Production of S-adenosyl-L-methionine by yeast Pichia stipitis

Abstract

Cilj ovog rada je bio ispitati mogućnost proizvodnje S-adenozil-L-metionina (SAMe) pomoću kvasca Pichia stipitis CBS 5776 (P. stipitis). Primjenom UV-mutageneze i nistatina selekcioniran je soj M12 kvasca P. stipitis koji ne eksprimira C-24 sterol metiltransferazu (Erg6p) čime je narušena biosinteza ergosterola te povećana proizvodnja SAMe. Soju M12 kvasca P. stipitis određen je genetski i proteomski profil te optimalan sastav podloge (vrsta osnovnog izvora ugljika, količina i oblik metionina, prisutnost soli) kao i optimalni uvjeti uzgoja (aeracija, temperatura i pH-vrijednost) za proizvodnju SAMe. Uzgojem soja M12 kvasca P. stipitis na glicerolu kao osnovnom izvoru ugljika uz pulsni dodatak metionina na početku stacionarne faze rasta ostvareno je povećanje sadržaja SAMe u biomasi soja M12 kvasca P. stipitis u odnosu na divlji soj kvasca P. stipitis.The aim of this work was to evaluate the possibility of S-adenosyl-L-methionine (SAM) production using yeast Pichia stipitis CBS 5776 (P. stipitis). P. stipitis M12 strain was selected using UV-mutagenesis and nystatin. M12 strain does not express C-24 sterol metyltransferase (Erg6p), and thus has decreased ergosterol biosynthesis and increased production of SAM. Genetic and proteomic profile for P. stipitis M12 strain, as well as optimal medium composition (a type of the main carbon source, amount and form of methionine, presence of salts) and growth conditions (aeration, temperature and pH) for SAM production were determined. An increase in SAM content in the biomass by growing M12 strain on glycerol as the main carbon source with a pulse addition of methionine at the beginning of the stationary growth phase, compared to the P. stipitis wild strain was achieved