Improved galactose fermentation of Saccharomyces cerevisiae through inverse metabolic engineering
ABSTRACT 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.
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- "The data (Fig. 3) revealed that hexoses are key carbon sources for the tested bacteria, and their utilizations have a sequence, such as glucose or/and galactose firstly, followed by the mannose as the alternative sugar . These results coincide with previous research, reporting that among the fermentable sugars in hydrolysates, glucose is considered as the desirable fermentation substrate for oleaginous microorganisms, while other hemicellulose-derived monosaccharides, such as mannose and galactose, can also be selectively utilized depending on the microbial species (Jin et al. 2015; Lee et al. 2011). Considering that xylose is the second most abundant sugar in hardwood, obtaining bacteria with xylose-degrading ability is of importance for better utilization of its hydrolysate. "
ABSTRACT: Metabolic synthesis of single cell oils (SCOs) for biodiesel application by heterotrophic oleaginous microorganisms is being hampered by the high cost of culture media. This study investigated the possibility of using loblolly pine and sweetgum autohydrolysates as economic feedstocks for microbial lipid production by oleaginous Rhodococcus opacus (R. opacus) PD630 and DSM 1069. Results revealed that when the substrates were detoxified by the removal of inhibitors (such as HMF-hydroxymethyl-furfural), the two strains exhibited viable growth patterns after a short adaptation/lag phase. R. opacus PD630 accumulated as much as 28.6 % of its cell dry weight (CDW) in lipids while growing on detoxified sweetgum autohydrolysate (DSAH) that translates to 0.25 g/l lipid yield. The accumulation of SCOs reached the level of oleagenicity in DSM 1069 cells (28.3 % of CDW) as well, while being cultured on detoxified pine autohydrolysate (DPAH), with the maximum lipid yield of 0.31 g/l. The composition of the obtained microbial oils varied depending on the substrates provided. These results indicate that lignocellulosic autohydrolysates can be used as low-cost fermentation substrates for microbial lipid production by wild-type R. opacus species. Consequently, the variety of applications for aqueous liquors from lignocellulosic pretreatment has been expanded, allowing for the further optimization of the integrated biorefinery.Applied Microbiology and Biotechnology 07/2015; DOI:10.1007/s00253-015-6752-5 · 3.81 Impact Factor
Metabolomics, 02/2012; , ISBN: 978-953-51-0046-1
- "Aside from rational design stochastic methods based on inverse metabolic engineering have been developed for S. cerevisiae to identify key target reactions and associated gene sequences enabling the desired new cellular property (Bailey et al. 2002; Bengtsson et al. 2008; Bro et al. 2005; Hong et al. 2010; Jin et al. 2005; Lee et al. 2010). Differently, methods targeting on the induction of a cellular property, such as growth, increase of substrate conversion rate or enhancing resistance to environmental stress, that is hardly to capture by in silico design because of its highly intricate metabolic relations that have to be satisfied, rely on the cellular adaptability to a certain environmental stress by evolution (Cakar et al. 2009; Cakar et al. 2005; Garcia Sanchez et al. 2010; Kuyper et al. 2005; Sonderegger and Sauer 2003; Wisselink et al. 2009). "
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ABSTRACT: A galactose-fermenting yeasts, Saccharomyces cerevisiae No. 9, was selected by screening their abilities to produce carbon dioxide gas when grown on galactose. The selected strain, No. 9 and the reference strains NRRL Y-1528 which was exceptionally resistant to high concentration of substrate, were acclimated on sugars such as glucose, mannose, and galactose, and then their ethanol productivities were investigated during fermentation on these three carbon sources. Ethanol productivity of the strain No. 9 reached to the maximum levels after 18 h of fermentation and the ethanol yield was from 36 to 38% when presented as , regardless of the conditions of acclimation. From the results obtained by acclimation and fermentation, it was concluded that the ethanol yields from galactose were not affected by the sugars acclimated. Improvements of the strain S. cerevisiae No. 9 were attempted to increase the fermentation efficiency and/or ethanol yields on high concentration of substrate by the conventional mutation methods employing methanesulfonic acid, ethyl ester (EMS). Mutants, Mut-5 (SJ1-40), -17 (LK4-25) and -24 (LK2-48) fermented galactose at the concentration of 20% in the levels of higher 39.9~51.6% than the mother strain, No. 9, however, their ethanol yields never exceeded those of the reference strain.Journal of Applied Biological Chemistry 03/2011; 54(1). DOI:10.3839/jabc.2011.007