Precursor-Directed Mutational Biosynthesis ... - ACS Publications

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Precursor-directed Mutational Biosynthesis Facilitates the Assignment of Two Cytochromes P450 in Thiostrepton Biosynthesis

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Functional

Qingfei Zhenga,#, Shoufeng Wanga,#,*, Rijing Liaoa, Wen Liua,b,* a

State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China. b Huzhou Center of Bio-Synthetic Innovation, 1366 Hongfeng Road, Huzhou 313000, China. #

These authors equally contributed to this work.

* To whom correspondence should be addressed: Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Rd., Shanghai 200032, China. Shoufeng Wang, Email: [email protected], Tel: +86-21-54925539; Wen Liu, Email: [email protected], Tel: +86-21-54925111, Fax: +86-21-64166128.

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ABSTRACT Side ring-modified thiostrepton (TSR) derivatives that vary in their quinaldic acid (QA) substitution possess more potent biological activities and better pharmaceutical properties than the parent compound. In this work, we sought to introduce fluorine onto C-7’ or C-8’ of the TSR QA moiety via precursor-directed mutational biosynthesis to obtain new TSR variants. Unexpectedly, instead of the target product, the exogenous chemical feeding of 7-F-QA into the △tsrT mutant strain resulted in a unique TSR analog with an incomplete side ring structure and an unoxidized QA moiety (1). Accordingly, two cytochrome P450 genes, tsrP and tsrR, were in-frame deleted to elucidate the candidate responsible for the monooxidation of the QA moiety in TSR. The unfluorinated analog of compound 1 that was thus isolated from △tsrP (2), and the abolishment of TSR production in △tsrR revealed not only the biosynthetic logic of the TSR side ring but also the essential checkpoint in TSR maturation before macro ring closure.



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Thiostrepton (TSR, Figure 1) is one of the most structurally complex, ribosomally synthesized and post-translationally modified peptides (RiPPs),1 and its biosynthetic pathway, which contains a variety of intriguing biochemical reactions, remains elusive.2 Although thiostrepton was discovered more than sixty years ago, the biosynthetic gene clusters of TSR and its analog, siomycin (SIO), were identified only in recent years.3,4 Since then, a number of in vivo and in vitro investigations of TSR biosynthesis have been reported, specifically regarding the formation of its quinaldic acid (QA) moiety5,6 and its C-terminal amidation.7 Recent advances in the chemo-enzymatic synthesis of a thiocillin analog8 and in vitro reconstitution of the thiomuracin core scaffold9 revealed a uniform paradigm for the biosynthesis of pyridine-containing thiopeptides, in which the leader peptides are removed during the aromatization of pyridine. However, the biosynthetic logic of the dehydropiperidine-containing thiopeptide TSR is not the same, and the removal of its leader peptide is proposed to occur during the closure of the side ring.3 Although the functional assignment of enzymes involved in the formation of the TSR side ring has not yet been accomplished, the side ring-modified TSR derivatives can still be obtained via either mutagenesis10-12 or precursor-directed mutational biosynthesis.5,13,14 In a previous study, we discovered that substituted quinolone ketones could serve as QA mimics and be incorporated into the TSR skeleton by being fed into the non-TSR-producing strain, Streptomyces laurentii △tsrT.13 In this mutant strain, the TsrT-mediated C-α methylation of tryptophan that serves as the first step for QA biosynthesis (Figure 4) has been blocked, and thus △tsrT loses the ability of producing TSR.5 The synthetic QA analogs could be used to replace multiple gene functions, including tsrT, tsrA, tsrE, tsrD and/or tsrU.14 The obtained side ring-modified TSR derivatives, which varied with respect to the QA moiety of the side ring, possessed improved antibacterial activities and pharmaceutical properties.13-15 The electronic effect attributed to the introduction of fluorine onto C-5’ or C-6’ of TSR’s QA moiety exhibits a positive impact on TSR’s antimicrobial activity.13-15 Thus, we sought to produce two additional TSR derivatives, 7’-fluoro-TSR and 8’-fluoro-TSR, via the same strategy, i.e., by feeding key building block analogs 7-F-QA and 8-F-QA into the mutant strain △tsrT. 7-F-QA and 8-F-QA were synthesized according to the robust protocol that we had previously developed16 (Supplementary data) and were then fed into the △tsrT strain during the fermentation process. The exogenous feeding of 7-F-QA into △tsrT resulted in the accumulation of a new TSR analog (1, Figure 2b) that has a similar ultraviolet absorption spectrum as the parent compound (Figure S1). However, the HPLC-MS analyses indicated that compound 1 (m/z 1483.05 [M + H]+) did not have the same molecular weight as the target compound, 7’-fluoro-TSR (m/z calcd. for 1682.02 [M + H]+). To elucidate the structure of compound 1, we performed the fermentation and chemical feeding on a large scale, and then the structure of compound 1 was characterized via 1H, 13C, 19F and 2D NMR analyses (Supplementary data). Comparative analysis of the NMR data from compound 1 and TSR showed that compound 1 is a side ring-unclosed TSR analog that lacks Ile1 and Ala2. Moreover, Dha3 in TSR is replaced by a 1, 2-diketone structure in compound 1


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(Figure 1). Meanwhile, C-7’ and C-8’ of the QA moiety in compound 1 are not epoxidized, suggesting that the corresponding monooxygenase cannot tolerate the C-7’-fluorinated substrate. The lack of the C-7’/C-8’ epoxide structure in the QA moiety abolishes the side ring closure, allowing hydrolysis of the Ile1 and Ala2 residues. The resulting enamine structure of Dha3 might prevent further nonspecific hydrolysis via isomerization and deamination (Figure 4). Although fluoro-olefins can be stereo-selectively epoxidized by chiral dioxirane,17 the fluorine still blocks the epoxidation of the QA moiety in compound 1 by affecting the recognition between the monooxygenase and the substrate. In contrast, there was no such TSR analog obtained from the feeding of 8-F-QA (Figure 2b) to △tsrT, suggesting that 8-F-QA could not be loaded onto the precursor peptide (TsrH) and thus did not restore the production of the TSR analogs. The structure of compound 1 and the results of the chemical feeding experiments provide insights into the biosynthetic logic of the TSR side ring: 1) QA is loaded onto the precursor peptide (TsrH) before the formation of dehydropiperidine, 2) the epoxidation of QA moiety occurs before the removal of the leader peptide, and 3) the dihydroxylation of Ile10 occurs before the side ring-closure. The successful heterologous production of TSR in other Streptomyces hosts (e.g., S. lividans and S. actuosus) indicates that all of the genes required for the maturation of TSR are involved in its biosynthetic gene cluster, tsr.18 There are only two genes in tsr coding for cytochromes P450, tsrP and tsrR, that are predicted to perform the function of oxidative modification and probably be responsible for the epoxidation of the QA moiety and the dihydroxylation of Ile10 in TSR (Figure 1a). Given the fact that the fluorization on C-7’ of QA moiety blocks the epoxidation function of TsrP or TsrR, we performed in-frame deletions of tsrP and tsrR to construct the mutant strains △tsrP and △tsrR. HPLC-MS analyses of the fermentation metabolites of △tsrP and △tsrR revealed that a new TSR analog (2) is produced by △tsrP (m/z 1465.02 [M + H]+), but no TSR analogs were produced by △tsrR (Figure 2c). The 1H, 13C, and 2D NMR analyses (Supplementary data) revealed that compound 2 possesses the same structure as unfluorinated compound 1 (Figure 1), indicating that TsrP catalyzes the epoxidation of the TSR QA moiety. Thus, the dihydroxylation of Ile10 is mediated by TsrR and serves as an essential checkpoint in TSR maturation before dehydropiperidine formation. Meanwhile, the in trans complementation of tsrP and tsrR restored the production of TSR in the △tsrP and △tsrR mutants (Figure 2c). Phylogenetic analysis of TsrP, TsrQ and their homologous proteins were conducted to predict and distinguish the epoxidation and dihydroxylation functions of other cytochromes P450 that may serve as monooxygenases or dioxygenases (Figure 3). The precursor-directed mutational biosynthesis and the genetic loss-of-function experiments showed that both the loading of QA onto precursor peptide (TsrH) and the dihydroxylation of Ile10 are checkpoints in TSR maturation prior to macro ring closure. The previous in vitro enzymatic assays8,9 and the isolation of linear shunt products from the [4+2] cyclase-gene knockout mutant strains10,19-21 indicate that the leader peptides of pyridine-containing thiopeptides can be either removed by the [4+2] cyclase or nonspecifically hydrolyzed. The deletion of genes that play roles upstream of pyridine formation can also result in linear shunt products.22 Unlike the



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biosynthetic logic of pyridine-containing thiopeptides, the leader peptide of TSR is not removed during the dehydropiperidine formation, and thus nonspecific hydrolysis may not occur before macro ring closure. The gene knockout of the homolog of [4+2] cyclase (TsrL) in the TSR-producing strain resulted in no linear shunt products (Figure S4), further suggesting this notion. Benefitting from the precursor-directed mutational biosynthesis, the desire to understand the biosynthetic logic of the bicyclic thiopeptide TSR has been renewed, specifically regarding its macro ring and side ring closure, as well as the timing of QA loading. QA or its analogs are loaded onto Thr12 of the core peptide before macro ring closure, and if the QA analog (e.g., 8-F-QA) cannot be incorporated, the formation of dehydropiperidine mediated by TsrL is abolished. Because compound 2 could not be further oxidized by being fed into △tsrT (Figure 2c) or by TsrP in vitro (Figures S3), the notion that TsrP recognizes the leader peptide-containing substrate is thereby suggested. Thus the resulting leader peptide (LP)-containing epoxy intimidate is predicted to simultaneously undergo the removal of LP and side ring-closure via SN2 nucleophilic attack of the Ile1 amino group (Figure 4). Intriguingly, compound 2 can also be obtained as a by-product via mutagenesis-induced modifications to the TSR precursor peptide.23 Antibacterial bioassays showed that compounds 1 and 2 both lost their remarkable biological activities (Table S4), most likely due to their incomplete side ring structures. However, the 1, 2-diketone structures of compounds 1 and 2 may serve as chemical handles for further modifications via semi-synthesis to develop new TSR-type chemical probes.24 The unexpected results of this study suggest that precursor-directed mutational biosynthesis can not only expand molecular diversity and utility25-27 but also provide insights and facilitate the understanding of complex biosynthetic pathways. Moreover, the function elucidation and phylogenetic analysis of TsrP and TsrR may facilitate understanding the multiple functions of cytochromes P450 and provide insights into the future studies of genome mining for epoxy- and/or dihydroxy-containing natural products. In conclusion, we performed precursor-directed mutational biosynthesis by feeding 7-F-QA and 8-F-QA into the mutant strain △tsrT, and a new fluorine-substituted TSR derivative (1) was obtained. Analyses of the chemical structure and biosynthetic process of compound 1 motivated us to determine the functions of the two cytochrome P450 genes in the tsr gene cluster. Accordingly, two mutant strains, △tsrP and △tsrR, were constructed, and then another TSR derivative (2) was isolated from △tsrP. Therefore, the functions of TsrP and TsrR are validated. TsrP meditates the epoxidation of the QA moiety, and TsrR catalyzes the dihydroxylation of Ile10. On the other hand, because TsrR functions before TsrP during the molecular maturation of TSR and no corresponding products were identified in the metabolites of △tsrR, the TsrR-meditated dihydroxylation of Ile10 should be an essential checkpoint in TSR maturation prior to macro ring closure. METHODS


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Detailed descriptions of experimental procedures and reagents are provided in the Supporting Information. ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxxxxxxxxxxx. AUTHOR INFORMATION Corresponding Authors *Tel.: +86-21-54925539. E-mail: [email protected]. *Tel.: +86-21-54925111. Fax: +86-21-64166128. E-mail: [email protected]. Author Contributions # These authors contributed equally to this work Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported in part by grants from NSFC (31430005 and 91413101), STCSM (13XD1404500 and 14JC1407700), “973 program” (2012CB721100), and MST (2012AA02A706) of China for W. Liu. This research was also funded in part by the Youth Innovation Foundation Fellowship, Chinese Academy of Sciences (2014228) for S. Wang. We also thank H.G. Floss at University of Washington, for providing the TSR-producing strain S. laurentii ATCC 31255. REFERENCES (1) Arnison, P. G., Bibb, M. J., Bierbaum, G., Bowers, A. A., Bugni, T. S., Bulaj, G., Camarero, J. A., Campopiano, D. J., Challis, G. L., Clardy, J., Cotter, P. D., Craik, D. J., Dawson, M., Dittmann, E., Donadio, S., Dorrestein, P. C., Entian, K. D., Fischbach, M. A., Garavelli, J. S., Goransson, U., Gruber, C. W., Haft, D. H., Hemscheidt, T. K., Hertweck, C., Hill, C., Horswill, A. R., Jaspars, M., Kelly, W. L., Klinman, J. P., Kuipers, O. P., Link, A. J., Liu, W., Marahiel, M. A., Mitchell, D. A., Moll, G. N., Moore, B. S., Müller, R., Nair, S. K., Nes, I. F., Norris, G. E., Olivera, B. M., Onaka, H., Patchett, M. L., Piel, J., Reaney, M. J., Rebuffat, S., Ross, R. P., Sahl, H. G., Schmidt, E. W., Selsted, M. E., Severinov, K., Shen, B., Sivonen, K., Smith, L., Stein, T., Süssmuth, R. D., Tagg, J. R., Tang, G. L., Truman, A. W., Vederas, J. C., Walsh, C. T., Walton, J. D., Wenzel, S. C., Willey, J. M., and van der Donk, W. A. (2013) Ribosomally synthesized and post-translationally modified peptide natural products: overview and



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FIGURE LEGENDS Figure 1 The biosynthetic gene cluster tsr (a) and the chemical structures of compound 1 and 2 in this study (b). The two genes coding for cytochromes P450, tsrP and tsrR, are indicated in blue and red respectively, while the genes responsible for quinaldic acid biosynthesis are indicated in green. The sites oxidized by TsrP and TsrR are also highlighted in blue and red in the chemical structures. Figure 2 The chemical structures of synthesized precursors (7-F-QA and 8-F-QA) for chemical feeding (a), HPLC analyses of the fermentation cultures of the wide type strain WT and mutant strain ∆tsrT, in the absence or presence of the exogenous QA analogs (b), and HPLC analyses of the fermentation cultures of the mutant strains ∆tsrP and ∆tsrR (c). The in trans complementation of tsrP and tsrR restores the production of TSR in the ∆tsrP and ∆tsrR, while compound 2 cannot be further biotransformed by ∆tsrT. λ = 254 nm. Figure 3 Phylogenetic analysis of TsrP, TsrR and other homologous cytochromes P450. The tree was constructed with the neighbor-joining method. TsrP and TsrR are indicated by blue ● and red ■, respectively. Scale bar, 0.2 substitutions per site. Figure 4 The different biosynthetic pathways for TSR side ring formation and leader peptide removal in WT, ∆tsrP and ∆tsrT strains in the presence of the exogenous QA analogs as feeding precursors.


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