Physiological effects of over-expressing compartment-specific components of the protein folding machinery in xylose-fermenting Saccharomyces cerevisiae

Abstract

BackgroundEfficient utilization of both glucose and xylose is necessary for a competitive ethanol production from lignocellulosic materials. Although many advances have been made in the development of xylose-fermenting strains of Saccharomyces cerevisiae, the productivity remains much lower compared to glucose. Previous transcriptional analyses of recombinant xylose-fermenting strains have mainly focused on central carbon metabolism. Very little attention has been given to other fundamental cellular processes such as the folding of proteins. Analysis of previously measured transcript levels in a recombinant XR/XDH-strain showed a wide down-regulation of genes targeted by the unfolded protein response during xylose fermentation. Under anaerobic conditions the folding of proteins is directly connected with fumarate metabolism and requires two essential enzymes: FADH2-dependent fumarate reductase (FR) and Ero1p. In this study we tested whether these enzymes impair the protein folding process causing the very slow growth of recombinant yeast strains on xylose under anaerobic conditions.ResultsFour strains over-expressing the cytosolic (FRD1) or mitochondrial (OSM1) FR genes and ERO1 in different combinations were constructed. The growth and fermentation performance was evaluated in defined medium as well as in a complex medium containing glucose and xylose. Over-expression of FRD1, alone or in combination with ERO1, did not have any significant effect on xylose fermentation in any medium used. Over-expression of OSM1, on the other hand, led to a diversion of carbon from glycerol to acetate and a decrease in growth rate by 39% in defined medium and by 25% in complex medium. Combined over-expression of OSM1 and ERO1 led to the same diversion of carbon from glycerol to acetate and had a stronger detrimental effect on the growth in complex medium.ConclusionsIncreasing the activities of the FR enzymes and Ero1p is not sufficient to increase the anaerobic growth on xylose. So additional components of the protein folding mechanism that were identified in transcription analysis of UPR related genes may also be limiting. This includes i) the transcription factor encoded by HAC1 ii) the activity of Pdi1p and iii) the requirement of free FAD during anaerobic growth.

Preparation of stable recombinant Osm1 noncovalently bound with flavin adenosine dinucleotide cofactor for structural study

“…In support of this assumption, S. cerevisiae does not utilize anaerobic respiration for ATP synthesis under anaerobic conditions and instead employs fermentation, which does not necessitate fumarate reduction. Osm1 and Frd1 appear to play roles in redox balancing and protein production during anaerobiosis (Bergdahl et al, 2014;Enomoto et al, 2002;Liu et al, 2013).…”