Biosynteza związków poliketydowych przez Aspergillus Terreus

• Autorzy: Bizukojć M.
• Języki publikacji: PL

Abstrakty

PL

Aspergillus terreus jest grzybem nitkowym należącym do gromady Ascomycota. Posiada on szczególną zdolność do biosyntezy poliketydowego metabolitu, będącego inhibitorem reduktazy (S)-3-hydroksy-3-metyloglutarylo-CoA, zwanego kwasem mewinolinowym. Związek ten w postaci laktonu, nazwanego oficjalnie lowastatyną, jest wykorzystywany w medycynie jako lek obniżający poziom endogennego cholesterolu w organizmie człowieka. Niniejsze opracowanie ma formę krótkiej monografii mającej na celu przybliżenie czytelnikowi zagadnień biochemicznych, fizjologicznych, morfologicznych i kinetycznych biosyntezy niektórych metabolitów wtórnych produkowanych przez A. terreus ze szczególnym uwzględnieniem najważniejszego metabolitu, czyli lowastatyny, oraz często jej towarzyszącej (+)-geodyny. W pracy omówione zostały w pierwszej kolejności mechanizmy biochemiczne rządzące biosyntezą lowastatyny oraz (+)-geodyny. Opisany został klaster genów odpowiedzialnych za syntezę enzymów niezbędnych do biosyntezy lowastatyny oraz przedstawione zostały zasady działania syntaz poliketydowych. Szczegółowo pokazano szlak biosyntezy lowastatyny oraz takich oktaketydowych metabolitów, jak sulochryna, (+)-geodyna i kwas astrowy. Podana została także pierwszorzędowa struktura enzymów odpowiedzialnych za biosyntezę lowastatyny. Zagadnienia biochemiczne biosyntezy metabolitów wtórnych A. terreus zostały opracowane w większości na podstawie danych literaturowych. Uzupełniono jednakże niektóre brakujące dane poprzez odtworzenie mechanizmu działania syntazy oktaketydowej antronu emodyny na podstawie ogólnej wiedzy na temat syntaz poliketydowych. Dodatkowo została przeprowadzona na podstawie anotacji częściowo zsekwencjonowanego genomu własna rekonstrukcja sieci metabolicznej dla pierwotnego metabolizmu A. terreus. Skorelowana ona została z danymi fizjologicznymi pochodzącymi z własnych eksperymentów i znalezionymi w literaturze przedmiotu, związanymi na przykład z asymilacją substratów i produkcją metabolitów pierwotnych, jak etanol. Przeprowadzona krótka analiza porównawcza sieci metabolicznej z danymi eksperymentalnymi pozwoliła także na sformułowanie propozycji uzupełnienia „dziur" (holes), czyli brakujących enzymów bądź reakcji w anotacji sieci metabolicznej, szczególnie tych związanych z metabolizmem laktozy. W dalszej części pracy skupiono się na aspektach fizjologicznych procesu biosyntezy metabolitów wtórnych i przedstawiono wpływ składu podłoża oraz warunków procesowych na biosyntezę lowastatyny i (+)-geodyny przez A. terreus. Dzięki wynikom badań eksperymentalnych określony został wpływ źródła węgla i azotu na jednoczesną biosyntezę lowastatyny i (+)-geodyny, co w dalszym etapie badań pozwoliło na sformułowanie modelu kinetycznego procesu biosyntezy lowastatyny przez A. terreus. Dodatkowo przebadany został wpływ takich śladowych substancji odżywczych, jak witaminy, na biosyntezę metabolitów A. terreus. Na podstawie własnych danych eksperymentalnych udowodniono, że niektóre witaminy z grupy B wpływają pozytywnie na biosyntezę lowastatyny. Opracowana została także strategia prowadzenia procesu biosyntezy lowastatyny w bioreaktorze. Udana okazała się próba zminimalizowania produkcji (+)-geodyny poprzez stosowanie odpowiedniej kontroli pH oraz suplementację podłóż witaminami. Kolejnym aspektem omawianym w pracy jest znaczenie morfologii i różnicowania się strzępek grzybni w biosyntezie lowastatyny i (+)-geodyny. Przedstawione zostały czynniki wpływające na morfologię grzybni, wielkość, kształt i formę peletek A. terreus. Przedstawiono także wyniki badań dotyczących wewnętrznej struktury peletek grzybni A. terreus, a różnicowanie się strzępek grzybni zostało skorelowane z biosyntezą lowastatyny i (+)-geodyny. Wreszcie przedstawiono dwa modele biosyntezy lowastatyny przez A. terreus, które można znaleźć w literaturze przedmiotu. Są to model morfologicznie strukturalny oraz sformułowany przez autora niestrukturalny model kinetyczny. Ten drugi model powstał na podstawie wyników badań z hodowli wstrząsanej i został pozytywnie zweryfikowany w procesach bioreaktorowych.

EN

Aspergillus terreus is a filamentous fungus that belongs to the phylum Ascomycota. It is able to biosynthesise a polyketide metabolite, which inhibits (S)-3-hydroxy-3-methylglutaryl-CoA reductase and is called mevinolinic acid. The lactone form of this compound, which was internationally named lovastatin, is used in medicine to decrease the level of the endogenous cholesterol in human organisms. This work is a short monograph, whose aim is to present the biochemical, physiological, morphological and kinetic issues of biosynthesis of selected secondary metabolites produced by A. terreus. The special attention is paid to the most important metabolite, i.e. the aforementioned lovastatin and to (+)-geodin, which frequently accompanies lovastatin in the cultivation processes. In this work the biochemical mechanisms governing lovastatin and (+)-geodin biosynthesis were first presented. The gene cluster, which is responsible for the expression of the enzymes required for lovastatin biosynthesis, was described. Also the action of polyketide synthases was presented. Metabolic pathways of lovastatin biosynthesis and of such octaketide metabolites as sulochrin, (+)-geodin, and asterric acid were described in detail. The biochemical issues of secondary metabolites biosynthesis by A. terreus were mostly elaborated upon the literature data. However, few lacking data were supplemented. The hypothetical action of emodin anthrone octaketide synthase was described on the basis of the general knowledge concerning polyketide synthases. Additionally, the reconstruction of the metabolic network to mainly describe the primary metabolism of A. terreus was made upon the annotation the partially sequenced genome. The results of this reconstruction were correlated with the physiological data originated from own experiments and selected literature data. They concerned the assimilation of substrates and primary metabolites formation, e.g. ethanol. The comparative analysis of the reconstructed metabolic network and the experimental data allowed for the proposal to fill the holes, i.e. lacking enzymes or reactions, in the annotated network, especially those connected with lactose metabolism. In the second part of the work the physiological issues of the biosynthesis of secondary metabolites were discussed. The influence of the medium composition and process conditions on lovastatin and (+)-geodin biosynthesis by A. terreus was presented. Owing to the experimental results the influence of carbon and nitrogen source on the simultaneous lovastatin and (+)-geodin biosynthesis was described. Next, it allowed for the formulation of the kinetic model of lovastatin biosynthesis by A. terreus. Additionally, the influence of such trace nutrients as vitamins on lovastatin and (+)-geodin biosynthesis by A. terreus was studied. On the basis of own experimental data, it was proved that some B-group vitamins positively influence lovastatin biosynthesis. Also the strategy of lovastatin biosynthesis in bioreactor was elaborated. The minimisation of (+)-geodin production was successfully achieved by means of the valid pH control procedure and media supplementation with B-group vitamins. Another issue discussed was the influence of morphology and hyphal differentiation on lovastatin and (+)-geodin biosynthesis. The factors influencing the hyphal morphology, i.e. the size and shape of A. terreus pellets were discussed. Also the experimental results concerning the infrastructure of the pellets were presented and hyphal differentiation was correlated with lovastatin and (+)-geodin biosynthesis. Finally, two kinetic models to describe lovastatin biosynthesis were shown. These are a morphologically structured and an unstructured kinetic models. The latter was formulated by the author of this work upon the experimental results from a shake flask cultures and successfully verified in the bioreactor cultivations.

Słowa kluczowe

PL

biosynteza poliketydowego matabolitu; zagadnienia biochemiczne; lowastatyna; metabolity wtórne; Aspergillus Terreus; grzyby nitkowate

EN

biosynthesise a polyketide metabolite; biochemical issues; lovastatin; secondary metabolites; Aspergillus terreus; filamentous fungi

• Wydawca: Wydawnictwo Politechniki Łódzkiej
• Czasopismo: Zeszyty Naukowe. Rozprawy Naukowe / Politechnika Łódzka
• Rocznik: 2009
• Tom: Z. 377
• Strony: 8--154
• Opis fizyczny: Bibliogr. 118 poz.

Twórcy

autor

Bizukojć M.

• Wydział Inżynierii Procesowej i Ochrony Środowiska Politechniki Łódzkiej, Katedra Inżynierii Bioprocesowej,

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• Strony internetowe:
• [1] BRENDA: http://www.brenda-enzymes.org
• [2] KEGG Kyoto Encyclopedia of Genes and Genomes: http://www.kegg.jp
• [3] PubMed: http://www.pubmedcentral.nih.gov
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