynthetic gene cluster by Gibson’s assemble method yielding pKU592A::tva cluster ΔtvaA::tvaA_X#Y or pKU592A::ntv cluster ΔntvA::ntvA_X#Y (see the experimental procedure described in the Supporting Information). When we designed the artificial core peptide, the core peptide sequences found in the cryptic gene clusters for TVAlike compounds were referred to generate natural productmimicking derivatives. Namely, 26 out of 42 designs (32 for TVA cluster, and 10 for NTV cluster) were inspired by genome mining (see Tables S2 and S3 in the Supporting Information). On the other hand, the 16 remaining designs were completely artificial, where the amino acid residue after substitution is not found in core peptides discovered from genome mining. All designs were constructed as described above, and each was introduced into S. avermitilis SUKA strain
Figure 3. Amino acid substitutions successfully produced by using TVA biosynthetic machinery. (A) The structure and MS/MS spectroscopic analysis of TVA_I8 V. The substituted position is colored in red. Deduced mass fragmentation pattern is depicted with arrows, and their calculated m/z values are shown. The corresponding masses observed experimentally are highlighted in red boxes. Those for the other derivatives are shown in the Supporting Information. (B) Core peptide sequences discussed in the main text. Red represents the substituted position; boldface font represents the artificial core peptide partially mimicking the core peptide found by genome mining. (C) An example of desthio-derivative produced as a byproduct of the designed derivative. (D) Substitutions resulted in the production of responsible derivatives of prethioviridamide are indicated (in red), as well as those caused the low yield (in blue). 1137
DOI: 10.1021/acschembio.9b00330 ACS Chem. Biol. 2019, 14, 1135−1140
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derivatives. This surely reflects the substrate recognition mechanism of TvaH (YcaO protein) and TvaI (TfuA-like protein), which are proposed to catalyze the polythioamide formation;14,15 however, further biochemical analyses of these proteins are required for in-depth understanding. The other case, compound NTV_F10Y, was produced with two minor components. The high-resolution mass spectrum indicated that the corresponding m/z values were 1277.4545 and 1291.4740, while that for the anticipated product was calculated to be 1321.4806. By taking these differences into consideration, the two minor components were deduced: N,N′-didemethyl-dehydroxy and N-demethyl-dehydroxy derivatives, respectively. The ratio of productivity was ∼1:1, which implied that F10Y substitution caused the inhibitory effect on N-methylation activity of NtvF, the conserved methyltransferase homologue. The dehydroxy derivatives were also observed with TVA_M2A, L10A, and NTV_V8I. Next, to assess the effect of substitutions on the biological activities of TVA, we selected and purified 20 unnatural TVA derivatives (see Figure S4 in the Supporting Information). A total of 16 derivatives, along with prethioviridamide and NTV, exhibited cytotoxic activities against SKOV-3 (human ovarian adenocarcinoma), Meso-1 (malignant pleural mesothelioma), and Jurkat (immortalized human T lymphocyte) cells with IC50 value of μM to sub-μM order, while 4 derivatives did not (see Table 1, as well as Figure S5A in the Supporting Information). Among the active derivatives, 8 derivatives (TVA_M2I, M2L, M2 V, M2I_I8 V, M2I_L10F, M2I_I8 V_L10F, A6 V, and A9 V) showed more than twice as high cytotoxicity as prethioviridamide (wild-type), suggesting that the substitution at Met2, Ala6, and Ala9 into aliphatic amino
substitutions (12 of them were inspired by genome mining), and 8 double amino acid substitutions (four of them were inspired by genome mining), could produce the corresponding compounds. In contrast, 9 triple and 2 quadruple substituents were designed, and 7 and 1 of them were produced, respectively. Such flexibility of the biosynthetic pathway of TVA has not been reported until our method enabled extensive construction and verification of the modified gene clusters. With regard to the designs copying, the core peptide retrieved from genome mining, TVA_S7T-I8 V-L10F (identical to NTV10 or thioholgamide A9), TVA_A5I-S7T-I8 VL10Y (identical to thiostreptamide S48), NTV_T7S-V8I-F10L (identical to prethioviridamide7) were detected, while those copying Salinispora pacifica CNT029 (octuple substituent) or Amycolatopsis alba DSM442628 (sextuple substituent) were not. Taken together, it was accomplished to produce at least quadruple substituents by using TVA biosynthetic systems. The productivity of each substituent was compared based on the peak area of UV chromatogram at 271 nm (see Tables S2 and S3). Most of the single substituents showed comparable (>10%) titer versus wild-type sequence; however, TVA_S7T, NTV_T7S, and TVA_H11T were far less produced. Ser7 of prethioviridamide and Thr7 of NTV comprises a vinylthioether-mediated macrocycle with C-terminal cysteine.12 One possible interpretation of the lesser productivity of substitutions at the seventh residue is the strict substrate specificity of enzyme(s) catalyzing the dehydration (uncharacterized protein(s)) or the following decarboxylative thioether bond formation (TvaF or NtvE, the EpiD13 homologues) (see Figure S3 in the Supporting Information). On the other hand, His11 is the residue, which is modified by both methyltransferase (TvaG) and hydroxylase (TvaJ) (Figure S3). Because the gene disruption experiments suggest that both TvaG and TvaJ work after the leader peptide cleavage (data not shown), the relationship between H11T substitution and less productivity is unclear. When we adopted the core amino acid sequence of NTV (VMAAAATVAFHC) to insert into the biosynthetic gene cluster for TVA, a novel compound that consisted of the sequential thioamide moiety (thioamideVMAAA, where the first alanine is not thioamidated in NTV itself) was produced. Therefore, we considered that TVA-type compounds, but not the NTV-type one, are produced when employing TVA biosynthetic gene cluster. Contrary to our prediction, unprecedented derivatives of TVA were detected besides the anticipated ones in four cases. In such cases, during the course of analyzing the UPLC chromatogram of TVA_M2A, I8A, I8H, and I8L (Figure 3B), we found minor peaks close to those of the anticipated products (sequential thioamide moiety). The high-resolution mass spectrum indicated that the corresponding m/z values were 1211.5020, 1229.4567, 1295.4797, and 1271.5110, while those for the anticipated products were calculated to be 1227.4781, 1245.4350, 1311.4568, and 1287.4820, respectively (Figure S1). Therefore, they were assigned to be the differences between sulfur and oxygen atoms (Δm = 15.9772) rather than that between OH and hydrogen (Δm = 15.9949), implying that one of the amide bonds was not converted to a thioamide bond (Figure 3C). In addition, the comparisons of the mass fragmentation patterns between the new “desthio” derivatives and the corresponding compounds revealed that Ala5 and Ala6 were amide-bonded (see Figure S2). Naturally occurring TVA derivatives are known to possess a thioamide bond at this position; thus, the artificial design led to a new type of TVA
Table 1. Cytotoxicity of New Thioviridamide Derivativesa IC50 (μM) compound
SKOV-3
MESO-1
Jurkat
prethioviridamide TVA_V1I TVA_M2I TVA_M2L TVA_M2 V TVA_A6 V TVA_I8A TVA_I8A (A5-amide) TVA_I8H TVA_I8H (A5-amide) TVA_I8L TVA_I8L (A5-amide) TVA_I8 V TVA_A9 V TVA_L10F TVA_M2I- I8 V TVA_M2I-L10F TVA_I8 V-L10F TVA_M2I−I8 V-L10F NTV NTV_F10Y NTV_F10Y(dOHdMe)
2.10 10.0 0.94 0.62 0.28 0.63 3.89 4.33 n/ab n/ab 2.46 2.89 2.11 0.49 1.70 0.62 0.49 1.31 1.06 5.9 n/ab n/a
2.00 8.59 0.70 0.77 0.36 0.73 10.2 5.81 n/ab n/ab 2.11 4.59 2.26 0.72 1.51 0.38 0.39 1.52 0.64 5.3 n/ab 20.3
0.80 3.78 0.52 0.38 0.23 0.20 1.07 1.24 n/ab n/ab 0.79 0.77 0.76 0.16 0.89 0.21 0.28 0.51 0.39 0.7 3.2 13.9
a
The cytotoxic activity was significantly improved by the compounds underlined. Compounds partially mimicking the core peptide found by genome mining are written in bold letters. bn/a = not available due to high viability. 1138
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RiPPs will help to generate designed RiPPs with improved properties.
acid led to higher cytotoxicity. The additional experiment using TVA_M2A further confirmed the importance of Met2 on cytotoxic activity (see Figure S5B in the Supporting Information). Interestingly, Met2 substitutions to aliphatic residues were relatively potent to solid tumor cell lines. These results demonstrated the possibility to design the unnatural TVA derivatives with improved activity. In contrast, 4 derivatives (TVA_I8H, I8H (A5-amide), NTV_F10Y, and F10Y (dOHdMe)) abolished cytotoxicity, suggesting the importance of aliphatic residue at the 8th position and phenyl group at the 10th position for the cytotoxicity. These observations provided the first SAR information on TVA. The targets of substitutions at the position of Met2 and Ala6 exchanging as M2I, M2 V, and A6 V were referenced from the core peptides of thioviridamide-like compounds that are deduced by the genome mining (Figure 1). Taking into consideration of the facts that these naturally learned substituents, but not most of randomized derivatives, showed more potent cytotoxic activities than that of naturally occurring prethioviridamide, nature-inspired designs may give us more beneficial designs than those of randomized19 or saturated20,21 mutation. In addition to the observation that Met2 substitution may improve the cytotoxic activity of TVA derivatives, we found that A9 V, which is not found in nature, also improved the cytotoxicity. This should provide a new aspect for SAR study of TVA. As a conclusion, a newly developed method that has been reported here sets a platform for producing the designed TVA derivatives. In a similar but different approach, Kelly and coworkers reported the amino acid substitutions of precursor peptide (TsrA) for thiostrepton biosynthesis.16,17 In their approach, modified tsrA genes were prepared via site-directed mutagenesis or PCR, using long synthetic ssDNA (∼150-mer, called “ultramer”) as a template. In another case, Onaka employed site-directed mutagenesis to produce goadsporin analogues in vivo.18 In contrast to these previous approaches, our method requires an oligomeric DNA pair to be ligated into the restriction sites introduced in advance by silent mutations. In combination with our substitution method and the formerly established heterologous expression system using clean host strain, the methodology for the derivatization of RiPPs has been a well-established in vivo system. According to the results of genome mining on public databases, biosynthetic gene clusters for TVA-like compounds are widely distributed in Actinomytales micro-organisms, which is not restricted in the genus Streptomyces, implying that several of these rare polythioamidated compounds remain to be explored. For the future design of TVA derivatives toward improved bioactivity, our observations set two policies; select either the TVA or NTV biosynthetic system, according to the seventh residue to be produced, and avoid changing His11 or Cys12 (Figure 3D). In addition, swapping of the gene(s) encoding the tailoring enzyme(s) may lead to the production of highly substituted TVA derivatives, including those not produced in our experiments. Our strategy to access the chemical space of TVA derivatives is also suitable for assessing SAR of the amino acid residue at the position of interest, because the method can provide appropriate titer and ready purification for the bioassay. For instance, the effect of Met2 substitution, which shows several varieties in the sequences based on genome mining, was first comparatively evaluated by applying our methodology. Thus, further application of our strategy on TVA but also other
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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/acschembio.9b00330. Strains and cultivations; experimental procedures; supplemental figures and tables (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (K. Shin-ya). *E-mail:
[email protected] (H. Ikeda). ORCID
Kei Kudo: 0000-0001-7500-9462 Makoto Nishiyama: 0000-0001-8143-8052 Tomohisa Kuzuyama: 0000-0002-7221-5858 Kazuo Shin-ya: 0000-0002-4702-0661 Haruo Ikeda: 0000-0003-3977-7856 Author Contributions ¶
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by a grant for “Project Focused on Developing Key Technology for Discovering and Manufacturing Drugs for Next-Generation Treatment and Diagnosis” from Japan Agency for Medical Research and Development (AMED), Japan, and from Ministry of Economy, Trade and Industry (METI), Japan.
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REFERENCES
(1) Pye, C. R., Bertin, M. J., Lokey, R. S., Gerwick, W. H., and Linington, R. G. (2017) Retrospective Analysis of Natural Products Provides Insights for Future Discovery Trends. Proc. Natl. Acad. Sci. U. S. A. 114, 5601−5606. (2) Hayakawa, Y., Sasaki, K., Adachi, H., Furihata, K., Nagai, K., and Shin-Ya, K. (2006) Thioviridamide, a Novel Apoptosis Inducer in Transformed Cells from Streptomyces olivoviridis. J. Antibiot. 59, 1−5. (3) Hayakawa, Y., Sasaki, K., Nagai, K., Shin-ya, K., and Furihata, K. (2006) Structure of Thioviridamide, a Novel Apoptosis Inducer from Streptomyces olivoviridis. J. Antibiot. 59, 6−10. (4) Izawa, M., Kawasaki, T., and Hayakawa, Y. (2013) Cloning and Heterologous Expression of the Thioviridamide Biosynthesis Gene Cluster from Streptomyces olivoviridis. Appl. Environ. Microbiol. 79, 7110−7113. (5) Izawa, M., Nagamine, S., Aoki, H., and Hayakawa, Y. (2018) Identification of Essential Biosynthetic Genes and a True Biosynthetic Product for Thioviridamide. J. Gen. Appl. Microbiol. 64, 50−53. (6) Lincke, T., Behnken, S., Ishida, K., Roth, M., and Hertweck, C. (2010) Closthioamide: An Unprecedented Polythioamide Antibiotic from the Strictly Anaerobic Bacterium Clostridium cellulolyticum. Angew. Chem., Int. Ed. 49, 2011−2013. (7) Izumikawa, M., Kozone, I., Hashimoto, J., Kagaya, N., Takagi, M., Koiwai, H., Komatsu, M., Fujie, M., Satoh, N., Ikeda, H., and Shin-ya, K. (2015) Novel Thioviridamide Derivative - JBIR-140: Heterologous Expression of the Gene Cluster for Thioviridamide Biosynthesis. J. Antibiot. 68, 533−536. (8) Frattaruolo, L., Lacret, R., Cappello, A. R., and Truman, A. W. (2017) A Genomics-Based Approach Identifies a Thioviridamide-Like
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ACS Chemical Biology Compound with Selective Anticancer Activity. ACS Chem. Biol. 12, 2815−2822. (9) Kjaerulff, L., Sikandar, A., Zaburannyi, N., Adam, S., Herrmann, J., Koehnke, J., and Müller, R. (2017) Thioholgamides: ThioamideContaining Cytotoxic RiPP Natural Products. ACS Chem. Biol. 12, 2837−2841. (10) Kawahara, T., Izumikawa, M., Kozone, I., Hashimoto, J., Kagaya, N., Koiwai, H., Komatsu, M., Fujie, M., Sato, N., Ikeda, H., and Shin-ya, K. (2018) Neothioviridamide, a Polythioamide Compound Produced by Heterologous Expression of a Streptomyces sp. Cryptic RiPP Biosynthetic Gene Cluster. J. Nat. Prod. 81, 264− 269. (11) Huang, Y., Ferrie, J. J., Chen, X., Zhang, Y., Szantai-Kis, D. M., Chenoweth, D. M., and Petersson, E. J. (2016) Electronic Interactions of i, i + 1 Dithioamides: Increased Fluorescence Quenching and Evidence for n-to-Π* Interactions. Chem. Commun. 52, 7798−7801. (12) Sit, C. S., Yoganathan, S., and Vederas, J. C. (2011) Biosynthesis of Aminovinyl-Cysteine-Containing Peptides and Its Application in the Production of Potential Drug Candidates. Acc. Chem. Res. 44, 261−268. (13) Kupke, T., Kempter, C., Jung, G., and Gotz, F. (1995) Oxidative Decarboxylation of Peptides Catalyzed by Flavoprotein EpiD. Determination of Substrate Specificity Using Peptide Libraries and Neutral Loss Mass Spectrometry. J. Biol. Chem. 270, 11282− 11289. (14) Nayak, D. D., Mahanta, N., Mitchell, D. A., and Metcalf, W. W. (2017) Post-Translational Thioamidation of Methyl-Coenzyme M Reductase, a Key Enzyme in Methanogenic and Methanotrophic Archaea. eLife 6, No. e29218. (15) Mahanta, N., Liu, A., Dong, S., Nair, S. K., and Mitchell, D. A. (2018) Enzymatic Reconstitution of Ribosomal Peptide Backbone Thioamidation. Proc. Natl. Acad. Sci. U. S. A. 115, 3030−3035. (16) Li, C., Zhang, F., and Kelly, W. L. (2011) Heterologous Production of Thiostrepton A and Biosynthetic Engineering of Thiostrepton Analogs. Mol. BioSyst. 7, 82−90. (17) Li, C., Zhang, F., and Kelly, W. L. (2012) Mutagenesis of the Thiostrepton Precursor Peptide at Thr7 Impacts Both Biosynthesis and Function. Chem. Commun. 48, 558−560. (18) Onaka, H. (2005) Cloning and Characterization of the Goadsporin Biosynthetic Gene Cluster from Streptomyces sp. TPA0584. Microbiology 151, 3923−3933. (19) Young, T. S., Dorrestein, P. C., and Walsh, C. T. (2012) Codon Randomization for Rapid Exploration of Chemical Space in Thiopeptide Antibiotic Variants. Chem. Biol. 19, 1600−1610. (20) Zhang, F., and Kelly, W. L. (2015) Saturation Mutagenesis of TsrA Ala4 Unveils a Highly Mutable Residue of Thiostrepton A. ACS Chem. Biol. 10, 998−1009. (21) Tran, H. L., Lexa, K. W., Julien, O., Young, T. S., Walsh, C. T., Jacobson, M. P., and Wells, J. A. (2017) Structure−Activity Relationship and Molecular Mechanics Reveal the Importance of Ring Entropy in the Biosynthesis and Activity of a Natural Product. J. Am. Chem. Soc. 139, 2541−2544.
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