Genetics of actinobacterial secondary metabolism
Actinobacteria are some of the most gifted chemists of Nature: they produce an impressive array of chemically and biologically diverse small molecules (see a few examples in the Figure below), collectively known as secondary metabolites. These compounds are not essential for the producing cells, yet they apparently confer selective advantage to the producer under certain growth conditions. Antibiotics are perhaps the most publicized and valuable class of secondary metabolites.
Regulation of antibiotic biosynthesis in genus Streptomyces is one of the main foci of my research activity. Particularly, my current research projects are centered on the elucidation of all aspects of transcriptional and post-transcriptional regulation of expression of adpA (also known as bldH), a gene for pleiotropic transcriptional factor which serves as a master switch for secondary metabolism and morphogenesis in streptomycetes .
Besides regulation of biosynthesis of secondary metabolites, we are interested in genes and proteins that control unusual biochemical reactions. For example, we revealed that several chemically different modifications of carboxy group of uronic acid residue within antibiotic moenomycin are catalyzed by a single enzyme, MoeH5 (see the Figure below). The latter represents a unique case of evolution of glutamine amidotransferases into promiscuous catalyst of amidoligase type (Ostash et al. 2013).
Evolution of codon usage
Coding sequences of genes consist of codons – three adjacent nucleotides that define an amino acid to be incorporated into growing polypeptide during translation. Many amino acids are encoded by more that one codon, and the latter are referred to as synonymous. Therefore, different nucleotide sequences may code for the same polypeptide. Synonymous codons are not used uniformly across species, within single genome and even within one gene – a phenomenon known as synonymous codon usage bias (CUB). CUB represents an additional layer of genetic information, hidden within coding sequences, which may influence, for example, protein synthesis rate, folding and stability. Hence, synonymous mutatiuons, long time considered silent, might have a phenotypic manifestations, including pathological ones (see Plotkin and Kudla 2011). At the moment there is no satisfactory explanation of the driving forces behind varying CUB across tree of Life. Current experimental models (yeast, E. coli) are GC-neutral, too complex and not amenable to study CUB via complete elimination of even single codon (or knockout of respective tRNA gene), genome-wide. Our research is a contribution to understanding CUB from “GC-rich” perspective. We use Streptomyces albus J1074 as a model to glean into the evolution of usage of extremely rare (80 occurences per 6.8-Mbp genome) UUA codon in this genus. Significant efforts are also invested into development of novel methods to visualize CUB – see Zaburannyi et al. 2009, and cTools on http://biotools.online/
Below are selected publications for the last 5 years (2014-2018 рр); full list – see https://www.researchgate.net/profile/Bohdan_Ostash/contributions and https://www.ncbi.nlm.nih.gov/pubmed/?term=ostash+b
1: Yushchuk O, Ostash I, Vlasiuk I, Gren T, Luzhetskyy A, Kalinowski J, Fedorenko V, Ostash B. Heterologous AdpA transcription factors enhance landomycin production in Streptomyces cyanogenus S136 under a broad range of growth conditions. Appl Microbiol Biotechnol. 2018 Jul 28. doi: 10.1007/s00253-018-9249-1. [Epub ahead of print] PubMed PMID: 30056513.
2: Kuzhyk Y, Mutenko H, Fedorenko V, Ostash B. Analysis of Streptomyces ghanaensis ATCC14672 gene SSFG_07725 for putative γ-butyrolactone synthase. Folia Microbiol. 2018. doi: 10.1007/s12223-018-0614-3. [Epub ahead of print] PubMed PMID: 29786765.
3: Rabyk M, Yushchuk O, Rokytskyy I, Anisimova M, Ostash B. Genomic Insights into Evolution of AdpA Family Master Regulators of Morphological Differentiation and Secondary Metabolism in Streptomyces. J Mol Evol. 2018 Apr;86(3-4):204-215. doi: 10.1007/s00239-018-9834-z. Epub 2018 Mar 13. PubMed PMID: 29536136.
4: Ostash I, Kolvenbach B, Corvini PF, Fedorenko V, Ostash B, Cichocka D. Gene cloning system for sulfonamide-mineralizing Microbacterium sp. strain BR1. J Appl Genet. 2018;59(1):119-121. doi: 10.1007/s13353-017-0427-0. Epub 2018 Jan 25. PubMed PMID: 29372514.
5: Gren T, Ostash B, Babiy V, Rokytskyy I, Fedorenko V. Analysis of Streptomyces coelicolor M145 genes SCO4164 and SCO5854 encoding putative rhodaneses. Folia Microbiol (Praha). 2018 Mar;63(2):197-201. doi: 10.1007/s12223-017-0551-6. Epub 2017 Sep 23. PubMed PMID: 28942582.
6: Tsypik O, Makitrynskyy R, Bera A, Song L, Wohlleben W, Fedorenko V, Ostash B. Role of GntR Family Regulatory Gene SCO1678 in Gluconate Metabolism in Streptomyces coelicolor M145. Biomed Res Int. 2017;2017:9529501. doi: 10.1155/2017/9529501. Epub 2017 Apr 27. PubMed PMID: 28536705; PubMed Central PMCID: PMC5425828.
7: Koshla O, Lopatniuk M, Rokytskyy I, Yushchuk O, Dacyuk Y, Fedorenko V, Luzhetskyy A, Ostash B. Properties of Streptomyces albus J1074 mutant deficient in tRNA(Leu)(UAA) gene bldA. Arch Microbiol. 2017 Oct;199(8):1175-1183. doi: 10.1007/s00203-017-1389-7. Epub 2017 May 20. PubMed PMID: 28528473.
8: Rokytskyy I, Koshla O, Fedorenko V, Ostash B. Decoding options and accuracy of translation of developmentally regulated UUA codon in Streptomyces: bioinformatic analysis. Springerplus. 2016 Jul 4;5(1):982. doi: 10.1186/s40064-016-2683-6. eCollection 2016. PubMed PMID: 27429891; PubMed Central PMCID: PMC4932002.
9: Horbal L, Ostash B, Luzhetskyy A, Walker S, Kalinowski J, Fedorenko V. A gene cluster for the biosynthesis of moenomycin family antibiotics in the genome of teicoplanin producer Actinoplanes teichomyceticus. Appl Microbiol Biotechnol. 2016 Sep;100(17):7629-38. doi: 10.1007/s00253-016-7685-3. Epub 2016 Jun 25. PubMed PMID: 27344593.
10: Yushchuk O, Ostash B, Pham TH, Luzhetskyy A, Fedorenko V, Truman AW, Horbal L. Characterization of the Post-Assembly Line Tailoring Processes in Teicoplanin Biosynthesis. ACS Chem Biol. 2016 Aug 19;11(8):2254-64. doi: 10.1021/acschembio.6b00018. Epub 2016 Jun 20. PubMed PMID: 27285718.
11: Shashkov AS, Streshinskaya GM, Tul’skaya EM, Senchenkova SN, Baryshnikova LM, Dmitrenok AS, Ostash BО, Fedorenko VA. Cell wall glycopolymers of Streptomyces albus, Streptomyces albidoflavus and Streptomyces pathocidini. Antonie Van Leeuwenhoek. 2016;109(7):923-36. doi: 10.1007/s10482-016-0691-8. Epub 2016 Apr 7. PubMed PMID: 27055525.
12: Tsypik O, Yushchuk O, Zaburannyi N, Flärdh K, Walker S, Fedorenko V, Ostash B. Transcriptional regulators of GntR family in Streptomyces coelicolor A3(2): analysis in silico and in vivo of YtrA subfamily. Folia Microbiol (Praha). 2016 May;61(3):209-20. doi: 10.1007/s12223-015-0426-7. Epub 2015 Oct 3. PubMed PMID: 26433722.
13: Medema MH, Kottmann R, Yilmaz P, [114 authors], Ostash B, et al. Minimum Information about a Biosynthetic Gene cluster. Nat Chem Biol. 2015 Sep;11(9):625-31. doi: 10.1038/nchembio.1890. PubMed PMID: 26284661; PubMed Central PMCID: PMC5714517.
14: Ostash B, Yushchuk O, Tistechok S, Mutenko H, Horbal L, Muryn A, Dacyuk Y, Kalinowski J, Luzhetskyy A, Fedorenko V. The adpA-like regulatory gene from Actinoplanes teichomyceticus: in silico analysis and heterologous expression. World J Microbiol Biotechnol. 2015 Aug;31(8):1297-301.
doi: 10.1007/s11274-015-1882-6. Epub 2015 Jun 4. PubMed PMID: 26041369.
15: Lopatniuk M, Ostash B, Makitrynskyy R, Walker S, Luzhetskyy A, Fedorenko V. Testing the utility of site-specific recombinases for manipulations of genome of moenomycin producer Streptomyces ghanaensis ATCC14672. J Appl Genet. 2015 Nov;56(4):547-550. doi: 10.1007/s13353-015-0283-8. Epub 2015 Mar 24. PubMed PMID: 25801470.
16: Mutenko H, Makitrinskyy R, Tsypik O, Walker S, Ostash B, Fedorenko V. [Genes for biosynthesis of butenolide-like signalling molecules in Streptomyces ghanaensis, their role in moenomycin production]. Genetika. 2014 Jun;50(6):645-51. Russian. PubMed PMID: 25715454.
17: Lopatniuk MM, Ostash BO, Luzhetskyy AN, Walker S, Fedorenko VA. [Generation and study of the strains of streptomycetes–heterologous hosts for the production of moenomycin]. Genetika. 2014 Apr;50(4):413-9. Russian. PubMed PMID: 25715443.
18: Horbal L, Kobylyanskyy A, Truman AW, Zaburranyi N, Ostash B, Luzhetskyy A, Marinelli F, Fedorenko V. The pathway-specific regulatory genes, tei15* and tei16*, are the master switches of teicoplanin production in Actinoplanes teichomyceticus. Appl Microbiol Biotechnol. 2014 Nov;98(22):9295-309.
doi: 10.1007/s00253-014-5969-z. Epub 2014 Aug 9. PubMed PMID: 25104028.
19: Rabyk MV, Ostash BO, Fedorenko VO. [Gene networks that regulate secondary metabolism in actinomycetes: pleiotropic regulators]. Tsitol Genet. 2014 Jan-Feb;48(1):67–82. Review. Ukrainian. PubMed PMID: 24791475.
20: Ostash B, Shashkov A, Streshinskaya G, Tul’skaya E, Baryshnikova L, Dmitrenok A, Dacyuk Y, Fedorenko V. Identification of Streptomyces coelicolor M145 genomic region involved in biosynthesis of teichulosonic acid-cell wall glycopolymer. Folia Microbiol (Praha). 2014 Jul;59(4):355-60. doi: 10.1007/s12223-014-0306-6.
Epub 2014 Feb 6. PubMed PMID: 24526589.
21: Zaburannyi N, Rabyk M, Ostash B, Fedorenko V, Luzhetskyy A. Insights into naturally minimised Streptomyces albus J1074 genome. BMC Genomics. 2014 Feb 5;15:97. doi: 10.1186/1471-2164-15-97. PubMed PMID: 24495463; PubMed Central PMCID: PMC3937824.
- B. Ostash, О. Yushchuk, М. Rabyk, І. Ostash, V. Fedorenko. Biology of antibiotics-inhibitors of bacterial cell wall. Lviv. LNU after I. Franko. L.: 2018. 235 p. ISBN 978-617-10-0459-7 (in Ukrainian)
- B. Ostash. Transcriptional factors of AdpA subfamily in Streptomyces: function and evolution. De Gruyter Publishing House: August 2018. 67 pages. ISBN 978-3-11-062777-0. Доступ онлайн: https://www.degruyter.com/view/product/512875
- Int Pat № WO/2008/021367 (PCT/US2007/01799) International application, C07K 14/00 (2006.01), C12N 15/52 (2006.01). Moenomycin biosynthesis-related compositions and methods of use thereof / B. Ostash, S. Walker, owner – Harvard University, USA. – № WO/2008/021367, filed 13.08.2007; publ. 25.08.20
Born and raised in Ukraine, at the foot of Carpathian Mountains, as per cultural norm I entered Ivan Franko National University of Lviv (IFNUL). I got my undergraduate degree in Genetics from IFNUL in 1999. I spent the next four years working towards a Ph.D. on understanding the biosynthesis of antitumor antibiotic landomycin E. This has been achieved by making and analyzing knockouts of individual genes for landomycin biosynthesis in actinobacterium Streptomyces globisporus 1912. Since 2003 I have worked in the field of actinobacterial genetics and genomics, first as a postdoctoral fellow in the lab of Suzanne Walker (Harvard Medical School), and then as an independent researcher at IFNUL. In 2011 I took part-time teaching position at the Department of Genetics and Biotechnology of IFNUL. I teach courses in genomics, molecular phylogenetics and bionformatics.
БГ-80Ф: “Induction of cryptic genes of actinobacteria as a method to discover novel biologically active compounds”, 2019-2021, Ministry of Education of Ukraine (MESU)
Бг-41Нр: “Universal genetic mechanism of control of production of biologically active compounds by actinomycetes”, 2016-2018, MESU
1/Бг4-18 “Genotyping of microbial isolates”, 2018, JSC “Galychfarm”
Ф80/2-2018 “Post-transcriptional modifications of tRNA as regulators of primary and secondary metabolism in actinobacteria”, 2018-2019, joint project of National Fund of Research, Ukraine and Japanese Society of Promotion of Science (JSPS)
М/18-2017+М/26-2018 “Pleiotropic transcriptional regulators of family AdpA as a tool to discover novel bioactive compounds”, 2017-2018, joint project of MESU and Bundesministerium fur Bildung and Forschung (BMBF), Germany
Ф60/2-2015+Ф60/52-2016 “Ribosome as a regulatory element of metabolism: insights from genome engineering approaches”, 2015-2016, joint project of National Fund of Research, Ukraine and Japanese Society of Promotion of Science (JSPS)
М256-2013+М83-2014 “Landomycin A – a platform for development of novel anticancer drugs”, 2013-2014, joint project of MESU and Bundesministerium fur Bildung and Forschung (BMBF), Germany
R03TW009424 “Enabling biotechnologies to generate novel phosphoglycolipid antibiotics”, 2013-2015 рр., funded by Fogarti International center at NIH. http://grantome.com/grant/NIH/R03-TW009424-03
INTAS – Ukraine 95-20: “Biotechnological improvement of novel potential anticancer antibiotic”. 1997-2000 рр., Germany.
БГ-46Ф: “Novel actinobacterial genes that control production of and resistance to antibiotics-inhibitors of peptidoglycan synthesis”, 2017-2019, MESU.
Бл-01Ф: “Са2+-transport systems and regulations of cell respiration of exocrine glands under normal and stress conditions”. 2014, MESU.
M/82-2016 “Engineering of secondary metabolism of streptomycetes using the mutations in gene for ribosomal protein S12”, 2016, MESU.
Бг-98Ф “Structural and functional genomics of biosynthesis of phosphoglycolipid family antibiotics”, 2011-2013, MESU
Бг-01Ф, “Genetic mechanisms of resistance of actinomycetes to moenomycins and role of rare codons in their biosynthesis”, 2008-2010, MESU
Бг-48П: “Biosynthesis of thiopeptides as a way of utilization of reduced sulfur species: novel energy-saving biotechnologies for mining industry: 2010-2011, MESU
Бг35-Ф “Actinomycete genes that control synthesis of aglycon part of angucycline antibiotics”, 2005-2007, MESU
1998 – Mary Lion award (Canada) for talented students from rural districts
2002 – INTAS award, for abroad training (Oviedo University, Spain)
2008 – award of Lviv state administration for junior scientists
2009 – scholarship of Cabinet of Ministries of Ukraine for talented young scientists
2011 – DAAD award (DAAD) A/12/04489, for two-month training in Helmholtz Institute for Pharmaceutical Research Saarland, Germany
2013 – award of Verkhovna rada of Ukraine for the most talented young scientists
2017 – DAAD award N91655938, for two-month training in Center for Biotechnology of Bielefeld University, Germany
2017 – award of Lviv city council for talented scientists
2019 – scholarship of Lviv system of researchers
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