Genetic and Biochemical Characterization of Halogenation and Drug Transportation Genes Encoded in the Albofungin Biosynthetic Gene Cluster

Overview

Journal Appl Environ Microbiol

Specialties Environmental Health
Microbiology

Date 2022 Aug 24

PMID 36000868

Authors

Zhe-Chong Wang

I-Wen Lo

Kuan-Hung Lin

An Ning Cheng

Saeid Malek Zadeh

Yen-Hua Huang

Tsung-Lin Li

Affiliations

Soon will be listed here.

Abstract

Albofungin, a hexacyclic aromatic natural product, exhibits broad-spectrum antimicrobial activity. Its biosynthesis, regulation, and resistance remain elusive. Here, we report the albofungin () biosynthetic gene cluster (BGC) from its producing strain Streptomyces tumemacerans JCM5050. The nascent BGC encodes 70 putative genes, including regulators, transporters, type II polyketide synthases (PKSs), oxidoreductase, and tailoring enzymes. To validate the intactness and functionality of the BGC, we developed an Escherichia coli- shuttle bacterial artificial chromosome system, whereby the BGC was integrated into the genome of a nonproducing host via heterologous conjugation, wherefrom albofungin can be produced, confirming that the BGC is in effect. We then delimited the boundaries of the BGC by means of CRISPR-Cas9 DNA editing, concluding a minimal but essential 60-kb BGC ranging from to . The gene encoding a reduced flavin adenine dinucleotide (FADH)-dependent halogenase was examined and is capable of transforming albofungin to halogen-substituted congeners and . The gene encoding a transporter was examined . The presence/absence of or demonstrated that the MIC of albofungin is subject to alteration when an extracellular polysaccharide intercellular adhesin was formed. Despite that halogenation of albofungin somewhat increases binding affinity to transglycosylase (TGase), albofungin with/without a halogen substituent manifests similar antimicrobial activity. Halogenation, however, limits overall dissemination and effectiveness given a high secretion rate, weak membrane permeability, and high hydrophobicity of the resulting products, whereby the functions of and are correlated with drug detoxification/resistance for the first time. Albofungin, a natural product produced from Streptomycetes, exhibits bioactivities against bacteria, fungi, and tumor cells. The biosynthetic logic, regulations, and resistance of albofungin remain yet to be addressed. Herein, the minimal albofungin () biosynthetic gene cluster (BGC) from the producing strain Streptomyces tumemacerans JCM5050 was precisely delimited using the Escherichia coli shuttle bacterial artificial chromosome system, of which the gene essentiality was established and . Next, we characterized two genes and encoded in the BGC, which act as a reduced flavin adenine dinucleotide (FADH)-dependent halogenase and an albofungin-congeners transporter, respectively. While each testing microorganism exhibited different sensitivities to albofungins, the MIC values of albofungins against testing strains with/without and/or were subject to considerable changes. Halogen-substituted albofungins mediated by OrfA manifested overall compromised dissemination and effectiveness, revealing for the first time that two functionally distinct proteins OrfA and OrfL are associated together, exerting a novel "belt and braces" mechanism in antimicrobial detoxification/resistance.

References

Yuan Y, Barrett D, Zhang Y, Kahne D, Sliz P, Walker S . Crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis. Proc Natl Acad Sci U S A. 2007; 104(13):5348-53. PMC: 1817829. DOI: 10.1073/pnas.0701160104. View

Ortega M, Cogan D, Mukherjee S, Garg N, Li B, Thibodeaux G . Two Flavoenzymes Catalyze the Post-Translational Generation of 5-Chlorotryptophan and 2-Aminovinyl-Cysteine during NAI-107 Biosynthesis. ACS Chem Biol. 2016; 12(2):548-557. PMC: 5315687. DOI: 10.1021/acschembio.6b01031. View

Lu Y, Wang Y, Zhu W . Nonbonding interactions of organic halogens in biological systems: implications for drug discovery and biomolecular design. Phys Chem Chem Phys. 2010; 12(18):4543-51. DOI: 10.1039/b926326h. View

Dong C, Flecks S, Unversucht S, Haupt C, van Pee K, Naismith J . Tryptophan 7-halogenase (PrnA) structure suggests a mechanism for regioselective chlorination. Science. 2005; 309(5744):2216-9. PMC: 3315827. DOI: 10.1126/science.1116510. View

Pasqua M, Bonaccorsi di Patti M, Fanelli G, Utsumi R, Eguchi Y, Trirocco R . Host - Bacterial Pathogen Communication: The Wily Role of the Multidrug Efflux Pumps of the MFS Family. Front Mol Biosci. 2021; 8:723274. PMC: 8350985. DOI: 10.3389/fmolb.2021.723274. View

Luckner P, Brandsch M . Interaction of 31 beta-lactam antibiotics with the H+/peptide symporter PEPT2: analysis of affinity constants and comparison with PEPT1. Eur J Pharm Biopharm. 2004; 59(1):17-24. DOI: 10.1016/j.ejpb.2004.07.008. View

Cobb R, Wang Y, Zhao H . High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system. ACS Synth Biol. 2014; 4(6):723-8. PMC: 4459934. DOI: 10.1021/sb500351f. View

May A, Ehrlich R, Balashov S, Ehrlich G, Shanmugam M, Fine D . Complete Genome Sequence of Aggregatibacter actinomycetemcomitans Strain IDH781. Genome Announc. 2016; 4(6). PMC: 5105115. DOI: 10.1128/genomeA.01285-16. View

Preisitsch M, Heiden S, Beerbaum M, Niedermeyer T, Schneefeld M, Herrmann J . Effects of Halide Ions on the Carbamidocyclophane Biosynthesis in Nostoc sp. CAVN2. Mar Drugs. 2016; 14(1):21. PMC: 4728517. DOI: 10.3390/md14010021. View

10.

Sevillano L, Vijgenboom E, van Wezel G, Diaz M, Santamaria R . New approaches to achieve high level enzyme production in Streptomyces lividans. Microb Cell Fact. 2016; 15:28. PMC: 4743123. DOI: 10.1186/s12934-016-0425-7. View

11.

Bister B, Bischoff D, Nicholson G, Stockert S, Wink J, Brunati C . Bromobalhimycin and chlorobromobalhimycins--illuminating the potential of halogenases in glycopeptide antibiotic biosyntheses. Chembiochem. 2003; 4(7):658-62. DOI: 10.1002/cbic.200300619. View

12.

Sung M, Lai Y, Huang C, Chou L, Shih H, Cheng W . Crystal structure of the membrane-bound bifunctional transglycosylase PBP1b from Escherichia coli. Proc Natl Acad Sci U S A. 2009; 106(22):8824-9. PMC: 2689995. DOI: 10.1073/pnas.0904030106. View

13.

Kong L, Zhang W, Chooi Y, Wang L, Cao B, Deng Z . A Multifunctional Monooxygenase XanO4 Catalyzes Xanthone Formation in Xantholipin Biosynthesis via a Cryptic Demethoxylation. Cell Chem Biol. 2016; 23(4):508-16. DOI: 10.1016/j.chembiol.2016.03.013. View

14.

Shah M, Liu J, Zhang Q, Stout C, Halpert J . Halogen-π Interactions in the Cytochrome P450 Active Site: Structural Insights into Human CYP2B6 Substrate Selectivity. ACS Chem Biol. 2017; 12(5):1204-1210. PMC: 5748325. DOI: 10.1021/acschembio.7b00056. View

15.

Imai Y, Inoue Y, Nakanishi I, Kitaura K . Cl-pi interactions in protein-ligand complexes. Protein Sci. 2008; 17(7):1129-37. PMC: 2442010. DOI: 10.1110/ps.033910.107. View

16.

Mahmoudi H, Pourhajibagher M, Chiniforush N, Soltanian A, Alikhani M, Bahador A . Biofilm formation and antibiotic resistance in meticillin-resistant and meticillin-sensitive Staphylococcus aureus isolated from burns. J Wound Care. 2019; 28(2):66-73. DOI: 10.12968/jowc.2019.28.2.66. View

17.

Hertweck C, Luzhetskyy A, Rebets Y, Bechthold A . Type II polyketide synthases: gaining a deeper insight into enzymatic teamwork. Nat Prod Rep. 2007; 24(1):162-90. DOI: 10.1039/b507395m. View

18.

Matsuda K, Tomita T, Shin-Ya K, Wakimoto T, Kuzuyama T, Nishiyama M . Discovery of Unprecedented Hydrazine-Forming Machinery in Bacteria. J Am Chem Soc. 2018; 140(29):9083-9086. DOI: 10.1021/jacs.8b05354. View

19.

Gomi S, Sezaki M, Hamada M, Kondo S, Takeuchi T . Biosynthesis of benanomicins. J Antibiot (Tokyo). 1989; 42(7):1145-50. DOI: 10.7164/antibiotics.42.1145. View

20.

Mori S, Pang A, Chandrika N, Garneau-Tsodikova S, Tsodikov O . Unusual substrate and halide versatility of phenolic halogenase PltM. Nat Commun. 2019; 10(1):1255. PMC: 6424973. DOI: 10.1038/s41467-019-09215-9. View