Abstract
Microbes can be metabolically engineered to produce biofuels and biochemicals, but rerouting metabolic flux toward products is a major hurdle without a systems-level understanding of how cellular flux is controlled. To understand flux rerouting, we investigated a panel of Saccharomyces cerevisiae strains with progressive improvements in anaerobic fermentation of xylose, a sugar abundant in sustainable plant biomass used for biofuel production. We combined comparative transcriptomics, proteomics, and phosphoproteomics with network analysis to understand the physiology of improved anaerobic xylose fermentation. Our results show that upstream regulatory changes produce a suite of physiological effects that collectively impact the phenotype. Evolved strains show an unusual co-activation of Protein Kinase A (PKA) and Snf1, thus combining responses seen during feast on glucose and famine on non-preferred sugars. Surprisingly, these regulatory changes were required to mount the hypoxic response when cells were grown on xylose, revealing a previously unknown connection between sugar source and anaerobic response. Network analysis identified several downstream transcription factors that play a significant, but on their own minor, role in anaerobic xylose fermentation, consistent with the combinatorial effects of small-impact changes. We also discovered that different routes of PKA activation produce distinct phenotypes: deletion of the RAS/PKA inhibitor IRA2 promotes xylose growth and metabolism, whereas deletion of PKA inhibitor BCY1 decouples growth from metabolism to enable robust fermentation without division. Comparing phosphoproteomic changes across ira2Δ and bcy1Δ strains implicated regulatory changes linked to xylose-dependent growth versus metabolism. Together, our results present a picture of the metabolic logic behind anaerobic xylose flux and suggest that widespread cellular remodeling, rather than individual metabolic changes, is an important goal for metabolic engineering.
Highlights
Engineering microbes for non-native metabolic capabilities is a major goal in strain engineering
Integrative modeling implicates causal events linked to growth versus metabolism and shows the hypoxic response is dependent on carbon sensing in yeast
We first compared the transcriptome and proteome responses of parental strain Y22-3 and evolved strains Y127 and Y128 growing on glucose or xylose, with or without oxygen
Summary
Engineering microbes for non-native metabolic capabilities is a major goal in strain engineering. A better understanding of cellular regulatory systems that can modulate metabolism without producing undesired off-target effects is an active area research for industrial microbiology [4,5,6] An example of this is seen in yeast fermentation of non-native sugars present in plant material. Many studies have attempted to improve xylose metabolism, for example by optimizing xylose metabolism proteins [9,10,11], mutating or overexpressing xylose transporters [12,13,14], inducing genes in the pentose-phosphate pathway [15,16,17,18,19,20], or deleting pathways that siphon intermediates [21,22,23,24] While these modifications improve the phenotype, many of the individual mutations often do so with relatively small effects [12,13,15,17,21,22,23,24,25]. In many cases the reason for improved xylose metabolism remains unknown, which does not advance strategies for rationale engineering
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