초록
<P><B>Abstract</B></P><P>Although <I>Saccharomyces cerevisiae</I> is capable of fermenting galactose into ethanol, ethanol yield and productivity from galactose are significantly lower than those from glucose. An inverse metabolic engineering approach was undertaken to improve ethanol yield and productivity from galactose in <I>S</I>. <I>cerevisiae</I>. A genome‐wide perturbation library was introduced into <I>S</I>. <I>cerevisiae</I>, and then fast galactose‐fermenting transformants were screened using three different enrichment methods. The characterization of genetic perturbations in the isolated transformants revealed three target genes whose overexpression elicited enhanced galactose utilization. One confirmatory (<I>SEC53</I> coding for phosphomannomutase) and two novel targets (<I>SNR84</I> coding for a small nuclear RNA and a truncated form of <I>TUP1</I> coding for a general repressor of transcription) were identified as overexpression targets that potentially improve galactose fermentation. Beneficial effects of overexpression of <I>SEC53</I> may be similar to the mechanisms exerted by overexpression of <I>PGM2</I> coding for phosphoglucomutase. While the mechanism is largely unknown, overexpression of <I>SNR84</I>, improved both growth and ethanol production from galactose. The most remarkable improvement of galactose fermentation was achieved by overexpression of the truncated <I>TUP1</I> (t<I>TUP1</I>) gene, resulting in unrivalled galactose fermentation capability, that is 250% higher in both galactose consumption rate and ethanol productivity compared to the control strain. Moreover, the overexpression of t<I>TUP1</I> significantly shortened lag periods that occurs when substrate is changed from glucose to galactose. Based on these results we proposed a hypothesis that the mutant Tup1 without C‐terminal repression domain might bring in earlier and higher expression of <I>GAL</I> genes through partial alleviation of glucose repression. mRNA levels of <I>GAL</I> genes (<I>GAL1</I>, <I>GAL4</I>, and <I>GAL80</I>) indeed increased upon overexpression of <I>tTUP</I>. The results presented in this study illustrate that alteration of global regulatory networks through overexpression of the identified targets (<I>SNR84</I> and t<I>TUP1</I>) is as effective as overexpression of a rate limiting metabolic gene (<I>PGM2</I>) in the galactose assimilation pathway for efficient galactose fermentation in <I>S</I>. <I>cerevisiae</I>. In addition, these results will be industrially useful in the biofuels area as galactose is one of the abundant sugars in marine plant biomass such as red seaweed as well as cheese whey and molasses. Biotechnol. Bioeng. 2011; 108:621–631. © 2010 Wiley Periodicals, Inc.</P>