Print Email Facebook Twitter Mutations in PMR1 stimulate xylose isomerase activity and anaerobic growth on xylose of engineered Saccharomyces cerevisiae by influencing manganese homeostasis Title Mutations in PMR1 stimulate xylose isomerase activity and anaerobic growth on xylose of engineered Saccharomyces cerevisiae by influencing manganese homeostasis Author Verhoeven, M.D. (TU Delft BT/Industriele Microbiologie) Lee, Misun (Rijksuniversiteit Groningen) Kamoen, L. (Student TU Delft) van den Broek, M.A. (TU Delft BT/Industriele Microbiologie) Janssen, Dick B. (Rijksuniversiteit Groningen) Daran, J.G. (TU Delft BT/Industriele Microbiologie) van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie) Pronk, J.T. (TU Delft BT/Industriele Microbiologie) Date 2017-04-12 Abstract Combined overexpression of xylulokinase, pentose-phosphate-pathway enzymes and a heterologous xylose isomerase (XI) is required but insufficient for anaerobic growth of Saccharomyces cerevisiae on d-xylose. Single-step Cas9-assisted implementation of these modifications yielded a yeast strain expressing Piromyces XI that showed fast aerobic growth on d-xylose. However, anaerobic growth required a 12-day adaptation period. Xylose-adapted cultures carried mutations in PMR1, encoding a Golgi Ca2+/Mn2+ ATPase. Deleting PMR1 in the parental XI-expressing strain enabled instantaneous anaerobic growth on d-xylose. In pmr1 strains, intracellular Mn2+ concentrations were much higher than in the parental strain. XI activity assays in cell extracts and reconstitution experiments with purified XI apoenzyme showed superior enzyme kinetics with Mn2+ relative to other divalent metal ions. This study indicates engineering of metal homeostasis as a relevant approach for optimization of metabolic pathways involving metal-dependent enzymes. Specifically, it identifies metal interactions of heterologous XIs as an underexplored aspect of engineering xylose metabolism in yeast. To reference this document use: http://resolver.tudelft.nl/uuid:9e41144d-6c4a-41ee-a178-ce78a6e7e5e6 DOI https://doi.org/10.1038/srep46155 ISSN 2045-2322 Source Scientific Reports, 7 Part of collection Institutional Repository Document type journal article Rights © 2017 M.D. Verhoeven, Misun Lee, L. Kamoen, M.A. van den Broek, Dick B. Janssen, J.G. Daran, A.J.A. van Maris, J.T. Pronk Files PDF srep46155.pdf 981.62 KB Close viewer /islandora/object/uuid:9e41144d-6c4a-41ee-a178-ce78a6e7e5e6/datastream/OBJ/view