Improved galactose fermentation of Saccharomyces cerevisiae through inverse metabolic engineering.
Although Saccharomyces cerevisiae 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 S. cerevisiae. A genome-wide perturbation library was introduced into S. cerevisiae, 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 (SEC53 coding for phosphomannomutase) and two novel targets (SNR84 coding for a small nuclear RNA and a truncated form of TUP1 coding for a general repressor of transcription) were identified as overexpression targets that potentially improve galactose fermentation. Beneficial effects of overexpression of SEC53 may be similar to the mechanisms exerted by overexpression of PGM2 coding for phosphoglucomutase. While the mechanism is largely unknown, overexpression of SNR84, improved both growth and ethanol production from galactose. The most remarkable improvement of galactose fermentation was achieved by overexpression of the truncated TUP1 (tTUP1) 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 tTUP1 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 GAL genes through partial alleviation of glucose repression. mRNA levels of GAL genes (GAL1, GAL4, and GAL80) indeed increased upon overexpression of tTUP. The results presented in this study illustrate that alteration of global regulatory networks through overexpression of the identified targets (SNR84 and tTUP1) is as effective as overexpression of a rate limiting metabolic gene (PGM2) in the galactose assimilation pathway for efficient galactose fermentation in S. cerevisiae. 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.
School of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon, Korea.
Ki-Sung
KS
Lee
Min-Eui
ME
Hong
Suk-Chae
SC
Jung
Suk-Jin
SJ
Ha
Byung Jo
BJ
Yu
Hyun Min
HM
Koo
Sung Min
SM
Park
Jin-Ho
JH
Seo
Dae-Hyuk
DH
Kweon
Jae Chan
JC
Park
Yong-Su
YS
Jin
D000431:Ethanol / Q000378:metabolism*; D005285:Fermentation; D005690:Galactose / Q000378:metabolism*; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D005818:Genetic Engineering; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D009687:Nuclear Proteins / Q000096:biosynthesis; D012342:RNA, Small Nuclear / Q000096:biosynthesis; D012097:Repressor Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis
D009687:Nuclear Proteins; D012342:RNA, Small Nuclear; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C066502:TUP1 protein, S cerevisiae; D000431:Ethanol; C072713:SEC53 protein, S cerevisiae; D005690:Galactose