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.