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Review
. 2023 Jul 26;9(8):786.
doi: 10.3390/jof9080786.

Advances in S. cerevisiae Engineering for Xylose Fermentation and Biofuel Production: Balancing Growth, Metabolism, and Defense

Ellen R Wagner  1   2   3 , Audrey P Gasch  1   2   3
Affiliations
Review

Advances in S. cerevisiae Engineering for Xylose Fermentation and Biofuel Production: Balancing Growth, Metabolism, and Defense

Ellen R Wagner et al. J Fungi (Basel). .

Abstract

Genetically engineering microorganisms to produce chemicals has changed the industrialized world. The budding yeast Saccharomyces cerevisiae is frequently used in industry due to its genetic tractability and unique metabolic capabilities. S. cerevisiae has been engineered to produce novel compounds from diverse sugars found in lignocellulosic biomass, including pentose sugars, like xylose, not recognized by the organism. Engineering high flux toward novel compounds has proved to be more challenging than anticipated since simply introducing pathway components is often not enough. Several studies show that the rewiring of upstream signaling is required to direct products toward pathways of interest, but doing so can diminish stress tolerance, which is important in industrial conditions. As an example of these challenges, we reviewed S. cerevisiae engineering efforts, enabling anaerobic xylose fermentation as a model system and showcasing the regulatory interplay's controlling growth, metabolism, and stress defense. Enabling xylose fermentation in S. cerevisiae requires the introduction of several key metabolic enzymes but also regulatory rewiring of three signaling pathways at the intersection of the growth and stress defense responses: the RAS/PKA, Snf1, and high osmolarity glycerol (HOG) pathways. The current studies reviewed here suggest the modulation of global signaling pathways should be adopted into biorefinery microbial engineering pipelines to increase efficient product yields.

Keywords: environmental stress response; protein kinase A; signal transduction; xylose fermentation; yeast.

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Conflict of interest statement

Our understanding of how growth, metabolism, and defense are integrated into cellular regulatory networks is only beginning to emerge, but recent studies have uncovered new insights into the balance between the growth and defense controls. Rapid growth and maximal stress tolerance are competing interests in the cell since both require significant resources to enact. When times are good and nutrients are plentiful, S. cerevisiae maximizes its growth rate, but to do so, cells decrease the defense systems to direct resources to biomass production and division. Thus, the fastest-growing cells are typically the most sensitive to acute stress [30,31,32,33,34,35,36]. In response to sudden stress, cells typically decrease their growth rate and transiently arrest their cell cycle while they redirect cellular resources to mounting the stress response, which includes mechanisms to defend against the imposing stress as well as what are likely protective mechanisms against future stresses.

A major component of the S. cerevisiae stress response is reorganizing the transcriptome. In addition to specialized responses triggered by specific stresses, stressed yeast mounts a common response to stress. The environmental stress response (ESR) comprises ~900 genes whose expression is altered in response to a variety of stresses, leading to massive physiological changes [37,38,39,40]. The ESR includes ~300 genes whose transcript abundance increases during stress and ~600 genes whose transcript abundance decreases. Induced genes are broadly involved in stress defense processes, including oxidation-reduction balancing, protein folding, the production of defense molecules like trehalose and glycerol, and specific regulators. The transcriptional induction of these genes is controlled by a variety of stress-specific factors in conjunction with the general stress transcription factors Msn2 and Msn4 [37,38,41,42,43]. In contrast, genes repressed in the ESR include genes that normally promote growth, including ribosomal protein (RP) and ribosomal biogenesis (RiBi) genes involved in ribosome production, RNA metabolism, protein synthesis, and cell growth [37].

Activation of the ESR can co-occur with the decreased growth rate of a culture, leading several studies to suggest that the ESR is intimately regulated with, and predictive of, the cellular growth rate [30,44,45,46]. However, work from our lab shows that the ESR is separable from growth and division: the ESR is still activated upon heat or salt stress, even in cells that are already arrested in their cell cycle with low biomass production [34]. Instead, we argue that the dramatic transcriptome changes associated with the ESR serve to accelerate a stress response. The transient repression of ribosome-related and growth-promoting genes during stress helps to redirect the transcriptional and translational capacity toward stress-induced transcripts [34,47,48]. Somewhat counterintuitively, cells that lack repressors of the repressed ESR genes, Dot6 and Tod6, grow well in the absence of stress but acclimate much slower to salt stress [48]. At least part of this mutant effect can be explained by the delayed production of defense proteins: ribosome- and growth-related transcripts stay associated with translating ribosomes in the mutant cells at the expense of stress-induced transcripts, leading to a delay in the production of stress defense proteins [34,48].

The ESR is regulated by multiple upstream signaling pathways, many of which are only activated by specific conditions [33,34,37,38]. Among the best studied of these are the protein kinase A (PKA), Snf1, and high osmolarity glycerol (HOG) pathways, all of which turn out to be important for engineered xylose fermentation (reviewed in more detail below). PKA inhibits the ESR in part by phosphorylating and inhibiting Msn2/4, Dot6/Tod6, and other regulators [49,50]; it also functions to modulate gene expression of specific genes by binding promoters and coding regions via interactions with chromatin proteins or the RNA polymerase [51,52,53,54]. Snf1 and HOG can also modulate downstream ESR regulators, including Msn2/4 and others, both directly and indirectly [41,55,56,57]. Interestingly, several independent studies found that modulating the activity of these broadly acting signaling pathways is necessary to promote robust anaerobic fermentation of xylose in engineered biofuel yeast strains (see below). The remainder of this review will discuss the potential roles of the PKA, Snf1, and HOG pathways in engineering xylose fermentation.

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Overview of different cellular regimes that prioritize growth, stress defense response, or engineered metabolic flux in biofuel-producing microorganisms. See text for details.
Figure 2
Figure 2
The Protein Kinase A pathway. A simplified view of the RAS/PKA pathway in yeast. HXTs, hexose transporters; HXK, hexokinase. See text for details [99].
Figure 3
Figure 3
The Snf1 pathway. The Snf1 complex can be regulated by one of three kinases (Tos1, Sak1, or Elm1). Active Snf1 complex translocates into the nucleus to modulate gene expression. When glucose concentration increases, the Glc7-Reg1 protein phosphatase complex is activated by hexokinase (HXK) to dephosphorylate and inactivate Snf1. See text for details [154].
Figure 4
Figure 4
The high osmolarity glycerol pathway. See text for details [103].
See this image and copyright information in PMC

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