doktorska disertacija
Špela Zver (Author), Hrvoje Petković (Mentor)

Abstract

Bakterije iz rodu Streptomyces so proizvajalci medicinsko in industrijsko pomembnih sekundarnih metabolitov. Genom streptomicet zapisuje biosintezne genske gruče (BGG) za več kot 20 različnih sekundarnih metabolitov, vendar večino teh metabolitov, ni mogoče odkriti pod standardnimi laboratorijskimi pogoji. Izražanje t.i. tihih BGG v heterolognih gostiteljih je privlačen pristop za reševanje tega problema. Številni streptomcetni gostitelji že obstajajo, vendar ne dosegajo zadostnih koncentracij ciljnega produkta. Streptomyces rimosus je pomemben industrijski proizvajalec, ki je znan po doseganju visokih koncentracij antibiotika oksitetraciklina (OTC). Naš cilj je bil oceniti sposobnost S. rimosus kot gostitelja za proizvodnjo heterologno izraženih produktov. Najprej smo uspešno odstranili dve BGG, ki kodirata OTC in izorenieraten, s čimer smo zmanjšali tekmovanje med nativnimi in heterolognimi BGG. Razvili smo integrativne vektorje, ki vsebujejo BGG, ki kodirajo biosintezo tetraciklina kelokardina (CHD), izoprenoidov ciklooktatina in β-karotena ter večkrat nenasičenih maščobnih kislin (PUFA). Vsi našteti metaboliti uporabljajo malonil-CoA kot glavni gradnik. Poleg nativne CHD BGG smo rekonstituirali tudi sintetično BGG. Poleg izoprenoidne BGG smo klonirali tudi mevalonatno pot in jo vnesli v S. rimosus, s čimer smo zagotovili boljšo razpoložljivost izoprenoidnega substrata. Za povečanje donosa PUFA smo v S. rimosus uvedli gen, ki kodira fosfopanteteinil transferazo. Ciklooktatin, β-karoten in PUFA BGG so bili uspešno izraženi v S. rimosus, medtem ko niti nativna niti sintetična BGG za kelokardin nista proizvajala ciljnega produkta.

Keywords

Streptomyces rimosus;heterologno izražanje;heterologni gostitelj;biosintezna genska gruča;sekundarni metaboliti;oksitetetraciklin;kelokardin;ciklooktatin;karatenoidi;večkrat nenasičene maščobne kisline;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UL BF - Biotechnical Faculty
Publisher: [Š. Zver]
UDC: 602.3:579.873.7:604.4:615.33(043.3)
COBISS: 106693891 Link will open in a new window
Views: 1042
Downloads: 15
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Other data

Secondary language: English
Secondary title: Production of heterologous metabolites in different strains of bacterium Streptomyces rimosus
Secondary abstract: Streptomyces are producers of medically and industrially important secondary metabolites. The genome of Streptomyces encodes biosynthesis of more than 20 different secondary metabolites. However, most of the secondary metabolites encoded by the corresponding biosynthetic gene clusters (BGCs) cannot be detected under standard laboratory conditions. Therefore, expression of these “silent” BGCs in a heterologous host seems an attractive approach to solve this problem. Several Streptomyces hosts already exists, but they do not achieve sufficient concentrations of the target product. Streptomyces rimosus is a producer of important antibiotic oxytetracycline (OTC) and is known to achieve high titers of it. Our goal was to evaluate ability of S. rimosus as a host to produce heterologous products. First, we successfully removed two BGCs encoding OTC and isorenieratene, reducing competition between native and heterologously expressed BGCs. We developed integrative vectors containing BGCs encoding tetracycline chelocardin (CHD), isoprenoids cyclooctatin and β-carotene, and polyunsaturated fatty acids (PUFA), all of which consume malonyl-CoA as a main building block. In addition to native CHD BGC, we also reconstituted synthetic BGC. In addition to the isoprenoid BGC, we also cloned the mevalonate pathway and introduced it into S. rimosus, which could ensure better availability of isoprenoid substrate. To increase yield of PUFA, the gene encoding phosphopantetheinyl-transferase was introduced into S. rimosus. Cyclooctatin, β-carotene and PUFA BGCs were successfully expressed in S. rimosus, whereas neither native nor synthetic chelocardin BGC resulted in the production of the target product.
Secondary keywords: heterologous expression;heterologous host;biosynthetic gene cluster;secondary metabolites;oxytetracycline;chelocardin;cyclooctation;caratenoids;polyunsaturated fatty acids;
Type (COBISS): Doctoral dissertation
Study programme: 0
Embargo end date (OpenAIRE): 2024-04-07
Thesis comment: Univ. v Ljubljani, Biotehniška fak.
Pages: XVII, 133 f., [4] f. pril.
ID: 14993626