Introduction of gene for highly active pyruvate kinase in yeast Saccharomyces cerevisiae

Kvasovka Saccharomyces cerevisiae je eden izmed najpogosteje uporabljenih mikroorganizmov za proizvodnjo bioetanola. Z namenom pospešitve rasti seva kvasovke sfPFKM z modificirano obliko 45 kDa humanega encima 6-fosfofrukto-1-kinaza kot edino obliko encima s fosfofruktokinazno aktivnostjo, smo v ta sev vnesli dodatne kopije gena PYK1, ki kodira visoko aktiven encim piruvat-kinazo 1 (Pyk1) ter tako pridobili dvojno mutanto sfPFKM nPYK1. Predhodni testi so namreč pokazali, da na mestu delovanja Pyk1 obstaja ozko grlo v metabolnem pretoku med rastjo na maltoznem gojišču. Dvojna mutanta sfPFKM nPYK1 je na 0,5-odstotnem maltoznem minimalnem dopolnitvenem gojišču rastla hitreje kot izhodiščna enojna mutanta sfPFKM. Hitrejšo rast na tem gojišču smo opazili tudi pri nPFKM nPYK1 sevu, ki nosi zapis za nativni 85 kDa humani PFKM gen in ima vnešene dodatne kopije gena PYK1. Z nadaljnjimi modifikacijami izražanja kvasnih PYK1 genov v transformanti sfPFKM bi lahko odpravili ozko grlo v metabolnem pretoku preko glikolize in pripravili uporaben modelni organizem za študij deregulirane glikolize, kot jo povzroči modifikacija Pfk-M encima v rakastih celicah.The yeast Saccharomyces cerevisiae is one of the most commonly used microorganisms for bioethanol production. In order to promote the growth of the yeast strain with modified 45 kDa human PFK (sfPFKM) as the only form of phosphofructokinase enzyme in cells, additional copies of the PYK1 gene encoding the highly active piruvate-kinase 1 (Pyk1) was introduced into the strain. Preliminary tests have shown that at the site of Pyk1 activity there is a bottleneck in the metabolic flow during growth in the maltose medium. The double mutant sfPFKM nPYK1 grew faster in the 0,5-percent maltose minimal supplementary medium than the original sfPFKM strain. Faster growth on the same medium was observed also in the nPFKM nPYK1 strain, which carries a native 85 kDa human PFKM gene and has additional copies of the PYK1 gene. Further modification of the expression of yeast PYK1 genes in the transformation of sfPFKM could remove the bottleneck in the metabolic flow through glycolysis and enable design of a useful model organism for deregulated glycolysis, similar as caused by the modification of the Pfk-M enzyme in cancer cells