TY - JOUR
T1 - Mutations in PMR1 stimulate xylose isomerase activity and anaerobic growth on xylose of engineered Saccharomyces cerevisiae by influencing manganese homeostasis
AU - Verhoeven, Maarten D.
AU - Lee, Misun
AU - Kamoen, Lycka
AU - Van Den Broek, Marcel
AU - Janssen, Dick B.
AU - Daran, Jean Marc G.
AU - Van Maris, Antonius J.A.
AU - Pronk, Jack T.
PY - 2017/4/12
Y1 - 2017/4/12
N2 - 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.
AB - 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.
UR - http://resolver.tudelft.nl/uuid:9e41144d-6c4a-41ee-a178-ce78a6e7e5e6
UR - http://www.scopus.com/inward/record.url?scp=85017464092&partnerID=8YFLogxK
U2 - 10.1038/srep46155
DO - 10.1038/srep46155
M3 - Article
AN - SCOPUS:85017464092
SN - 2045-2322
VL - 7
JO - Scientific Reports
JF - Scientific Reports
M1 - 46155
ER -