Heterologous cross-expression of oxygenase and glycosyltransferase genes in streptomycetes, producing angucyclic antibiotics

The heterologous expression of oxygenase genes urdM, ovmOIII, lanM2, lndZ5 and glycosyltransferase gene urdGT2 in different angucycline producers of genus Streptomyces has been carried out. The introduction of genes urdM, lanM2 and urdGT2 results in the accumulation of new glycosylated compounds in...

Ausführliche Beschreibung

Gespeichert in:

Bibliographische Detailangaben
Datum:	2009
Hauptverfasser:	Kobylyanskyy, A., Ostash, B., Fedorenko V.
Format:	Artikel
Sprache:	English
Veröffentlicht:	Інститут клітинної біології та генетичної інженерії НАН України 2009
Schriftenreihe:	Цитология и генетика
Schlagworte:	Оригинальные работы
Online Zugang:	http://dspace.nbuv.gov.ua/handle/123456789/66645
Tags:	Tag hinzufügen Keine Tags, Fügen Sie den ersten Tag hinzu!
Назва журналу:	Digital Library of Periodicals of National Academy of Sciences of Ukraine
Zitieren:	Heterologous cross-expression of oxygenase and glycosyltransferase genes in streptomycetes, producing angucyclic antibiotics / A. Kobylyanskyy, B. Ostash, V. Fedorenko // Цитология и генетика. — 2009. — Т. 43, № 3. — С. 55-62. — Бібліогр.: 15 назв. — англ.

Institution

Digital Library of Periodicals of National Academy of Sciences of Ukraine

id	irk-123456789-66645
record_format	dspace
spelling	irk-123456789-666452014-07-20T03:01:49Z Heterologous cross-expression of oxygenase and glycosyltransferase genes in streptomycetes, producing angucyclic antibiotics Kobylyanskyy, A. Ostash, B. Fedorenko V. Оригинальные работы The heterologous expression of oxygenase genes urdM, ovmOIII, lanM2, lndZ5 and glycosyltransferase gene urdGT2 in different angucycline producers of genus Streptomyces has been carried out. The introduction of genes urdM, lanM2 and urdGT2 results in the accumulation of new glycosylated compounds in several strains under investigation. A number of processed recombinant strains display streptomycin sensitivity and decrease in total antibacterial activity. The obtained data is an evidence of changes in antibiotics production, resulting from post polyketide synthase (postPKS) tailoring gene expression across streptomycete strains. Our study demonstrates the potential of post PKS tailoring genes for generation of novel bioactive metabolites. Здійснено гетерологічну експресію генів оксигеназ urdM, ovmOIII, lanM2, lndZ5 та глікозилтрансферази urdGT2 у різних видів продуцентів ангуциклінів з роду Streptomyces. Введення генів urdM, lanM2 та urdGT2 у деякі з досліджуваних штамів зумовлює накопичення ними нових глікозильованих сполук. Виявлено виникнення чутливості до стрептоміцину та різке зниження загальної антибактерійної активності у деяких із одержаних рекомбінантних штамів. Отримані дані є свідченням модифікації продукції антибіотиків, яка є наслідком експресії генів постполікетидного синтезу (пост-ПКС) у штамах стрептоміцетів. Наші дослідження демонструють потенціал генів пост-ПКС у створенні нових біологічно активних метаболітів. Проведена гетерологическая экспрессия генов оксигеназ urdM, ovmOIII, lanM2, lndZ5 и гликозилтрансферазы urdGT2 в разных видах продуцентов ангуциклинов из рода Streptomyces. Введение генов urdM, lanM2 и urdGT2 в некоторые из исследуемых штаммов обусловливает накопление ими новых гликозилированных соединений. Обнаружено появление чувствительности к стрептомицину и резкое снижение общей антибактериальной активности у некоторых из полученных рекомбинантных штаммов. Представленные данные свидетельствуют о модификации продукции антибиотиков, которая является следствием экспрессии генов пост-поликетидного синтеза (пост-ПКС) в штаммах стрептомицетов. Наши исследования демонстрируют потенциал генов пост-ПКС в создании новых биологически активных метаболитов. 2009 Article Heterologous cross-expression of oxygenase and glycosyltransferase genes in streptomycetes, producing angucyclic antibiotics / A. Kobylyanskyy, B. Ostash, V. Fedorenko // Цитология и генетика. — 2009. — Т. 43, № 3. — С. 55-62. — Бібліогр.: 15 назв. — англ. 0564-3783 http://dspace.nbuv.gov.ua/handle/123456789/66645 579.25:579.842.11 en Цитология и генетика Інститут клітинної біології та генетичної інженерії НАН України
institution	Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection	DSpace DC
language	English
topic	Оригинальные работы Оригинальные работы
spellingShingle	Оригинальные работы Оригинальные работы Kobylyanskyy, A. Ostash, B. Fedorenko V. Heterologous cross-expression of oxygenase and glycosyltransferase genes in streptomycetes, producing angucyclic antibiotics Цитология и генетика
description	The heterologous expression of oxygenase genes urdM, ovmOIII, lanM2, lndZ5 and glycosyltransferase gene urdGT2 in different angucycline producers of genus Streptomyces has been carried out. The introduction of genes urdM, lanM2 and urdGT2 results in the accumulation of new glycosylated compounds in several strains under investigation. A number of processed recombinant strains display streptomycin sensitivity and decrease in total antibacterial activity. The obtained data is an evidence of changes in antibiotics production, resulting from post polyketide synthase (postPKS) tailoring gene expression across streptomycete strains. Our study demonstrates the potential of post PKS tailoring genes for generation of novel bioactive metabolites.
format	Article
author	Kobylyanskyy, A. Ostash, B. Fedorenko V.
author_facet	Kobylyanskyy, A. Ostash, B. Fedorenko V.
author_sort	Kobylyanskyy, A.
title	Heterologous cross-expression of oxygenase and glycosyltransferase genes in streptomycetes, producing angucyclic antibiotics
title_short	Heterologous cross-expression of oxygenase and glycosyltransferase genes in streptomycetes, producing angucyclic antibiotics
title_full	Heterologous cross-expression of oxygenase and glycosyltransferase genes in streptomycetes, producing angucyclic antibiotics
title_fullStr	Heterologous cross-expression of oxygenase and glycosyltransferase genes in streptomycetes, producing angucyclic antibiotics
title_full_unstemmed	Heterologous cross-expression of oxygenase and glycosyltransferase genes in streptomycetes, producing angucyclic antibiotics
title_sort	heterologous cross-expression of oxygenase and glycosyltransferase genes in streptomycetes, producing angucyclic antibiotics
publisher	Інститут клітинної біології та генетичної інженерії НАН України
publishDate	2009
topic_facet	Оригинальные работы
url	http://dspace.nbuv.gov.ua/handle/123456789/66645
citation_txt	Heterologous cross-expression of oxygenase and glycosyltransferase genes in streptomycetes, producing angucyclic antibiotics / A. Kobylyanskyy, B. Ostash, V. Fedorenko // Цитология и генетика. — 2009. — Т. 43, № 3. — С. 55-62. — Бібліогр.: 15 назв. — англ.
series	Цитология и генетика
work_keys_str_mv	AT kobylyanskyya heterologouscrossexpressionofoxygenaseandglycosyltransferasegenesinstreptomycetesproducingangucyclicantibiotics AT ostashb heterologouscrossexpressionofoxygenaseandglycosyltransferasegenesinstreptomycetesproducingangucyclicantibiotics AT fedorenkov heterologouscrossexpressionofoxygenaseandglycosyltransferasegenesinstreptomycetesproducingangucyclicantibiotics
first_indexed	2025-07-05T16:50:56Z
last_indexed	2025-07-05T16:50:56Z
_version_	1836826500327800832
fulltext	УДК 579.25:579.842.11 A. KOBYLYANSKYY, B. OSTASH, V. FEDORENKO Ivan Franko National University of Lviv, Department of Genetics and Biotechnology Hrushevskyy str. 4, Lviv 79005, Ukraine, E�mail: natureson@rambler.ru HETEROLOGOUS CROSS�EXPRESSION OF OXYGENASE AND GLYCOSYLTRANSFERASE GENES IN STREPTOMYCETES, PRODUCING ANGUCYCLIC ANTIBIOTICS The heterologous expression of oxygenase genes urdM, ovmOIII, lanM2, lndZ5 and glycosyltransferase gene urdGT2 in different angucycline producers of genus Streptomyces has been carried out. The introduction of genes urdM, lanM2 and urdGT2 results in the accumulation of new glycosylated compounds in several strains under investigation. A number of processed recombinant strains display strepto� mycin sensitivity and decrease in total antibacterial activity. The obtained data is an evidence of changes in antibiotics production, resulting from post�polyketide synthase (post� PKS) tailoring gene expression across streptomycete strains. Our study demonstrates the potential of post�PKS tailoring genes for generation of novel bioactive metabolites. Introduction. The angucyclic polyketide antibi� otics are microbial quinone natural products bear� ing a characteristic four�ring frame of the aglycon moiety, which is assembled in an angular manner [1]. The classification of the angucyclines, which numbers more than a hundred different compounds, is related to the tetracyclic benz[a]anthracene sys� tem and its derived compounds. Angucyclines show a multitude of valuable biological activities such as anticancer, antibacterial, antiviral, enzyme inhibitory etc. Angucycline producing organisms exclusively belong to order Actinomycetales, basi� cally to genus Streptomyces – Gram�positive, mycelial, sporulating bacteria. Though all the angucyclines possess antibiotic, particularly anticancer, activities, the range of their activity greatly depends on the presence of functional groups and glycosyl residues in their molecules [2]. Namely, bioactivity is usually defined by the arrangement and quantity of keto�, hydroxygroups and sugars. These functionalities are introduced by oxygenases and glycosyltrans� ferases (GT’s). The need for new polyketides is caused by the emergence of pathogenic strains and tumor cells resistant to traditional chemicals. Modern methods of genetic engineering allow the construction of artificial secondary metabolic pathways by combining the directed mutagenesis and heterologous gene expression approaches in foreign cells (of other species). Combinatorial biosynthesis anticipates system� atic modification and interchange of genes involved in biosynthesis of natural products with the consequential production of ‘unnatural’ or ‘hybrid’ natural products [2, 3]. Here, we report the use of combinatorial biosyn� thesis approach for modification of the biosynthe� sis pathways of five angucyclic antibiotics, i.e. urdamycin A (1), landomycins A and E (2), simocy� clinone D8 (3) and oviedomycin (4) (Fig. 1). The aim of current work was to perform heterologous cross�expression of four oxygenase genes urdM, ovmOIII, lanM2, lndZ5, descending, respectively, from S. fradiae Tu2717, S. antibioticus ATCC 11891, S. cyanogenus S136, S. globisporus 1912 and one gly� cosyltransferase gene urdGT2 from S. fradiae Tu2717 in these strains and in two S. globisporus mutants deficient oxygenase genes. This approach has been used to obtain the collection of recombi� nants producing novel compounds with altered bioactivity and to investigate the changes in their secondary metabolism. ІSSN 0564–3783. Цитология и генетика. 2009. № 3 55 © A. KOBYLYANSKYY, B. OSTASH, V. FEDORENKO, 2009 Materials and methods. Bacterial strains and plasmids used in this work are listed in accompa� nying table. Escherichia coli strains were grown at 37 °С in LB for routine applications [4]. Streptomyces strains were grown at 30 °С. Solid oatmeal medium was used for streptomycetes sporulation and plating of E. coli – Streptomyces matings [5]. Growth dynamics and antibiotic pro� duction of Streptomyces strains were examined in TSB (120 h of incubation). Where required, strains were grown in the presence of antibiotics and chromogenic substrates as described elsewhere [6]. Bacillus subtilis, B. cereus, S. albus and Sarcina flava were used as test�cultures for determining antibiotic activity of obtained mutants. Agar plugs were cut from solid media and put on the surface of test culture. The diameter of growth inhibition zones was measured on 16th, 24th and 48th hour of the cultivation. The resistance spectra were analysed by means of antibiotic disc diffusion method. Plasmid DNA from E. coli was isolated using standard protocols [4]. E. coli transformation and intergeneric matings (using E. coli ET12567 (pUB307) as a donor) were performed as described previously [5, 6]. Enzymes and kits for molecular biological manipulations were purchased from stan� dard commercial sources and used as described by the manufacturer. Secondary metabolites from the culture medi� um were extracted with equal volume of ethyl acetate on the 48–72th hour of Streptomyces strains growth in 25 ml of SG at 28 °С in a rotary shaker (120 rpm) as described [4]. The combined organic extracts were dried and dissolved in 30 μl of methanol for thin layer chromatography (TLC). TLC analysis was carried out on SilG�25 silica gel plates («Merck», Germany) with chloroform/methanol (9 : 1) as solvent. Rf values are not presented as appearance of blots different from control was cru� cial and sufficient in these experiments. In each separate experiment of heterologous gene expression we analysed few exconjugants to ensure unambiguous result. Results and discussion. Heterologous expression of urdM. Introduction of angular hydroxyl in 12b posi� tion of urdamycin A (1) molecule of S. fradiae Tu2717 is the unique reaction in angucycline biosyn� thesis (Fig. 1). Moreover, additional olivose is attached to this group, obviously, intensifying anti� cancer properties of the antibiotic [10]. The protein UrdM consists of two parts: oxygenase and reduc� tase. It is involved in oxygenation at 12b position of urdamycin precursor. In order to provide effective heterologous expression of urdM a 1,5�kb XbaI� EcoRI fragment of S. fradiae Tu2717 DNA contain� ing this gene was cloned into the conjugative expres� sion vector of pKC1218E [6] next to erythromycin resistance gene promoter giving pKC1218EurdM (Fig. 2, a). By means of intergeneric E. coli – Streptomyces conjugation pKC1218EurdM was ISSN 0564–3783. Цитология и генетика. 2009. № 356 A. Kobylynskyy, B. Ostash, V. Fedorenko Fig. 1. Structural formulae of urdamycin A (1), landomycin E (2), simocyclinone D8 (3), oviedomycin (4) transferred to the cultures S. globisporus Smy622, S. globisporus E7, S. globisporus M12 and S. cyanogenus S136. These cultures were selected on purpose, because none of their secondary metabo� lites comprises angular hydroxyl in 12b position and such experiments would help us understand better the substrate specificity of UrdM. The analysis of secondary metabolites extracts from obtained four recombinant strains by means of TLC discovered distinct changes in spectra of syn� thesized colored compounds for S. globisporus E7urdM and S. globisporus M12 urdM. Apparently, E7+urdM strain no longer produced 2,3�dehydro� UWM6 typical for initial E7 strain. Instead, there are three new compounds present on TLC (Fig. 2, b). M12 + urdM, generates no aromatic secondary metabolites at all, at least in detectable quantities. E7 + urdM strain most likely produces compounds with one, two and three sugars, as judged from changes in mobility and color of compounds. The experiment shows that 11�hydroxylation is advan� tageous for the glycosyltransfer and this can serve ІSSN 0564–3783. Цитология и генетика. 2009. № 3 57 Heterologous cross�expression of oxygenase and glycosyltransferase genes in streptomycetes Bacterial strains and plasmids used in this study Strains/plasmids E. coli DH5α E. coli ET12567 (pUB307) S. antibioticus ATCC 11891 S. antibioticus Tu6040 S. cyanogenus S136 S. globisporus SMY622 S. globisporus M12 S. fradiae Tu2717 S. globisporus E7 pSET152 pKC1139 pKC1218E pUWL201 pKC1218E�BX3 pKC1218EurdM pSETovmOIII pSETurdGT2 pUWLlanM2 pUWLurdGT2 Source or reference MBI Fermentas C. Smith, UMIST Manchester, UK J. Salas, University of Oviedo, Spain A. Bechtold, Albert�Ludwigs� University of Freiburg, Germany A. Bechtold, Albert�Ludwigs� University of Freiburg, Germany [7] [8] A. Bechtold, Albert�Ludwigs� University of Freiburg, Germany [9] P. Leadlay, Cambridge university, England [6] C. Olano, University of Oviedo, Spain [6] This work » » » » » Relevant characteristics F�(ϕ80d�(lacZ)M15 recA1 endA1 gyrA96 thi1 deoR (lacZYA�argF) U169) dam�13::Tn9 (Cmr) dcm�6 hsdM; contains RK2�based conjugative plasmid pUB307 Oviedomycin producer Symocyclinone producer Landomycin A producer Landomycin E overproducer lndM2 disruption mutant of S. globisporus 1912 Urdamycins producer S. globisporus SMY622 with mutated lndE E. coli – Streptomyces conjugative vector (ori ColE1, Amr, lacZ, oriT RK2, intPϕC31, attPϕC31) pUWL201 E. coli – Streptomyces shuttle expression vector with ermE promotor and pIJ101 replicon, Thr E. coli – Streptomyces shuttle expression vector with ermE promotor and SCP2 replicon, Amr E. coli – Streptomyces shuttle expression vector with ermE promotor and pIJ101 replicon, Thr pKC1218E, where lndZ4Z5 cloned downstream to ermE promotor pKC1218E, where urdM cloned downstream to ermE promotor pSET152, carries ovmOIII pSET152, carries urdGT2 pUWL201, where lanM2 cloned downstream to ermE promotor pUWL201, where urdGT2 cloned downstream to ermE promotor an evidence that the 11�hydroxylation normally precedes the glycosyltransfer in landomycins biosynthesis. It also shows that LndGT2 possesses broadened substrate specificity regarding its agly� con acceptor substrate. Heterologous expression of lanM2. Over the past decade, two gene clusters encoding landomycin bio� syntheses, namely, the trisaccharidal landomycin E (2) produced by S. globisporus 1912 (Fig. 1) and the hexasaccharidal landomycin A produced by S. cyanogenus S136 have been cloned and sequenced [11]. The studies revealed that both clusters display an extraordinarily high degree of identity. Previous investigation of lndM2 gene descending from S. glo� bisporus 1912 [8] asserted the role of its correspon� ding flavoprotein as oxidoreductase, responsible for the attachment 6�hydroxyl during the biosyn� thesis of landomycins precursor. With the purpose of lanM2 heterologous expression the amplifying replicative shuttle plasmid pUWLlanM2, where lanM2 is cloned downstream to erythromycin resist� ance gene promoter (Fig. 3, a) was transferred to the cells of three strains. We selected thiostrepton resistant exconjugants in the matings of E. coli (pUWLlanM2) with S. fradiae Tu2717, S. antibioti� cus Tu6040 and S. globisporus E7. 6�hydroxyl group is absent in the molecule of urdamycin A (1), pro� duced by S. fradiae Tu2717, so we intended to introduce this hydroxyl function, aiming to redis� tribute electron density in ring B. Unfortunately, we did not reveal changes in the secondary metabo� lites spectrums for this recombinant, as well as for E7 + lanM2. Concerning S. antibioticus Tu6040 car� rying pUWLlanM2, the spectrum differs in princi� ple (Fig. 3, b). S. antibioticus Tu6040 is the produc� er of a complex angucyclic antibiotic simocycli� ISSN 0564–3783. Цитология и генетика. 2009. № 358 A. Kobylynskyy, B. Ostash, V. Fedorenko Fig. 2. Recombinant plasmid pKC1218EurdM map (a) and change in spectrum of secondary metabolites for S. globis� porus E7urdM (b); Amr – apramycin�resistance gene; LaE – landomycin E Fig. 3. Recombinant plasmid pUWLlanM2 map (a) and change in spectrum of secondary metabolites for S. antibi� oticus lanM2 (b); Tsrr– thiostrepton�resistance gene; Apr – ampicillin�resistance gene; Sym – symocyclinone D8 none D8 (3), which biosynthetic cluster has been cloned and sequenced [16], still little is known about the mechanisms and timing of oxygenation� reduction reactions. One of simocyclinone’s dras� tic peculiarities is a unique epoxyfunction in aro� matic ring B (Fig. 1). We surmise that oxygenase domain of lanM2, due to its low substrate speci� ficity could facilitate aromatization of this ring and the recombinant strain produces as�yet�unidenti� fied aglyca along with the final compound. lndZ4/Z5 heterologous expression. The pair of genes lndZ4 and lndZ5, was declared to be respon� sible for the 11�hydroxylation that occurs during landomycin E (2) biosynthesis [13]. The authors also showed that hydroxylation at position 11 is not dependent on the length of the side chain and may occur at different stages during landomycin A biosynthesis. This opportunity encouraged us to introduce this pair of genes, encoding hydroxylase and reductase to S. fradiae Tu2717, as urdamycins just lack 11�hydroxyl. Interestingly, landomycin F, was approximately threefold less active against the MCF�7 breast cancer cell line than its correspon� ding 11�hydroxy analogue, landomycin D [13]. We brought lndZ4/Z5 consisting of the plasmid рКС1218Е�ВХ3 (Fig. 4, a) to the culture S. fradiae Tu2717. TLC analysis detected new major metabo� lite in comparison to wild type strain (Fig. 4, b). Previously we concluded that all oxygenations steps except the 11�hydroxylation occur before Lan/ LndGT2 adds the first sugar moiety to landomyci� none precursor [9], in contrast to urdamycins biosynthesis where first glycosylation step precedes all oxygenations. Therefore, it is interesting to know how the presence of additional hydroxyl would affect UrdGT2’s ability to attach first olivose to urdamycinone precursor molecule. ovmOIII heterologous expression. ovmOIII gene originates from Streptomyces antibioticus ATCC 11891 oviedomycin (4) biosynthesis genes cluster [14]. At least three oxygenations take place in oviedomycin formation. They are the result of ovmOI, OII and OIII expression. OvmOIII reveals similarity to flavin�type hydroxylases of Rhodococcus sp., that modify 7�ethoxycumarin and rifampin [14]. Consequently, it is considered that ovmOIII is involved in hydroxylation, or ketogroup attachment in the first or fourth positions of the oviedomycin pre� cursor. The presence of oxygen in 4th position of ovi� edomycin is an unprecedented case among angucy� clines. For heterologous expression of ovmOIII we engineered an integrating bifunctional plasmid pSETovmOIII. The plasmid was heterologously expressed in four cultures S. globisporus Smy622, S. globisporus E7, S. globisporus M12 and S. cyanogenus S136. No legible change in the spectra of secondary metabolites was discovered by TLC analysis. It is, obviously, predefined by inability of ovmOIII oxyge� nase to recognize heterologous substrates, or the fact that gene descends from a «taciturn» cluster and is naturally expressed only at very specific conditions. As it is well known, that most oxygenases taking part in aromatic polyketyde antibiotics biosynthe� sis are not cytochrome P450�dependent (for exam� ple LndE/LanE and UrdM are flavin�dependent), this, obviously stipulates for their low substrate specificity, while cytochrome P450�dependent oxy� genases are highly tailored [2]. These qualities are very important for the combinatorial biosynthesis of polyketide antibiotics. ІSSN 0564–3783. Цитология и генетика. 2009. № 3 59 Heterologous cross�expression of oxygenase and glycosyltransferase genes in streptomycetes Fig. 4. Recombinant plasmid pKC1218E�BX3 map (a) and change in spectrum of secondary metabolites for S. fra� diae lndZ4/Z5 (b) Heterologous expression of urdGT2. Bioactive natural products frequently include saccharide chains, which contribute to specific interactions with the biological target. Combinatorial biosyn� thesis approaches are being used in antibiotic�pro� ducing actinomycetes to generate derivatives with novel sugars in their architecture. Recent advances in this area indicate that glycosyltransferases involved in the biosynthesis of natural products have substrate flexibility regarding the sugar donor and also, less frequently, with respect to the agly� con acceptor [2, 15]. The first glycosyltransfer step in urdamycins biosynthesis is carried by UrdGT2, which is close� ly related to Lan/LndGT2. However, the acceptor substrates of these glycosyltransferases appear to be quite different (one is a carbon�GT, and the other is an oxygen�GT). Previously, we have shown that protein UrdGT2 reveals remarkably broadened substrate specificity, as its heterologous expression in the lndE�minus mutant of S. globis� porus 1912 (E7) yielded three novel prejadomycin analogues that differ in their C�glycosidically bound moieties [9]. Hence, urdGT2 turned out to be a promising candidate for combinatorial biosynthesis. Generated in advance pUWLurdGT2 plasmid (Fig. 5, a) was employed to introduce urdGT2 into three strains – S. globisporus Smy622, M12 and S. antibioticus Tu6040. The changes in aromatic secondary metabolites spectra took place in two cases (Fig. 5, b). Concer� ning S. antibioticus + urdGT2, its spectrum also differs radically from the original strain, but more profound investigation is needed to elucidate the structures of its secondary metabolites. Traditional studies on antibiotic resistance, antibacterial activity, and mutation maintenance examination for the obtained strains with obvious changes in antibiotic biosynthetic pathways were carried, using wild type strains as controls. To con� firm effective replication and passing of heterolo� gously expressed recombinant plasmids that carry urdM, lanM2 and urdGT2 genes we cultivated the strains in the course of five generations in nonse� lective conditions with consequent verification of antibiotic resistance maintenance on media con� taining selective agents. Approximately 85 % of colonies inherited replicative plasmids after five passages under nonselective conditions ascer� tained that new features of these recombinants are quite stable. We also analyzed the antibiotic resist� ance spectra of the mentioned strains against twelve antibiotics of different classes and observed unanticipated streptomycin sensitivity in case of E7 + urdGT2. In order to test the changes in anti� bacterial activity of recombinant strains in com� parison with landomycin E (2) we used four bacte� rial species as test�cultures – Bacillus subtilis, B. cereus, Streptomyces albus and Sarcina flava. The diminution of antibacterial activity against S. albus in case of S. globisporus E7 carrying urd�genes was found. These results apparently point at the deep� er changes in physiology of the strains and resist� ance mechanisms caused by overexpression of urdGT2 and urdM genes under ermE gene promot� er and undoubtedly can be a subject of future work. The heterologous expression of Streptomyces oxygenase and glycosyltransferase genes is known ISSN 0564–3783. Цитология и генетика. 2009. № 360 A. Kobylynskyy, B. Ostash, V. Fedorenko Fig. 5. Recombinant plasmid pUWLurdGT2 map (a); change in spectrums of secondary metabolites for S. antibioti� cus urdGT2 and M12 urdGT2 (b) to be an effective and beneficial approach to obtain recombinant strains with altered secondary metabo� lism. Here we report the generation of collection of angucycline�producing strains that carry various heterologous oxygenase and GT genes. We demon� strated here that introduction of certain post�PKS tailoring genes into actinomycete species provokes them to produce modified compounds that could possess valuable activities. The production of these antibiotics testifies low degree of substrate�speci� ficity of oxygenases UrdM, LanM2 and glycosyl� transferase UrdGT2. Their genetic determinants are promising candidates for combinatorial biosynthe� sis of ‘hybrid’ angucyclines. More detailed chemi� cal analysis and biological assays of mentioned compounds will also clarify the linkage between their structure and bioactivity. А.Н. Кобылянский, Б.О. Осташ, В.О. Федоренко ПЕРЕКРЕСТНАЯ ГЕТЕРОЛОГИЧЕСКАЯ ЭКСПРЕССИЯ ГЕНОВ ОКСИГЕНАЗ И ГЛИКОЗИЛТРАНСФЕРАЗЫ У СТРЕПТОМИЦЕТОВ, ПРОДУЦИРУЮЩИХ АНТИБИОТИКИ АНГУЦИКЛИНОВОГО РЯДА Проведена гетерологическая экспрессия генов ок� сигеназ urdM, ovmOIII, lanM2, lndZ5 и гликозилтранс� феразы urdGT2 в разных видах продуцентов ангуцик� линов из рода Streptomyces. Введение генов urdM, lanM2 и urdGT2 в некоторые из исследуемых штаммов обусловливает накопление ими новых гликозилиро� ванных соединений. Обнаружено появление чувстви� тельности к стрептомицину и резкое снижение общей антибактериальной активности у некоторых из полу� ченных рекомбинантных штаммов. Представленные данные свидетельствуют о модификации продукции антибиотиков, которая является следствием экспрес� сии генов постполикетидного синтеза (пост�ПКС) в штаммах стрептомицетов. Наши исследования демон� стрируют потенциал генов пост�ПКС в создании но� вых биологически активных метаболитов. А.М. Кобилянський, Б.О. Осташ, В.О. Федоренко ПЕРЕХРЕСНА ГЕТЕРОЛОГІЧНА ЕКСПРЕСІЯ ГЕНІВ ОКСИГЕНАЗ ТА ГЛІКОЗИЛТРАНСФЕРАЗИ У СТРЕПТОМІЦЕТІВ, ЩО ПРОДУКУЮТЬ АНТИБІОТИКИ АНГУЦИКЛІНОВОГО РЯДУ Здійснено гетерологічну експресію генів оксигеназ urdM, ovmOIII, lanM2, lndZ5 та глікозилтрансферази urdGT2 у різних видів продуцентів ангуциклінів з роду Streptomyces. Введення генів urdM, lanM2 та urdGT2 у деякі з досліджуваних штамів зумовлює накопичення ними нових глікозильованих сполук. Виявлено виник� нення чутливості до стрептоміцину та різке зниження загальної антибактерійної активності у деяких із одер� жаних рекомбінантних штамів. Отримані дані є свід� ченням модифікації продукції антибіотиків, яка є на� слідком експресії генів постполікетидного синтезу (пост�ПКС) у штамах стрептоміцетів. Наші дослід� ження демонструють потенціал генів пост�ПКС у створенні нових біологічно активних метаболітів. REFERENCES 1. Rohr J., Thiericke R. Angucycline group antibiotics // Nat. Prod. Rep. – 1992. – 9. – P. 103–137. 2. Rix U., Fischer C., Remsing L., Rohr J. Modification of post�PKS tailoring steps through combinatorial bio� synthesis // Nat. Prod. Rep. – 2002. – 19. – P. 542– 580. 3. Pfeifer B., Khosla C. Biosynthesis of polyketides in hete� rologous hosts // Mol. Biol. Rev. – 2001. – 65. – P. 106– 118. 4. Sambrook J., Fristch E., Maniatis T. Molecular cloning: a laboratory manual. 2nd edition. CSH, Laboratory Press. – New York: Cold Spring Harbor, 1989. – 450 p. 5. Mazodier P., Petter R., Thompson C. Intergeneric conju� gation between Escherichia coli and Streptomyces species // J. Bacteriology. – 1989. – 171(6). – P. 3583– 3585. 6. Kieser T., Bibb M., Buttner M., Chater K., Hopwood D. Practical Streptomyces genetics. – Norwich: John Innes Foundation, 2000. – 634 p. 7. Gromyko O., Rebets Yu., Ostash B., Luzhetskyy A., Fukuhara M., Bechthold A., Nakamura T., Fedorenko V. Generation of Streptomyces globisporus SMY622 strain with increased landomycin E production and its initial characterization // J. Antibiot. – 2004. – 57. – P. 383– 389. 8. Zhu L., Ostash B., Rix U., Nur�e�Alam M., Mayers A., Luzhetskyy A., Mendez C., Salas J., Bechthold A., Fedorenko V., Rohr J. Identification of the function of gene lndM2 encoding a bifunctional oxygenase�reduc� tase involved in the biosynthesis of the antitumor antibiotic landomycin E by Streptomyces globisporus 1912 supports the originally assigned structure for lan� domycinone // J. Org. Chem. – 2004. – 70(2). – P. 631– 638. 9. Baig I., Kharel M., Kobylyanskyy A., Zhu L., Rebets Y., Ostash B., Luzhetskyy A., Bechthold A., Fedorenko V., Rohr J. On the acceptor substrate of C�glycosyltrans� ferase UrdGT2: three prejadomycin C�glycosides from an engineered mutant of Streptomyces globisporus 1912�lndE (urdGT2) // Angew. Chem. Int. Ed. – 2006. – 45(46). – P. 7842–7846. 10. Rix U., Remsing L., Hoffmeister D., Bechthold A., Rohr ІSSN 0564–3783. Цитология и генетика. 2009. № 3 61 Heterologous cross�expression of oxygenase and glycosyltransferase genes in streptomycetes J. Urdamycin L, a novel metabolic shunt product that provides evidence for the role of the urdM gene in the urdamycin A biosynthetic pathway of Streptomyces fradiae Tu 2717 // Chem. Bio. Chem. – 2003. – 4. – P. 109–111. 11. Henkel T., Rohr J., Beale J., Schwenen L. Lando� mycins, new angucycline antibiotics from Streptomyces sp. Structural studies on landomycins A�D // J. Antibiot. – 1990. – 43. – P. 492–503. 12. Ruth H., Flatman L., Alison J., Howells L., Lutz H., Fiedler H., Maxwell H. Simocyclinone D8, an inhibitor of DNA gyrase with a novel mode of action // Antimicr. agents and chemother. – 2005. – 49(3). – P. 1093– 1100. 13. Luzhetskyy A., Zhu L., Gibson M., Fedoryshyn M., Clemens D., Hofmann C., Hoffmeister D., Ostash Mattingly B., Adams V., Fedorenko V., Rohr J., Bechthold A. Generation of novel landomycins M and O through targeted gene disruption // Chem. Bio. Chem. – 2005. – 6(4). – P. 675–678. 14. Mendez C., Kunzel E., Lipata F., Lombo F., Cotham W., Walla M., Bearden D., Brana A., Salas J., Rohr J. Oviedomycin, an unusual angucyclinone encoded by genes of the oleandomycin producer Streptomyces antibioticus ATCC11891 // J. Nat. Prod. – 2002. – 65. – P. 779–782. 15. Hoffmeister D., Wilkinson B., Foster G., Sidebottom P.J., Ichinose K., Bechthold A. Engineered urdamycin glyco� syltransferases are broadened and altered in substrate specificity // Chem. Biol. – 2002. – 9. – P. 287–295. Received 12.05.08 ISSN 0564–3783. Цитология и генетика. 2009. № 362 A. Kobylynskyy, B. Ostash, V. Fedorenko

Heterologous cross-expression of oxygenase and glycosyltransferase genes in streptomycetes, producing angucyclic antibiotics

Institution

Ähnliche Einträge