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Vpliv prekomernega izražanja membranskih črpalk na produkcijo tetraciklinskih analogov pri bakteriji Streptomyces rimosus
ID Peternel, Helena (Author), ID Petković, Hrvoje (Mentor) More about this mentor... This link opens in a new window, ID Avbelj, Martina (Co-mentor)

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Abstract
Nekatere bakterije so sposobne proizvodnje raznovrstnih sekundarnih metabolitov, vendar pa jih običajno proizvajajo v nizkih koncentracijah. Če želimo doseči višje titre, je ena izmed možnosti izboljšava proizvodnega seva s povečanjem odpornosti na sekundarni metabolit. To lahko dosežemo npr. s povečanim izražanjem membranskih črpalk. Namen naloge je bil vstaviti dodatne gene za membranski črpalki OtrB in OtrC v tri izbrane seve Streptomyces rimosus in na ta način doseči povečano izražanje izbrane membranske črpalke. V nalogi smo želeli ovrednotiti, kako povečano izražanje membranskih črpalk OtrB in OtrC nato vpliva na produkcijo TC analogov OTC in ATC. Po konstrukciji plazmidov z genom za membransko črpalko in konstitutivnim promotorjem smo plazmide z metodo konjugacije vstavili v tri različne seve S. rimosus. Po pridobitvi konjugant je sledila kultivacija v produkcijskem gojišču in primerjava titra tarčnih spojin konstruiranih sevov s kontrolnimi sevi. Ugotovili smo, da vnos membranskih črpalk OtrB in OtrC v divji sev S. rimosus ATCC 10970 in industrijski sev S. rimosus HP0508 ni vplival na povečan titer OTC v primerjavi s kontrolnimi sevi. Izjema so konjugante S. rimosus ATCC 10970::OtrC, pri katerih je vnos membranske črpalke OtrC vplival na 32 % povečanje OTC titra. Pri sevu S. rimosus BF_7S pa smo ugotovili, da povečano izražanje obeh omenjenih membranskih črpalk pripomore k znatnemu, do 18-kratnemu izboljšanju produkcije ATC.

Language:Slovenian
Keywords:Streptomyces rimosus, membranske črpalke, sekundarni metaboliti, oksitetraciklin, anhidrotetraciklin, otrB, otrC
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Publisher:[H. Peternel]
Year:2023
PID:20.500.12556/RUL-151503 This link opens in a new window
UDC:602.3+602.6:579.873.7
COBISS.SI-ID:167614979 This link opens in a new window
Publication date in RUL:08.10.2023
Views:236
Downloads:0
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Secondary language

Language:English
Title:Overexpression of exporters and its influence on the production of tetracycline analogues in Streptomyces rimosus
Abstract:
Some bacterial species are capable of producing a variety of secondary metabolites, but usually only in small amounts. One way to achieve higher yields is to improve the producing strain. One possible option is to enhance its self-resistance to the metabolite it produces, for example, by overexpressing efflux proteins. Our aim was to insert an additional copy of the genes otrB and otrC, representing membrane efflux transporters, into three strains of Streptomyces rimosus bacteria. The aim of this work was to investigate the effect of overexpression of these genes on the production of TC analogues, more specifically OTC and ATC in different strains of S. rimosus. After construction of plasmids containing a membrane efflux transporter gene and a constitutive promoter, the plasmids were introduced into four S. rimosus strains by conjugation. The newly obtained exconjugants were then compared with controls for secondary metabolite production. We found that introduction of membrane pumps OtrB and OtrC into the wild type strain S. rimosus ATCC 10970 and the industrial strain S. rimosus HP0508 did not increase the yield of OTC compared to the control strains with the exception of S. rimosus ATCC 10970::OtrC, which showed up to 32% improvement in the production of OTC. However, in the case of S. rimosus BF _7S strain, we found that increased expression of OtrB and OtrC membrane pumps contributed to a significant, up to 18-fold improvement in ATC production.

Keywords:Streptomyces rimosus, membrane exporters, secondary metabolites, oxytetracycline, anhydrotetracycline, otrB, otrC

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