doktorska disertacija
Povzetek
Bakterija Streptomyces rimosus je poznana predvsem zaradi produkcije široko spektralnega antibiotika oksitetraciklina (OTC). Z namenom boljšega razumevanja biosinteze OTC in drugih naravnih tetraciklinov (TC) smo naredili bioinformacijsko primerjavo gruč biosinteznih genov (GBG), ki kodirajo biosintezo: OTC, klorotetraciklina, kelokardina, daktilociklina in SF2575. Nato smo se osredotočili na izbrane gene udeležene v zgodnje in pozne stopnje biosinteze OTC, katerih vloga še ni poznana. Izbili smo izbrane gene iz otc GBG (oxyI, oxyH, oxyG, oxyM, oxyO) v visoko donosnem sevu bakterije S. rimosus. Sevom z izbitimi geni smo ovrednotili donos OTC ter z analizo netarčnega metaboloma poskušali identificirati nove intermediate/stranske produkte. Pokazali smo, da geni oxyI, oxyG, oxyM in oxyO niso nujno potrebni v biosintezi OTC. Vendar ob izbitju genov oxyI, oxyG in oxyM donos OTC upade, zaradi česar domnevamo, da imajo pomožno vlogo v biosintezi. V sevu z izbitim genom oxyH je prišlo do prekinitve produkcije OTC in ob tem smo detektirali nov metabolit. V drugem sklopu smo se osredotočili na preostali sekundarni metabolizem bakterije S. rimosus, tako da smo s pomočjo bioinformacijskih orodij identificirali in analizirali GBG na kromosomu in plazmidu. Po kultivaciji bakterije S. rimosus v različnih gojiščih in LC-MS analizi smo detektirali produkcijo devetih sekundarnih metabolitov. Poleg tega smo poskušali identificirati GBG v genomu bakterije S. rimosus, ki kodira biosintezo rdeče obarvane spojine in smo jo detektirali le ob kokultivaciji z bakterijo Bacillus subtilis. Ob izbitju izbranih GBG je bakterija S. rimosus ob kokultivaciji z bakterijo B. subtilis še vedno proizvajala rdeče obarvano spojino.
Ključne besede
Streptomyces rimosus;gruča biosinteznih genov;sekundarni metaboliti;aromatski poliketidi;tetraciklini;oksitetraciklin;biosintezna pot;bioinformacijska analiza;kokultivacija;
Podatki
Jezik: |
Slovenski jezik |
Leto izida: |
2022 |
Tipologija: |
2.08 - Doktorska disertacija |
Organizacija: |
UL BF - Biotehniška fakulteta |
Založnik: |
[L. Slemc] |
UDK: |
604.4:579.873.7 |
COBISS: |
135468035
|
Št. ogledov: |
293 |
Št. prenosov: |
20 |
Ocena: |
0 (0 glasov) |
Metapodatki: |
|
Ostali podatki
Sekundarni jezik: |
Angleški jezik |
Sekundarni naslov: |
Analysis of oxytetracycline biosynthesis and other secondary metabolites in Streptomyces rimosus |
Sekundarni povzetek: |
The bacterium Streptomyces rimosus is mainly known for the production of broad-spectrum antibiotic oxytetracycline (OTC). To gain deeper understanding of the biosynthesis of OTC and other natural tetracyclines (TC) we initialy performed a bioinformatic comparison of the biosynthetic gene clusters (BGCs) encoding the biosynthesis of OTC, chlortetracycline, chelocardin, dactylocycline and SF2575. In the second step, we focused on selected genes involved in the early and late stages of OTC biosynthesis whose roles is not yet understood. We deleted selected genes from otc BGC (oxyI, oxyH, oxyG, oxyM, oxyO) in the S. rimosus high-producer strain. We then evaluated the titer of OTC produced by engineered strains and we attempted to identify potential new intermediates or side products by analyzing the non-targeted metabolome. We showed that the oxyI, oxyG, oxyM and oxyO genes are not essential for the OTC biosynthesis. However, when the oxyI, oxyG, and oxyM genes were deleted the OTC titer decreased suggesting that these genes play an auxiliary role. OTC production was entirely disrupted in the strain with deletion of oxyH gene, instead a new metabolite was detected. In the second part of the thesis, we focused on the analysis of all BGCs encoded in the S. rimosus genom by applying bioinformatics tools. After cultivation of S. rimosus in selected media and followed by LC-MS analysis, we detected the production of nine secondary metabolites. Finally, we aimed to identify BGCs in the S. rimosus genome encoding the biosynthesis of a red-colored metabolite, which only appears in co-cultivation with Bacillus subtilis. After deletion of selected BGCs, engineered S. rimosus strains in co-cultivation with B. subtilis still produced red-colored metabolite. |
Sekundarne ključne besede: |
Streptomyces rimosus;biosynthetic gene cluster;secondary metabolites;aromatic polyketides;tetracycline;oxytetracycline;biosynthetic pathway;bioinformatic analysis;cocultivation; |
Vrsta dela (COBISS): |
Doktorsko delo/naloga |
Študijski program: |
0 |
Konec prepovedi (OpenAIRE): |
2025-01-06 |
Komentar na gradivo: |
Univ. v Ljubljani, Biotehniška fak. |
Strani: |
1 spletni vir (1 datoteka PDF (XVII, 188 f., [7] f. pril.) |
ID: |
17509556 |