Transcriptome changes in adaptive evolution of xylose-fermenting industrial Saccharomyces cerevisiae strains with δ-integration of different xylA genes.

It is of utmost importance to construct industrial xylose-fermenting Saccharomyces cerevisiae strains for lignocellulosic bioethanol production. In this study, two xylose isomerase-based industrial S. cerevisiae strains, O7 and P5, were constructed by δ-integration of the xylose isomerase (XI) gene xylA from the fungus Orpinomyces sp. and from the bacterium Prevotella ruminicola, respectively. The xylose consumption of the strains O7 and P5 at 48-h fermentation was 17.71 and 26.10 g/L, respectively, in synthetic medium with xylose as the sole sugar source. Adaptive evolution further improved the xylose fermentation capacity of the two strains to 51.0 and 28.9% in average, respectively. The transcriptomes of these two strains before and after evolution were analyzed using RNA-Seq. The expression levels of the genes involved in cell integrity, non-optimal sugar utilization, and stress response to environment were significantly up-regulated after evolution and did not depend on the origin of xylA; the expression levels of the genes involved in transmembrane transport, rRNA processing, cytoplasmic translation, and other processes were down-regulated. The expression of genes involved in central carbon metabolism was fine-tuned after the evolution. The analysis of transcription factors (TFs) indicated that most of the genes with significant differential expression were regulated by the TFs related to cell division, DNA damage response, or non-optimal carbon source utilization. The results of this study could provide valuable references for the construction of efficient xylose-fermenting XI strains.

Comparative transcriptomes reveal novel evolutionary strategies adopted by Saccharomyces cerevisiae with improved xylose utilization capability.

Production of ethanol from xylose by recombinant Saccharomyces cerevisiae is suboptimal with slow fermentation rate, compared with that from glucose. In this study, a strain-expressing Scheffersomyces stipitis xylose reductase-xylitol dehydrogenase (XR-XDH) pathway was subjected to adaptive evolution on xylose; this approach generated populations with the significantly improved cell growth and ethanol production rate. Mutants were isolated, and the best one was used for sporulation to generate eight stable mutant strains with improved xylose fermentation ability. They were used in a microarray assay to study the molecular basis of the enhanced phenotype. The enriched transcriptional differences among the eight mutant strains and the native strain revealed novel responses to xylose, which likely contributes to the improved xylose utilization. The upregulated vitamin B1 and B6 biosynthesis indicated that thiamine served as an important cofactor in xylose metabolism and may alleviate the redox stress. The increased expression of genes involved in sulfur amino acid biosynthesis and the decreased expression of genes related to Fe(II) transport may alleviate redox stress as well. Meanwhile, it was remarkable that several glucose-repressible genes, including genes of the galactose metabolism, gluconeogenesis, and ethanol catabolism, had a lower expression level after adaptive evolution. Concomitantly, the expression levels of two regulators of the glucose signaling pathway, Rgs2 and Sip4, decreased, indicating a reshaped signaling pathway to xylose after adaptive evolution. Our findings provide new targets for construction of a superior bioethanol producing strain through inverse metabolic engineering.

Transcriptomes of a xylose-utilizing industrial flocculating Saccharomyces cerevisiae strain cultured in media containing different sugar sources.

Lignocellulosic hydrolysates used for bioethanol production contain a mixture of sugars, with xylose being the second most abundant after glucose. Since xylose is not a natural substrate for Saccharomyces cerevisiae, recombinant S. cerevisiae strongly prefers glucose over xylose, and the fermentation rate and ethanol yield with xylose are both lower than those with glucose. To determine the molecular basis for glucose and xylose fermentation, we used microarrays to investigate the transcriptional difference of a xylose-utilizing industrial strain cultured in both single sugar media and a mixed sugar medium of glucose and xylose. The transcriptomes were nearly identical between glucose metabolizing cells in the glucose alone medium and those in the glucose fermentation phase in the mixed-sugar medium. Whereas the transcriptomes highly differed between the xylose metabolizing cells in the xylose alone medium and those in the xylose fermentation phase in the mixed sugar medium, and the differences mainly involved sulfur metabolism. When the transcriptional profiles were compared between glucose fermentation state and xylose fermentation state, we found the expression patterns of hexose transporters and glucose signaling pathway differed in response to different sugar sources, and the expression levels of the genes involved in gluconeogenesis, the glyoxylate and tricarboxylic acid cycles and respiration increased with xylose, indicating that the xylose-metabolizing cells had high requirements for maintenance energy and lacked the carbon catabolite repression capability. The effect of carbon catabolite repression by glucose lasted after glucose depletion for specific genes to different extents.