20.500.12556/RUL-99012 Anaerobna produkcija bioetanola s transformantami kvasovke Saccharomyces cerevisiae z vključenim genom pfk1. Anaerobic production of bioethanol with transformant yeast Saccharomyces cerevisiae with inserted PFK1 gene Encim fosfofruktokinaza 1 (Pfk1) je osrednji regulatorni encim v glikolizi. Posttranslacijska modifikacija encima Pfk1 omogoča nastanek aktivnih krajših fragmentov Pfk1, ki imajo bistveno višjo aktivnost od izvornih encimov. Humani kratek fragment Pfk-M, velikosti 47 kDa, je bolj nagnjen k aktivaciji, hkrati pa nedovzeten za inhibitorje delovanja encimov Pfk1. Z vnosom gena sfPFKM za sintezo fragmentov Pfk-M v kvasovko S. cerevisiae pride do povišanega metabolnega pretoka preko glikolize, žal pa tudi do porušenega ravnotežja NADH/NADPH, kar pa preprečuje rast celic v gojiščih ob anaerobnih pogojih. Rast je mogoča z vključenim heterolognim genom za malični encim, ki omogoča dodatno produkcijo NADPH. Namen dela je bilo spremljanje rasti kvasovk S. cerevisiae z geni PFK različnega izvora in z genom MAEA za malični encim v fermentativnih gojiščih ob anaerobnih pogojih. S poskusi smo dokazali, da so sevi z vključenimi geni za kratke fragmente in maličnim encimom sposobni rasti na pentoznih sladkorjih, kar pa ne velja za divje tipe kvasovk. To pa je mogoče doseči le s spremembo alosterične regulacije pri encimu Pfk1. V gojiščih smo z upadanjem koncentracije ksilitola opazili kopičenje hlapnega produkta 2-feniletanola. Tako razgradnja pentoz ne pomeni le možnost pridobivanja bioetanola iz lignoceluloznih materialov, temveč tudi drugih industrijsko pomembnih fermentativnih produktov. Enzyme phosphofructokinase 1 (Pfk1) is a key regulatory enzyme of the glycolysis. Posttranslational modification of the Pfk1 enzyme allows the formation of an active Pfk1 fragments which have significantly higher activity than original enzymes. Shorter human 47 kDa Pfk-M fragments are more prone to activation but also resistant to feedback inhibition. Increased metabolic flow through glycolysis results in an unbalance of the NADH/NADPH ratio. This prevents the growth of the cells with shorter Pfk-M fragment in the media under anaerobic conditions. Growth can be re-established with included heterologic malic enzyme, which enables additional production of the NADPH in the cells. The growth of S. cerevisiae yeast with the PFK genes of different origins and the MAEA gene encoding the malic enzyme was observed in fermentative media under anaerobic conditions. We demonstrated experimentally that the strains with genes for short fragments and the malic enzyme were able to grow on pentose sugars which is not possible for the wild type of yeast strains. Therefore, only by modifying the regulatory Pfk1 enzyme, the yeast can utilize the pentose sugars. In the media, the accumulation of the volatile product a 2-phenylethanol was observed with decreasing xylitol concentration. Thus, the degradation of pentose sugars does not only present the possibility of obtaining bioethanol from lignocellulosic materials but also other industrially important fermentative products. kvasovke Saccharomyces cerevisiae transformirane celice bioetanol gen PFK1 yeast Saccharomyces cerevisiae transformed cells bioethanol PFK1 gene true false false [M. Dragan] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2017-12-21 07:15:08 2017-12-21 07:15:19 2022-08-16 10:27:33 0000-00-00 00:00:00 2017 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 602.3:602.6:582.282.23:604.2:661.722(043.2) 150607 8895097 1666.pdf 1666.pdf 1 282C0F700DD02729F2D7AA38EECDA6A5 f80b511ca0cab6ebe1b4951ca3e607af0e4f1291cfa7e1b81ef9cc719c51dd0d 79db96a7-a1b4-11eb-a523-00155dcfd717 20.500.12556/rul/723210b3-7fae-4934-add8-b2e2f0d3056d https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=108358 Biotehniška fakulteta 0 0 0