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.
Article: Comparative evaluation and selection of a method for lipid and fatty acid extraction from macroalgae.[show abstract] [hide abstract]
ABSTRACT: A comparative evaluation of Bligh and Dyer, Folch, and Cequier-Sánchez methods for quantitative determination of total lipids (TLs) and fatty acids (FAs) was accomplished in selective green (Ulva fasciata), red (Gracilaria corticata), and brown algae (Sargassum tenerrimum) using a full factorial categorical design. Applications of sonication and buffer individually on lipid extraction solvent systems were also evaluated. The FA recoveries obtained from the aforementioned methods were compared with those of direct transesterification (DT) methods to identify the best extraction methods. The experimental design showed that macroalgal matrix, extraction method, and buffer were key determinants for TL and FA recoveries (P≤0.05), exhibiting significant interactions. But sonication gave erratic results with no interaction with any of the factors investigated. The buffered solvent system of Folch rendered the highest TL yield in U. fasciata and G. corticata while the buffered system of Bligh and Dyer gave the highest yield in S. tenerrimum. DT methods were more convenient and accurate for FA quantification and rendered 1.5-2 times higher yields when compared with the best conventional method, minimizing the use of chlorinated solvents, their cost of analysis, and disposal. The buffered solvent system was found to be the most appropriate for lipid research in macroalgae.Analytical Biochemistry 08/2011; 415(2):134-44. · 3.00 Impact Factor