4. Discussion
In the present study, GCR2 encoding a transcriptional activator of glycolytic genes was identified as a novel deletion target (gcr2 ) to improve the xylose fermentation of S. cerevisiaeexpressing a heterologous xylose pathway. RNA-seq results revealed thatgcr2 results in not only the down-regulation of glycolytic genes but also the up-regulation of PPP genes, which explains the improved xylose metabolism by gcr2 . Specifically, gcr2 triggers the up-regulation of oxidative and non-oxidative PPP genes contributing to NADPH production for NAD(P)H-specific xylose reductase (XYL1 ) and direct metabolism of xylose.
Deleting both pho13 and gcr2 did not synergistically accelerate xylose fermentation. This outcome leads to a hypothesis that both genes might share similar molecular mechanism. Indeed, considering previous studies on pho13 (Kim et al., 2015; Xu et al., 2016), bothpho13 and gcr2 result in the up-regulation of TAL1gene, the essential overexpression target to improve xylose fermentation, as well as other genes in PPP. However, gcr2results in more global transcriptional changes compared to pho13 . The number of DE genes by pho13 was 12 and 277 on glucose and xylose, respectively, which was one order of magnitude lower than that by gcr2 . Also, some transcriptional changes induced bygcr2 were opposite directions from that by pho13 ; especially, genes in the lower glycolytic pathway were repressed bygcr2 but activated by pho13 during xylose fermentation. Therefore, it is difficult to ignore the possibility that pho13 - and gcr2 -mediated metabolic regulation are independent of each other but share TAL1 activation by chance.
Some native regulatory systems of S. cerevisiae might act negatively to heterologous metabolism. However, it is challenging to systematically investigate all native regulatory genes to identify inhibitory ones toward introduced pathways. One of the most practical solutions for metabolic engineering is to use adaptive evolution to induce spontaneous mutations favorable to heterologous metabolism. Identification of PHO13 and GCR2 is a successful example of such metabolic engineering strategy. Assisted with genome sequencing and omics approaches, such as RNA-seq, spontaneous mutations inPHO13 (intended) or GCR2 (by chance) were identified independently and led to the discovery of native inhibitory factors against the heterologous xylose pathway in our previous and present studies.