Leveraging transcription factors to speed cellobiose fermentation by

Abstract

Background: Saccharomyces cerevisiae, a key organism used for the manufacture of renewable fuels and chemicals, has been engineered to utilize non-native sugars derived from plant cell walls, such as cellobiose and xylose. However, the rates and efficiencies of these non-native sugar fermentations pale in comparison with those of glucose. Systems biology methods, used to understand biological networks, hold promise for rational microbial strain development in metabolic engineering. Here, we present a systematic strategy for optimizing non-native sugar fermentation by recombinant S. cerevisiae, using cellobiose as a model. Results: Differences in gene expression between cellobiose and glucose metabolism revealed by RNA deep sequencing indicated that cellobiose metabolism induces mitochondrial activation and reduces amino acid biosynthesis under fermentation conditions. Furthermore, glucose-sensing and signaling pathways and their target genes, including the cAMP-dependent protein kinase A pathway controlling the majority of glucose-induced changes, the Snf3-Rgt2-Rgt1 pathway regulating hexose transport, and the Snf1-Mig1 glucose repression pathway, were at most only partially activated under cellobiose conditions. To separate correlations from causative effects, the expression levels of 19 transcription factors perturbed under cellobiose conditions were modulated, and the three strongest promoters under cellobiose conditions were applied to fine-tune expression of the heterologous cellobiose-utilizing pathway. Of the changes in these 19 transcription factors, only overexpression of SUT1 or deletion of HAP4 consistently improved cellobiose fermentation. SUT1 overexpression and HAP4 deletion were not synergistic, suggesting that SUT1 and HAP4 may regulate overlapping genes important for improved cellobiose fermentation. Transcription factor modulation coupled with rational tuning of the cellobiose consumption pathway significantly improved cellobiose fermentation.

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

“…The genes encoding these transcription factors were systematically overexpressed or deleted and was screened for increased cellobiose assimilation. Overexpression of SUT1 or deletion of HAP4 were found to improve cellobiose fermentation . More recently, overexpression or downregulation of endogenous genes for increasing xylose consumption and growth were carried out.…”

“…These studies also underscore the need to analyze the problem of catabolic engineering from a systems‐level, rather than analyzing it from a pathway level, and the need for transcriptional reprogramming. Although strategies like transcription factor engineering have taken the initial steps in that direction, more research on systems‐level understanding of catabolism, regulatory mechanisms involved, and how metabolic, regulatory, structural, and phenotypic genes are interlinked is warranted. Moreover, rather than altering a few genes or transcription factors, evidence point to the need for a more systems‐level re‐wiring to achieve efficient pentose assimilation.…”

Distinct Proteome Remodeling of Industrial Saccharomyces cerevisiae in Response to Prolonged Thermal Stress or Transient Heat Shock

“…Glucose and ethanol were eluted from the column by 5 mM sulfuric acid solution at a flow rate of 0.6 mL/min. Fermentation rates including maximum growth rates (μ max ), maximum glucose-consumption rates ( q s max), and ethanol productivities ( P EtOH ) were calculated as described previously …”