Saccharomyces cerevisiaeIMX696, IMS0488 and IMS0489 Raw sequence reads. Saccharomyces cerevisiae strain:IMX696 | breed:CEN.PK113-5D

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 engineering yielded a strain based on Piromyces XI that showed fast aerobic growth on D-xylose (IMX696). However, anaerobic growth only occurred after a 12-day adaptation period. Xylose-adapted cultures carried mutations in PMR1, encoding a Golgi Ca2+/Mn2+ ATPase (IMS0488 and 0489). Deleting PMR1 in the parental strain enabled instantaneous anaerobic growth on D-xylose. In pmr1 strains, intracellular Mn2+ concentration and the fraction of Mn2+-bound XI were much higher than in the parental strain. Reconstitution experiments with purified XI apoenzyme showed superior enzyme kinetics with Mn2+ relative to other divalent metal ions. This study highlights engineering of metal homeostasis as a relevant approach in optimization of metabolic pathways involving metal-dependent enzymes. Specifically, it identifies metal interactions of heterologously expressed XIs as an underexplored aspect of engineering xylose metabolism in S. cerevisiae.