title: Mutations in PMR1 stimulate xylose isomerase activity and anaerobic growth on xylose of engineered Saccharomyces cerevisiae by influencing manganese homeostasis.
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.
authors.affiliation: Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.
authors.forename: Maarten D
authors.initials: MD
authors.lastname: Verhoeven
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Misun
authors.initials: M
authors.lastname: Lee
authors.affiliation: Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.
authors.forename: Lycka
authors.initials: L
authors.lastname: Kamoen
authors.affiliation: Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.
authors.forename: Marcel
authors.initials: M
authors.lastname: van den Broek
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Dick B
authors.initials: DB
authors.lastname: Janssen
authors.affiliation: Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.
authors.forename: Jean-Marc G
authors.initials: JG
authors.lastname: Daran
authors.affiliation: Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.
authors.forename: Antonius J A
authors.initials: AJ
authors.lastname: van Maris
authors.affiliation: Department of Biotechnology, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.
authors.forename: Jack T
authors.initials: JT
authors.lastname: Pronk
mesh_terms: D000222:Adaptation, Physiological / Q000187:drug effects; D000332:Aerobiosis; D019747:Aldose-Ketose Isomerases / Q000378:metabolism*; D000483:Alleles; D000693:Anaerobiosis; D055162:Biocatalysis / Q000187:drug effects; D000252:Calcium-Transporting ATPases / Q000235:genetics*; D005818:Genetic Engineering; D006706:Homeostasis; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology*; D018832:Molecular Chaperones / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000254:growth & development*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D014994:Xylose / Q000494:pharmacology*
chemical_list: D018832:Molecular Chaperones; C101767:SSC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D008345:Manganese; D014994:Xylose; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D000252:Calcium-Transporting ATPases