Print Email Facebook Twitter Reassessment of requirements for anaerobic xylose fermentation by engineered, non-evolved Saccharomyces cerevisiae strains Title Reassessment of requirements for anaerobic xylose fermentation by engineered, non-evolved Saccharomyces cerevisiae strains Author Bracher, J.M. (TU Delft BT/Industriele Microbiologie) Martinez-Rodriguez, Oscar A. (Genomatica, San Diego) Dekker, W.J.C. (TU Delft BT/Industriele Microbiologie) Verhoeven, M.D. (DSM) van Maris, A.J.A. (AlbaNova University Center) Pronk, J.T. (TU Delft BT/Industriele Microbiologie) Date 2018 Abstract 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. To reference this document use: http://resolver.tudelft.nl/uuid:b04fc945-12b9-4e14-983f-553127176662 DOI https://doi.org/10.1093/femsyr/foy104 ISSN 1567-1356 Source FEMS Yeast Research, 19 (1) Part of collection Institutional Repository Document type journal article Rights © 2018 J.M. Bracher, Oscar A. Martinez-Rodriguez, W.J.C. Dekker, M.D. Verhoeven, A.J.A. van Maris, J.T. Pronk Files PDF foy104.pdf 1.34 MB Close viewer /islandora/object/uuid%3Ab04fc945-12b9-4e14-983f-553127176662/datastream/OBJ/view