A semi-synthetic regulon enables rapid growth of yeast on xylose

Abstract

Nutrient assimilation is the first step that allows biological systems to proliferate and produce value-added products. Yet, implementation of heterologous catabolic pathways has so far relied on constitutive gene expression without consideration for global regulatory systems that may enhance nutrient assimilation and cell growth. In contrast, natural systems prefer nutrient-responsive gene regulation (called regulons) that control multiple cellular functions necessary for cell survival and growth. Here, in Saccharomyces cerevisiae, by partially- and fully uncoupling galactose (GAL)-responsive regulation and metabolism, we demonstrate the significant growth benefits conferred by the GAL regulon. Next, by adapting the various aspects of the GAL regulon for a non-native nutrient, xylose, we build a semi-synthetic regulon that exhibits higher growth rate, better nutrient consumption, and improved growth fitness compared to the traditional and ubiquitous constitutive expression strategy. This work provides an elegant paradigm to integrate non-native nutrient catabolism with native, global cellular responses to support fast growth.

Pentose Metabolism in Saccharomyces cerevisiae: The Need to Engineer Global Regulatory Systems

“…Multiple aspects of the native GAL‐system were re‐engineered to realize the XYL regulon that exhibited better growth rate, biomass accumulation, and substrate consumption than a strain that was engineered using the conventional strategy. Genome‐wide expression profiles of strains that grew with regulon assistance in both galactose and xylose had several growth‐related genes as well as transcription factors upregulated when compared to strains that constitutively expressed galactose or xylose metabolic genes . Similarly, activation of galactose regulon along with removing HXK2 ‐controlled carbon catabolite repression seemed to have resulted in the fastest arabinose‐growing S. cerevisiae strain, stressing the importance for higher systems‐level regulation for growth on a non‐native sugar …”

d‐Xylose consumption by nonrecombinant Saccharomyces cerevisiae: A review

“…Thus, in general, the genetically engineered yeast strains are transformed with the required genes under the control of strong and constitutive promoters to ensure the proper expression of the enzymes/transporters, which in general are not totally appropriate for the desired phenotype. This issue has been recently addressed by Endalur‐Gopinarayanan and Nair () using the galactose regulatory system in S. cerevisiae . For the efficient utilization of galactose, the sensor/regulator GAL3 , that in the presence of galactose and ATP binds to GAL80 , relieves GAL80 inhibition of GAL4 (a transcriptional activator of the zinc cluster family), allowing the induction of all GAL genes of the Leloir pathway, and several other genes required for growth/fermentation, but not directly involved in assimilating galactose (Malakar & Venkatesh, ; Peng & Hopper, ).…”

“…They further show the importance of regulatory systems by screening a library of mutant GAL3 regulators that can recognize xylose and analyse the impact of such “new” regulatory system on xylose utilization by yeast cells. Not surprisingly, when the required genes for xylose utilization (xylose isomerase encoded by XYLA , the GAL2 permease, and XKS1 and TAL1 ) were placed under the control of GAL1 and GAL10 promoters in GAL4 cells, the strain harbouring the mutated GAL3 regulator (that recognizes xylose) grew faster and consumed all xylose in the medium, and a strain with constitutive expression of these genes grew at lower specific rates, and it could not completely consume all the xylose (Endalur‐Gopinarayanan & Nair, ). To better understand the effects of the synthetic xylose regulon, a transcriptomic analysis using RNA‐seq was performed, revealing over 3.314 genes differentially expressed when comparing the cells xylose‐“regulated” with the xylose‐constitutive strain during growth on xylose.…”

“…and TAL1) were placed under the control of GAL1 and GAL10 promoters in GAL4 cells, the strain harbouring the mutated GAL3 regulator (that recognizes xylose) grew faster and consumed all xylose in the medium, and a strain with constitutive expression of these genes grew at lower specific rates, and it could not completely consume all the xylose (Endalur-Gopinarayanan & Nair, 2018). To better understand the effects of the synthetic xylose regulon, a transcriptomic analysis using RNA-seq was performed, revealing over 3.314 genes differentially expressed when comparing the cells xylose-"regulated"…”

Modular Engineering of Biomass Degradation Pathways

“…The development of a cross-species expression system using a T7 RNA polymerase and a self-regulating circuit has been shown to work reliably in B. subtilis, P. putida, and E. coli [25]. Similarly, the reuse of native regulatory mechanisms for heterologous pathways has been shown to improve reliability [178].…”