Efficient Production of Bacterial Antibiotics Aminoriboflavin and Roseoflavin in Eukaryotic Microorganisms, Yeasts

Overview

Journal Microb Cell Fact

Publisher Biomed Central

Specialties Biotechnology
Microbiology

Date 2023 Jul 20

PMID 37474952

Authors

Kostyantyn V Dmytruk

Justyna Ruchala

Liubov R Fayura

Grzegorz Chrzanowski

Olena V Dmytruk

Andriy O Tsyrulnyk

Yuliia A Andreieva

Daria V Fedorovych

Olena I Motyka

Diethard Mattanovich

Hans Marx

Andriy A Sibirny

Affiliations

Soon will be listed here.

Abstract

Background: Actinomycetes Streptomyces davaonensis and Streptomyces cinnabarinus synthesize a promising broad-spectrum antibiotic roseoflavin, with its synthesis starting from flavin mononucleotide and proceeding through an immediate precursor, aminoriboflavin, that also has antibiotic properties. Roseoflavin accumulation by the natural producers is rather low, whereas aminoriboflavin accumulation is negligible. Yeasts have many advantages as biotechnological producers relative to bacteria, however, no recombinant producers of bacterial antibiotics in yeasts are known.

Results: Roseoflavin biosynthesis genes have been expressed in riboflavin- or FMN-overproducing yeast strains of Candida famata and Komagataella phaffii. Both these strains accumulated aminoriboflavin, whereas only the latter produced roseoflavin. Aminoriboflavin isolated from the culture liquid of C. famata strain inhibited the growth of Staphylococcus aureus (including MRSA) and Listeria monocytogenes. Maximal accumulation of aminoriboflavin in shake-flasks reached 1.5 mg L (C. famata), and that of roseoflavin was 5 mg L (K. phaffii). Accumulation of aminoriboflavin and roseoflavin by K. phaffii recombinant strain in a bioreactor reached 22 and 130 mg L, respectively. For comparison, recombinant strains of the native bacterial producer S. davaonensis accumulated near one-order less of roseoflavin while no recombinant producers of aminoriboflavin was reported at all.

Conclusions: Yeast recombinant producers of bacterial antibiotics aminoriboflavin and roseoflavin were constructed and evaluated.

References

Gassler T, Sauer M, Gasser B, Egermeier M, Troyer C, Causon T . The industrial yeast Pichia pastoris is converted from a heterotroph into an autotroph capable of growth on CO. Nat Biotechnol. 2019; 38(2):210-216. PMC: 7008030. DOI: 10.1038/s41587-019-0363-0. View

Pedrolli D, Jankowitsch F, Schwarz J, Langer S, Nakanishi S, Mack M . Natural riboflavin analogs. Methods Mol Biol. 2014; 1146:41-63. DOI: 10.1007/978-1-4939-0452-5_3. View

Marx H, Mecklenbrauker A, Gasser B, Sauer M, Mattanovich D . Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus. FEMS Yeast Res. 2009; 9(8):1260-70. DOI: 10.1111/j.1567-1364.2009.00561.x. View

Gidijala L, Kiel J, Douma R, Seifar R, van Gulik W, Bovenberg R . An engineered yeast efficiently secreting penicillin. PLoS One. 2009; 4(12):e8317. PMC: 2789386. DOI: 10.1371/journal.pone.0008317. View

Blount K, Megyola C, Plummer M, Osterman D, OConnell T, Aristoff P . Novel riboswitch-binding flavin analog that protects mice against Clostridium difficile infection without inhibiting cecal flora. Antimicrob Agents Chemother. 2015; 59(9):5736-46. PMC: 4538501. DOI: 10.1128/AAC.01282-15. View

Otani S, Kasai S, Matsui K . Isolation, chemical synthesis, and properties of roseoflavin. Methods Enzymol. 1980; 66:235-41. DOI: 10.1016/0076-6879(80)66464-7. View

Pedrolli D, Matern A, Wang J, Ester M, Siedler K, Breaker R . A highly specialized flavin mononucleotide riboswitch responds differently to similar ligands and confers roseoflavin resistance to Streptomyces davawensis. Nucleic Acids Res. 2012; 40(17):8662-73. PMC: 3458559. DOI: 10.1093/nar/gks616. View

Kasai S, Kubo Y, Yamanaka S, Hirota T, Sato H, TSUZUKIDA Y . Anti-riboflavin activity of 8N-alkyl analogues of roseoflavin in some Gram-positive bacteria. J Nutr Sci Vitaminol (Tokyo). 1978; 24(4):339-50. DOI: 10.3177/jnsv.24.339. View

Tsyrulnyk A, Andreieva Y, Ruchala J, Fayura L, Dmytruk K, Fedorovych D . Expression of yeast homolog of the mammal BCRP gene coding for riboflavin efflux protein activates vitamin B production in the flavinogenic yeast Candida famata. Yeast. 2020; 37(9-10):467-473. DOI: 10.1002/yea.3470. View

10.

Pedrolli D, Nakanishi S, Barile M, Mansurova M, Carmona E, Lux A . The antibiotics roseoflavin and 8-demethyl-8-amino-riboflavin from Streptomyces davawensis are metabolized by human flavokinase and human FAD synthetase. Biochem Pharmacol. 2011; 82(12):1853-9. DOI: 10.1016/j.bcp.2011.08.029. View

11.

Pena D, Gasser B, Zanghellini J, Steiger M, Mattanovich D . Metabolic engineering of Pichia pastoris. Metab Eng. 2018; 50:2-15. DOI: 10.1016/j.ymben.2018.04.017. View

12.

Dmytruk K, Yatsyshyn V, Sybirna N, Fedorovych D, Sibirny A . Metabolic engineering and classic selection of the yeast Candida famata (Candida flareri) for construction of strains with enhanced riboflavin production. Metab Eng. 2010; 13(1):82-8. DOI: 10.1016/j.ymben.2010.10.005. View

13.

Nguyen H, Gaillardin C, Neuveglise C . Differentiation of Debaryomyces hansenii and Candida famata by rRNA gene intergenic spacer fingerprinting and reassessment of phylogenetic relationships among D. hansenii, C. famata, D. fabryi, C. flareri (=D. subglobosus) and D. prosopidis:.... FEMS Yeast Res. 2009; 9(4):641-62. DOI: 10.1111/j.1567-1364.2009.00510.x. View

14.

Awan A, Blount B, Bell D, Shaw W, Ho J, McKiernan R . Biosynthesis of the antibiotic nonribosomal peptide penicillin in baker's yeast. Nat Commun. 2017; 8:15202. PMC: 5418595. DOI: 10.1038/ncomms15202. View

15.

Abbas C, Sibirny A . Genetic control of biosynthesis and transport of riboflavin and flavin nucleotides and construction of robust biotechnological producers. Microbiol Mol Biol Rev. 2011; 75(2):321-60. PMC: 3122625. DOI: 10.1128/MMBR.00030-10. View

16.

Wang H, Mann P, Xiao L, Gill C, Galgoci A, Howe J . Dual-Targeting Small-Molecule Inhibitors of the Staphylococcus aureus FMN Riboswitch Disrupt Riboflavin Homeostasis in an Infectious Setting. Cell Chem Biol. 2017; 24(5):576-588.e6. DOI: 10.1016/j.chembiol.2017.03.014. View

17.

Zhu T, Sun H, Wang M, Li Y . Pichia pastoris as a Versatile Cell Factory for the Production of Industrial Enzymes and Chemicals: Current Status and Future Perspectives. Biotechnol J. 2019; 14(6):e1800694. DOI: 10.1002/biot.201800694. View

18.

Dmytruk K, Sibirny A . Candida famata (Candida flareri). Yeast. 2012; 29(11):453-8. DOI: 10.1002/yea.2929. View

19.

Schneider C, Konjik V, Kissling L, Mack M . The novel phosphatase RosC catalyzes the last unknown step of roseoflavin biosynthesis in Streptomyces davaonensis. Mol Microbiol. 2020; 114(4):609-625. DOI: 10.1111/mmi.14567. View

20.

Jankowitsch F, Schwarz J, Ruckert C, Gust B, Szczepanowski R, Blom J . Genome sequence of the bacterium Streptomyces davawensis JCM 4913 and heterologous production of the unique antibiotic roseoflavin. J Bacteriol. 2012; 194(24):6818-27. PMC: 3510588. DOI: 10.1128/JB.01592-12. View