Fluctuations in glucose availability prevent global proteome changes and physiological transition during prolonged chemostat cultivations of Saccharomyces cerevisiae.

Chemostat cultivation mode imposes selective pressure on the cells, which may result in slow adaptation in the physiological state over time. We applied a two-compartment scale-down chemostat system imposing feast-famine conditions to characterize the long-term (100 s of hours) response of Saccharomyces cerevisiae to fluctuating glucose availability. A wild-type strain and a recombinant strain, expressing an insulin precursor, were cultured in the scale-down system, and analyzed at the physiological and proteomic level. Phenotypes of both strains were compared with those observed in a well-mixed chemostat. Our results show that S. cerevisiae subjected to long-term chemostat conditions undergoes a global reproducible shift in its cellular state and that this transition occurs faster and is larger in magnitude for the recombinant strain including a significant decrease in the expression of the insulin product. We find that the transition can be completely avoided in the presence of fluctuations in glucose availability as the strains subjected to feast-famine conditions under otherwise constant culture conditions exhibited constant levels of the measured proteome for over 250 hr. We hypothesize possible mechanisms responsible for the observed phenotypes and suggest experiments that could be used to test these mechanisms.

Linking hydrolysis performance to Trichoderma reesei cellulolytic enzyme profile.

Trichoderma reesei expresses a large number of enzymes involved in lignocellulose hydrolysis and the mechanism of how these enzymes work together is too complex to study by traditional methods, for example, by spiking with single enzymes and monitoring hydrolysis performance. In this study, a multivariate approach, partial least squares regression, was used to see whether it could help explain the correlation between enzyme profile and hydrolysis performance. Diverse enzyme mixtures were produced by T. reesei Rut-C30 by exploiting various fermentation conditions and used for hydrolysis of washed pretreated corn stover as a measure of enzyme performance. In addition, the enzyme mixtures were analyzed by liquid chromatography-tandem mass spectrometry to identify and quantify the different proteins. A multivariate model was applied for the prediction of enzyme performance based on the combination of different proteins present in an enzyme mixture. The multivariate model was used for identification of candidate proteins that are correlated to enzyme performance on pretreated corn stover. A very large variation in hydrolysis performance was observed and this was clearly caused by the difference in fermentation conditions. Besides ß-glucosidase, the multivariate model identified several xylanases, Cip1 and Cip2, as relevant proteins to study further.