Reassessment of requirements for anaerobic xylose fermentation by engineered, non-evolved Saccharomyces cerevisiae strains

• Published in: FEMS yeast research 2019-01, Vol.19 (1), p.1
• Main Authors: Bracher, Jasmine M, Martinez-Rodriguez, Oscar A, Dekker, Wijb J C, Verhoeven, Maarten D, van Maris, Antonius J A, Pronk, Jack T
• Format: Article
• Language: English
• Subjects: Aldoses; Ammonium; Aspartate; Biomass energy; Bioreactors; Carbon dioxide; Carboxylation; CRISPR; Enzymes; Fermentation; Gene deletion; Genetic research; Genetically modified organisms; Inoculum; Lag phase; Metabolic engineering; Methods; Mimicry; Monosaccharides; Observations; Phosphates; Physiological aspects; Reductase; Saccharomyces cerevisiae; Transaldolase; Transketolase; Xylose; Xylose isomerase; Xylulokinase; Yeast; Yeasts (Fungi)
• ISSN: 1567-1364; 1567-1356; 1567-1364
• DOI: 10.1093/femsyr/foy104

Expression of a heterologous xylose isomerase, deletion of the GRE3 aldose-reductase gene and overexpression of genes encoding xylulokinase (XKS1) and non-oxidative pentose-phosphate-pathway enzymes (RKI1, RPE1, TAL1, TKL1) enables aerobic growth of Saccharomyces cerevisiae on d-xylose. However, literature reports differ on whether anaerobic growth on d-xylose requires additional mutations. Here, CRISPR-Cas9-assisted reconstruction and physiological analysis confirmed an early report that this basic set of genetic modifications suffices to enable anaerobic growth on d-xylose in the CEN.PK genetic background. Strains that additionally carried overexpression cassettes for the transaldolase and transketolase paralogs NQM1 and TKL2 only exhibited anaerobic growth on d-xylose after a 7-10 day lag phase. This extended lag phase was eliminated by increasing inoculum concentrations from 0.02 to 0.2 g biomass L-1. Alternatively, a long lag phase could be prevented by sparging low-inoculum-density bioreactor cultures with a CO2/N2-mixture, thus mimicking initial CO2 concentrations in high-inoculum-density, nitrogen-sparged cultures, or by using l-aspartate instead of ammonium as nitrogen source. This study resolves apparent contradictions in the literature on the genetic interventions required for anaerobic growth of CEN.PK-derived strains on d-xylose. Additionally, it indicates the potential relevance of CO2 availability and anaplerotic carboxylation reactions for anaerobic growth of engineered S. cerevisiae strains on d-xylose.