Functional analysis of the whole CYPome and Fdxome of Streptomyces venezuelae ATCC 15439

Abstract

Cytochrome P450 enzymes (CYPs or P450s) and ferredoxins (Fdxs) are ubiquitously distributed in all domains of life. Bacterial P450s are capable of catalyzing various oxidative reactions with two electrons usually donated by Fdxs. Particularly in Streptomyces, there are abundant P450s that have exhibited outstanding biosynthetic capacity of bioactive metabolites and great potential for xenobiotic metabolisms. However, no systematic study has been conducted on physiological functions of the whole cytochrome P450 complement (CYPome) and ferredoxin complement (Fdxome) of any Streptomyces strain to date, leaving a significant knowledge gap in microbial functional genomics. Herein, we functionally analyze the whole CYPome and Fdxome of Streptomyces venezuelae ATCC 15439 by investigating groups of single and sequential P450 deletion mutants, single P450 overexpression mutants, and Fdx gene deletion or repression mutants. Construction of an unprecedented P450-null mutant strain indicates that none of P450 genes are essential for S. venezuelae in maintaining its survival and normal morphology. The non-housekeeping Fdx1 and housekeeping Fdx3 not only jointly support the cellular activity of the prototypic P450 enzyme PikC, but also play significant regulatory functions. These findings significantly advance the understandings of the native functionality of P450s and Fdxs as well as their cellular interactions.

Keywords

Streptomyces / Cytochrome P450 / enzymes / CYPome / Ferredoxin / Fdxome / PikC

Data Availability Statement

The whole-genome sequence of Streptomyces venezuelae ATCC 15439 wild type (Sv-WT) and the P450-free strain Sv-Del23 were deposited into GenBank under Accession Nos. CP140569 (for Sv-WT) and CP140665 (for Sv-Del23). All other relevant data supporting the findings of this study are available in this manuscript and Supplementary Materials.

Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Given their roles as Executive Editor and Editorial Board Member, respectively, Dr. Shengying Li and Dr. Vlada B. Urlacher had no involvement in the peer-review of this article, and had no access to information regarding its peer-review. Full responsibility for the editorial process for this article was delegated to Dr. Linquan Bai.

CRediT authorship contribution statement

Shuai Li: Writing - review & editing, Writing - original draft, Validation, Investigation, Formal analysis, Conceptualization. Zhong Li: Investigation, Funding acquisition. Guoqiang Zhang: Formal analysis. Vlada B. Urlacher: Writing - review & editing. Li Ma: Writing - review & editing, Funding acquisition. Shengying Li: Writing - review & editing, Supervision, Resources, Funding acquisition, Conceptualization.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (32025001, 32071266 and 32200017), the Shandong Provincial Natural Science Foundation (ZR2022QC070 and ZR2019ZD20).

References

[1] D.A. Hopwood, Soil to genomics: the Streptomyces chromosome, Annu. Rev. Genet. 40 (2006) 1-23.

[2] K. Alam, A. Mazumder, S. Sikdar, Y. Zhao, J. Hao, C. Song, Y. Wang, R. Sarkar, S. Islam, Y. Zhang, A. Li, Streptomyces: the biofactory of secondary metabolites, Front. Microbiol. 13 (2022) 968053.

[3] S.B. Zotchev, Marine actinomycetes as an emerging resource for the drug develop- ment pipelines, J. Biotechnol. 158 (2012) 168-175.

[4] E.A. Barka, P. Vatsa, L. Sanchez, N. Gaveau-Vaillant, C. Jacquard, J.P. Meier-Kolthoff, H.P. Klenk, C. Clément, Y. Ouhdouch, G.P. van Wezel, Taxonomy, physiology, and natural products of Actinobacteria, Microbiol. Mol. Biol. Rev 80 (2016) 1-43.

[5] D.R. Nelson, T. Kamataki, D.J. Waxman, F.P. Guengerich, R.W. Estabrook, R. Fey- ereisen, F.J. Gonzalez, M.J. Coon, I.C. Gunsalus, O. Gotoh, K. Okuda, D.W. Nebert, The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature, DNA Cell Biol. 12 (1993) 1-51.

[6] J.D. Rudolf, C.Y. Chang, M. Ma, B. Shen, Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function, Nat. Prod. Rep. 34 (2017) 1141-1172.

[7] T. Sakaki, Practical application of cytochrome P450, Biol. Pharm. Bull. 35 (2012) 844-849.

[8] L.M. Podust, D.H. Sherman, Diversity of P450 enzymes in the biosynthesis of natural products, Nat. Prod. Rep 29 (2012) 1251-1266.

[9] N.M. Zondo, T. Padayachee, D.R. Nelson, K. Syed, Saprophytic to pathogenic my- cobacteria: loss of cytochrome P450s vis a vis their prominent involvement in natural metabolite biosynthesis, Int. J. Mol. Sci. 24 (2022) 149.

[10] J. Shin, J.E. Kim, Y.W. Lee, H. Son, Fungal cytochrome P450s and the P450 comple- ment (CYPome) of Fusarium graminearum, Toxins 10 (2018) 112 (Basel).

[11] D.C. Lamb, T. Skaug, H.L. Song, C.J. Jackson, L.M. Podust, M.R. Waterman, D.B. Kell, D. E. Kelly, S.L. Kelly, The cytochrome P450 complement (CYPome) of Streptomyces coelicolor A3(2), J. Biol. Chem. 277 (2002) 24000-24005.

[12] D.C. Lamb, H. Ikeda, D.R. Nelson, J. Ishikawa, T. Skaug, C. Jackson, S. Omura, M. R. Waterman, S.L. Kelly, Cytochrome P450 complement (CYPome) of the aver- mectin-producer Streptomyces avermitilis and comparison to that of Streptomyces coeli- color A3(2), Biochem. Biophys. Res. Commun. 307 (2003) 610-619.

[13] J.Y. Song, Y.J. Yoo, S.K. Lim, S.H. Cha, J.E. Kim, J.H. Roe, J.F. Kim, Y.J. Yoon, Complete genome sequence of Streptomyces venezuelae ATCC 15439, a promising cell factory for production of secondary metabolites, J. Biotechnol. 219 (2016) 57-58.

[14] D.C. Lamb, M.R. Waterman, B. Zhao, Streptomyces cytochromes P450: applications in drug metabolism, Expert Opin. Drug Metab. Toxicol. 9 (2013) 1279-1294.

[15] M.A. Cho, S. Han, Y.R. Lim, V. Kim, H. Kim, D. Kim, Streptomyces cytochrome P450 enzymes and their roles in the biosynthesis of macrolide therapeutic agents, Biomol. Ther. 27 (2019) 127-133 (Seoul).

[16] S.C. Moody, E.J. Loveridge, CYP105-diverse structures, functions and roles in an intriguing family of enzymes in Streptomyces, J. Appl. Microbiol. 117 (2014) 1549-1563.

[17] F.C. Mnguni, T. Padayachee, W. Chen, D. Gront, J.-H. Yu, D.R. Nelson, K. Syed, More P450s are involved in secondary metabolite biosynthesis in Streptomyces compared to Bacillus, Cyanobacteria, and Mycobacterium, Int. J. Mol. Sci. 21 (2020) 4814-4832.

[18] L.E. Mortenson, J.E. Carnahan, R.C. Valentine, An electron transport factor from Clostridium Pasteurianum, Biochem. Biophys. Res. Commun. 7 (1962) 448-452.

[19] S. Li, L. Du, R. Bernhardt, Redox partners: function modulators of bacterial P450 enzymes, Trends Microbiol. 28 (2020) 445-454.

[20] Z.E. Chiliza, J. Martínez-Oyanedel, K. Syed, An overview of the factors playing a role in cytochrome P450 monooxygenase and ferredoxin interactions, Biophys. Rev. 12 (2020) 1217-1222.

[21] K.J. McLean, M. Sabri, K.R. Marshall, R.J. Lawson, D.G. Lewis, D. Clift, P.R. Balding, A.J. Dunford, A.J. Warman, J.P. McVey, A.M. Quinn, M.J. Sutcliffe, N.S. Scrutton, A.W. Munro, Biodiversity of cytochrome P450 redox systems, Biochem. Soc. Trans. 33 (2005) 796-801.

[22] H. Mustila, Y. Allahverdiyeva, J. Isojärvi, E.M. Aro, M. Eisenhut, The bacterial-type [4Fe-4S] ferredoxin 7 has a regulatory function under photooxidative stress con- ditions in the cyanobacterium Synechocystis sp. PCC 6803, Biochim. Biophys. Acta Bioenerg. 1837 (2014) 1293-1304.

[23] Y. Takahashi, M. Nakamura, Functional assignment of the ORF2-iscS-iscU-iscA-hscB-hscA-fdx-ORF3 gene cluster involved in the assembly of Fe-S clusters in Escherichia coli, J. Biochem. 126 (1999) 917-926.

[24] S. Elsen, G. Efthymiou, P. Peteinatos, G. Diallinas, P. Kyritsis, J.M. Moulis, A bacte- ria-specific 2[4Fe-4S] ferredoxin is essential in Pseudomonas aeruginosa, BMC Micro- biol. 10 (2010) 271.

[25] Y.J. Chun, T. Shimada, R. Sanchez-Ponce, M.V. Martin, L. Lei, B. Zhao, S.L. Kelly, M. R. Waterman, D.C. Lamb, F.P. Guengerich, Electron transport pathway for a Strep- tomyces cytochrome P450: cytochrome P450 105D5-catalyzed fatty acid hydroxyla- tion in Streptomyces coelicolor A3(2), J. Biol. Chem. 282 (2007) 17486-17500.

[26] W. Zhang, L. Du, F. Li, X. Zhang, Z. Qu, L. Hang, Z. Li, J. Sun, F. Qi, Q. Yao, Y. Sun, C. Geng, S. Li, Mechanistic insights into interactions between bacterial class I P450 enzymes and redox partners, ACS Catal. 8 (2018) 9992-10003.

[27] X. Liu, F. Li, T. Sun, J. Guo, X. Zhang, X. Zheng, L. Du, W. Zhang, L. Ma, S. Li, Three pairs of surrogate redox partners comparison for class I cytochrome P450 enzyme activity reconstitution, Commun. Biol. 5 (2022) 791.

[28] Y. Xue, D. Wilson, L. Zhao, H. Liu, D.H. Sherman, Hydroxylation of macrolactones YC-17 and narbomycin is mediated by the pikC -encoded cytochrome P450 in Strep- tomyces venezuelae, Chem. Biol. 5 (1998) 661-667.

[29] H. Huang, G. Zheng, W. Jiang, H. Hu, Y. Lu, One-step high-efficiency CRISPR/Cas9-mediated genome editing in Streptomyces, Acta Biochim. Biophys. Sin. 47 (2015) 231-243.

[30] Y. Zhao, L. Li, G. Zheng, W. Jiang, Z. Deng, Z. Wang, Y. Lu, CRISPR/dCas9-mediated multiplex gene repression in Streptomyces, Biotechnol. J. 13 (2018) e1800121.

[31] K. Blin, S. Shaw, A.M. Kloosterman, Z. Charlop-Powers, G.P. van Wezel, M. H. Medema, T. Weber, antiSMASH 6.0: improving cluster detection and compar- ison capabilities, Nucleic Acids Res. 49 (2021) W29-W35.

[32] P. Gouet, E. Courcelle, D.I. Stuart, F. Métoz, ESPript: analysis of multiple sequence alignments in PostScript, Bioinformatics 15 (1999) 305-308.

[33] T. Kieser, M.J. Bibb, K.F. Chater, M.J. Butter, D.A. Hopwood, M. Bittner, Practical Streptomyces genetics: a laboratory manual, John Innes Foundation Norwich 291 (2000).

[34] Z. Li, Y. Jiang, X. Zhang, Y. Chang, S. Li, X. Zhang, S. Zheng, C. Geng, P. Men, L. Ma, Y. Yang, Z. Gao, Y. Tang, S. Li, Fragrant venezuelaenes A and B with a 5-5-6-7 tetracyclic skeleton: discovery, biosynthesis, and mechanisms of central catalysts, ACS Catal. 10 (2020) 5846-5851.

[35] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method, Methods 25 (2001) 402-408.

[36] J.E. Kim, J.S. Choi, J.H. Roe, Growth and differentiation properties of pikromycin-producing Streptomyces venezuelae ATCC 15439, J. Microbiol. 57 (2019) 388-395.

[37] Y. Xue, L. Zhao, H. Liu, D.H. Sherman, A gene cluster for macrolide antibiotic biosyn- thesis in Streptomyces venezuelae: architecture of metabolic diversity, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 12111-12116.

[38] Y. Xue, D.H. Sherman, Biosynthesis and combinatorial biosynthesis of pikromycin-related macrolides in Streptomyces venezuelae, Metab. Eng. 3 (2001) 15-26.

[39] Z. Li, L. Zhang, K. Xu, Y. Jiang, J. Du, X. Zhang, L. Meng, Q. Wu, L. Du, X. Li, Y. Hu, Z. Xie, X. Jiang, Y. Tang, R. Wu, R. Guo, S. Li, Molecular insights into the catalytic promiscuity of a bacterial diterpene synthase, Nat. Commun. 14 (2023) 4001.

[40] S. Li, L. Chi, Z. Li, M. Liu, R. Liu, M. Sang, X. Zheng, L. Du, W. Zhang, S. Li, Discovery of venediols by activation of a silent type I polyketide biosynthetic gene cluster in Streptomyces venezuelae ATCC 15439, Tetrahedron 126 (2022) 133072.

[41] R.J. Gurbiel, C.J. Batie, M. Sivaraja, A.E. True, J.A. Fee, B.M. Hoffman, D.P. Ballou, Electron-nuclear double resonance spectroscopy of 15N-enriched phthalate dioxy- genase from Pseudomonas cepacia proves that two histidines are coordinated to the [2Fe-2S] Rieske-type clusters, Biochemistry 28 (1989) 4861-4871.

[42] Z. Chen, Y. Yu, Z. Zuo, J.B. Nelson, G.k. Michalopoulos, S. Monga, S. Liu, G. Tseng, J. Luo, Targeting genomic rearrangements in tumor cells through Cas9-mediated insertion of a suicide gene, Nat. Biotechnol. 35 (2017) 543-550.

[43] G.P. van Wezel, K.J. McDowall, The regulation of the secondary metabolism of Streptomyces: new links and experimental advances, Nat. Prod. Rep. 28 (2011) 1311-1333.

[44] G. Liu, K.F. Chater, G. Chandra, G. Niu, H. Tan, Molecular regulation of antibiotic biosynthesis in Streptomyces, Microbiol, Mol. Biol. Rev. 77 (2013) 112-143.

[45] Z. Tian, Q. Cheng, F.K. Yoshimoto, L. Lei, D.C. Lamb, F.P. Guengerich, Cytochrome P450 107U1 is required for sporulation and antibiotic production in Streptomyces coelicolor, Arch. Biochem. Biophys. 530 (2013) 101-107.

[46] R.H. Lambalot, D.E. Cane, Isolation and characterization of 10-deoxymethynolide produced by Streptomyces venezuelae, J. Antibiot. 45 (1992) 1981-1982.

[47] M. Klingenberg, Pigments of rat liver microsomes, Arch. Biochem. Biophys. 75 (1958) 376-386.

[48] F.P. Guengerich, Rate-limiting steps in cytochrome P450 catalysis, Biol. Chem. 383 (2002) 1553-1564.

[49] T. Sagadin, J.L. Riehm, M. Milhim, M.C. Hutter, R. Bernhardt, Binding modes of CYP106A2 redox partners determine differences in progesterone hydroxylation product patterns, Commun. Biol. 1 (2018) 99.

[50] F. Kern, T.K.F. Dier, Y. Khatri, K.M. Ewen, J.P. Jacquot, D.A. Volmer, R. Bernhardt, Highly efficient CYP167A1 (EpoK) dependent epothilone B formation and produc- tion of 7-ketone epothilone D as a new epothilone derivative, Sci. Rep. 5 (2015) 14881.

[51] L. Ma, L. Du, H. Chen, Y. Sun, S. Huang, X. Zheng, E.S. Kim, S. Li, Reconstitution of the in vitro activity of the cyclosporine-specific P450 hydroxylase from Sebekia benihana and development of a heterologous whole-cell biotransformation system, Appl. Environ. Microbiol. 81 (2015) 6268-6275.

[52] W. Zhang, Y. Liu, J. Yan, S. Cao, F. Bai, Y. Yang, S. Huang, L. Yao, Y. Anzai, F. Kato, L. M. Podust, D.H. Sherman, S. Li, New reactions and products resulting from alter- native interactions between the P450 enzyme and redox partners, J. Am. Chem. Soc. 136 (2014) 3640-3646.

[53] Q. Lu, K. Chen, Y. Long, X. Liang, B. He, L. Yu, J. Ye, Benzo(a)pyrene degradation by cytochrome P450 hydroxylase and the functional metabolism network of Bacillus thuringiensis, J. Hazard. Mater. 366 (2019) 329-337.

[54] M. Qi, H. Huang, Y. Zhang, H. Wang, H. Li, Z. Lu, Novel tetrahydrofuran (THF) degradation-associated genes and cooperation patterns of a THF-degrading mi- crobial community as revealed by metagenomic, Chemosphere 231 (2019) 173-183.

[55] I. Nagy, F. Compernolle, K. Ghys, J. Vanderleyden, R. Demot, A single cy- tochrome-P-450 system is involved in degradation of the herbicides Eptc (S-Ethyl Dipropylthiocarbamate) and atrazine by Rhodococcus Sp strain Ni86/21, Appl. Env- iron. Microbiol. 61 (1995) 2056-2060.

[56] J.N. Volff, J. Altenbuchner, Genetic instability of the Streptomyces chromosome, Mol. Microbiol. 27 (1998) 239-246.

[57] P. Dyson, H. Schrempf, Genetic Instability and DNA Amplification in Streptomyces lividans 66, J. Bacteriol. 169 (1987) 4796-4803.

[58] U. Hornemann, C.J. Otto, X.Y. Zhang, DNA amplification in Streptomyces achromo- genes subsp. rubradiris is accompanied by a deletion, and the amplified sequences are conditionally stable and can be eliminated by two pathways, J. Bacteriol. 171 (1989) 5817-5822.

[59] P. Leblond, B. Decaris, New insights into the genetic instability of Streptomyces, FEMS Microbiol. Lett. 123 (1994) 225-232.

[60] E.T. Ralph, C. Scott, P.A. Jordan, A.J. Thomson, J.R. Guest, J. Green, Anaerobic acquisition of [4Fe 4S] clusters by the inactive FNR(C20S) variant and restora- tion of activity by second-site amino acid substitutions, Mol. Microbiol. 39 (2001) 1199-1211.

[61] C.J. Schwartz, J.L. Giel, T. Patschkowski, C. Luther, F.J. Ruzicka, H. Beinert, P.J. Ki- ley, IscR, an Fe-S cluster-containing transcription factor, represses expression of Es- cherichia coli genes encoding Fe-S cluster assembly proteins, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 14895-14900.

[62] B. Demple, H. Ding, M. Jorgensen, Escherichia coli SoxR protein: sensor/transducer of oxidative stress and nitric oxide, Methods Enzymol. 348 (2002) 355-364.

[63] Y. Xue, D. Wilson, D.H. Sherman, Genetic architecture of the polyketide syn- thases for methymycin and pikromycin series macrolides, Gene 245 (2000) 203-211.

[64] Y. Xue, D.H. Sherman, Alternative modular polyketide synthase expression controls macrolactone structure, Nature 403 (2000) 571-575.

[65] D.J. Wilson, Y. Xue, K.A. Reynolds, D.H. Sherman, Characterization and analysis of the PikD regulatory factor in the pikromycin biosynthetic pathway of Streptomyces venezuelae, J. Bacteriol. 183 (2001) 3468-3475.

[66] N.H. Bhat, R.H. Vass, P.R. Stoddard, D.K. Shin, P. Chien, Identification of ClpP sub- strates in Caulobacter crescentus reveals a role for regulated proteolysis in bacterial development, Mol. Microbiol. 88 (2013) 1083-1092.

[67] W.K. Smits, C.C. Eschevins, K.A. Susanna, S. Bron, O.P. Kuipers, L.W. Hamoen, Strip- ping Bacillus: comK auto-stimulation is responsible for the bistable response in com- petence development, Mol. Microbiol. 56 (2005) 604-614.

[68] A. Battesti, N. Majdalani, S. Gottesman, The RpoS-mediated general stress response in Escherichia coli, Annu. Rev. Microbiol. 65 (2011) 189-213.

[69] A. Küberl, T. Polen, M. Bott, The pupylation machinery is involved in iron home- ostasis by targeting the iron storage protein ferritin, Proc. Natl. Acad. Sci. U. S. A. 113 (2016) 4806-4811.

[70] M.R. Jacobson, K.E. Brigle, L.T. Bennett, R.A. Setterquist, M.S. Wilson, V.L. Cash, J. Beynon, W.E. Newton, D.R. Dean, Physical and genetic map of the ma- jor nif gene cluster from Azotobacter vinelandii, J. Bacteriol. 171 (1989) 1017-1027.

[71] J. Pérard, S. Ollagnier de Choudens, Iron-sulfur clusters biogenesis by the SUF ma- chinery: close to the molecular mechanism understanding, J. Biol. Inorg. Chem. 23 (2018) 581-596.

[72] D.R. Crooks, N. Maio, A.N. Lane, M. Jarnik, R.M. Higashi, R.G. Haller, Y. Yang, T.W. Fan, W.M. Linehan, T.A. Rouault, Acute loss of iron-sulfur clusters results in metabolic reprogramming and generation of lipid droplets in mammalian cells, J. Biol. Chem. 293 (2018) 8297-8311.

[73] D. Missiakas, S. Raina, The extracytoplasmic function sigma factors: role and regu- lation, Mol. Microbiol. 28 (1998) 1059-1066.

[74] R.P. Swift, K. Rajaram, R. Elahi, H.B. Liu, S.T. Prigge, Roles of ferredoxin-depen- dent proteins in the apicoplast of Plasmodium falciparum parasites, mBio 13 (2021) e0302321.