magistrsko delo
Barbara Ramšak (Author), Ines Mandić-Mulec (Reviewer), Hrvoje Petković (Mentor), Maja Paš (Co-mentor)

Abstract

FK506 (takrolimus) je makrolakton poliketidnega izvora z imunosupresivno aktivnostjo, ki ga proizvajajo nekatere streptomicetne vrste. Uporablja se kot imunosupresivno zdravilo za preprečevanje zavrnitve organov po presaditvi in zdravljenje vnetnih kožnih bolezni. Pri uporabi FK506 so pogosti neželeni učinki kot so nefrotoksičnost, nevrotoksičnost in arterijska hipertenzija. Z uporabo Streptomyces tsukubaensis NRRL 18488, ki je industrijski producent FK506, smo razvili robusten bioproces za produkcijo FK506 na nivoju stresalnika. Vzpostavljen bioproces smo uporabili za optimizacijo kemobiosinteznega postopka, kjer smo v procesno brozgo S. tsukubaensis s prekinjenim genom allR (homologom krotonil-CoA-reduktaze/karboksilaze) dodajali prekurzor propargilmalonil-SNAC in na ta način pridobili nov analog FK506. Sev S. tsukubaensis s prekinjenim genom allR ne more sintetizirati FK506, niti FK520 in propil-FK506, ki nastajata kot stranska produkta med bioprocesom. Prisotnost novega analoga FK506, propargil-FK506 smo potrdili z metodo HPLC in MS. Produkcija propargil-FK506 je bila nižja v primerjavi s produkcijo FK506 pri izhodnem naravnem sevu S. tsukubaensis in pri sevu ∆allR po dohranjevanju z naravno podaljševalno enoto alilmalonil-SNAC. Nadalje smo zato optimizirali kemobiosintezni proces z dodajanjem propargilmalonil-SNAC v različnih koncentracijah. Ugotovili smo, da imajo višje koncentracije nenaravne podaljševalne enote propargilmalonil-SNAC inhibitorni učinek na produkcijo FK506 pri sevu S. tsukubaensis NRRL 18488. Kemobiosintezni bioproces je deloval najbolj optimalno pri koncentraciji prekurzorja propargilmalonil-SNAC v končni koncentraciji 0,5 g/L in temperaturi 24 °C.

Keywords

streptomicete;Streptomyces tsukubaensis;poliketidi;FK506;takrolimus;biosinteza poliketidov;kemobiosinteza;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [B. Ramšak]
UDC: 604.4:615.332
COBISS: 4805496 Link will open in a new window
Views: 1178
Downloads: 427
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Other data

Secondary language: English
Secondary title: Chemobiosynthetic approach for production of FK506 analogues by Streptomyces sp.
Secondary abstract: FK506 (tacrolimus) is a 23-membered macrolide of poliketide origin with a powerful immunosuppressive activity produced by several Streptomyces spp. It is a clinically important therapeutic drug used for preventing rejection of transplanted organs and in combating inflammatory skin diseases. FK506 can causes serious side effects primarily nephrotoxicity, neurotoxicity and arterial hypertension. Firstly, we have used the FK506-producing strain Streptomyces tsukubaensis NRRL 18488 to develop robust bioproces for FK506 production at the shaker level. However, the goal of this study was to develop a chemobiosinthetic process for production of a novel FK506 analogue by using allR inactivated mutant strain of S. tsukubaensis. The allR deletion mutant produced neither FK506, nor FK520 or propyl-FK506. The non-natural synthetic unit propargylmalonyl-SNAC was supplemented to the cultivation broth during the fermentation process of ∆allR mutant strain, which resulted in production of the new FK506 analogue propargyl-FK506. The presence of a new analogue propargyl-FK506 was confirmed by HPLC and MS. However, production of propargyl-FK506 was low as compared to the production of FK506 in wild type strain (S. tsukubaensis NRRL 18488) and production of FK506 after chemobiosinthesis with natural extender unit allylmalonly-SNAC when using the ∆allR mutant strain. Therefore, we tested different proparglymalonyl-SNAC feeding regimes. We observed significant inhibitory effect of proparglymalonyl-SNAC on the production of FK506 in wild type strain. We demonstrated that the most effective feeding regime was when supplementation of extender unit proparglymalonyl-SNAC was carried at the final concentration 0, 5 g/L at 24 °C.
Secondary keywords: streptomycetes;Streptomyces tsukubaensis;polyketide;FK506;tacrolimus;polyketide biosynthesis;chemobiosynthesis;
Type (COBISS): Master's thesis/paper
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Študij mikrobiologije
Pages: XIV, 70 f., [3] f. pril.
ID: 10850552