Mutations in PMR1 stimulate xylose isomerase activity and anaerobic growth on xylose of engineered Saccharomyces cerevisiae by influencing manganese homeostasis

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 expre...

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Bibliographic Details
Published in:Scientific reports 2017-04, Vol.7 (1), p.46155, Article 46155
Main Authors: Verhoeven, Maarten D., Lee, Misun, Kamoen, Lycka, van den Broek, Marcel, Janssen, Dick B., Daran, Jean-Marc G., van Maris, Antonius J. A., Pronk, Jack T.
Format: Article
Language:English
Subjects:
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Adaptation, Physiological - drug effects
Adenosine triphosphatase
Aerobiosis
Aldose-Ketose Isomerases - metabolism
Alleles
Anaerobiosis
Biocatalysis - drug effects
Calcium
Calcium-Transporting ATPases - genetics
Enzymes
Genetic Engineering
Golgi apparatus
Homeostasis
Humanities and Social Sciences
Kinetics
Manganese
Manganese - pharmacology
Metabolic pathways
Metals
Molecular Chaperones - genetics
multidisciplinary
Mutation
Mutation - genetics
Saccharomyces cerevisiae - drug effects
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - growth & development
Saccharomyces cerevisiae Proteins - genetics
Science
Xylose
Xylose - pharmacology
Xylose isomerase
Xylulokinase
Yeast
Yeasts
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Summary: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 Ca 2+ /Mn 2+ ATPase. Deleting PMR1 in the parental XI-expressing strain enabled instantaneous anaerobic growth on d -xylose. In pmr1 strains, intracellular Mn 2+ 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 Mn 2+ 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.
ISSN:2045-2322
2045-2322
DOI:10.1038/srep46155