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

Maarten D Verhoeven,Antonius J A Van Maris,Jean-Marc G Daran,Jack T Pronk,Misun Lee,Dick B Janssen,Marcel Van Den Broek,Lycka Kamoen

doi:10.1038/srep46155

Maarten D Verhoeven, Antonius J A Van Maris + Show 6 more

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https://doi.org/10.1038/srep46155

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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.

Highlights

In conventional feedstocks for fermentative production of fuel ethanol, such as corn starch and cane sugar, carbohydrates predominantly occur as dimers or polymers of hexose sugars
Kuyper et al (2005a)[19] reported that expression of xylose isomerase gene (xylA) combined with constitutive overexpression of the genes encoding the native S. cerevisiae xylulokinase (XKS1, EC 2.7.1.17), ribulose 5-phosphate epimerase (RPE1, EC 5.3.1.1), ribulose 5-phosphate isomerase (RKI1, EC 5.3.1.6), transketolase (TKL1, EC 2.2.1.1) and transaldolase (TAL1, EC 2.2.1.2) was sufficient to enable anaerobic growth on d-xylose, at a specific growth rate of 0.07 h−1
The nine copies of the xylA cassette were introduced as tandem repeats to facilitate adaptation of the xylA copy number by homologous recombination

Summary

Introduction

In conventional feedstocks for fermentative production of fuel ethanol, such as corn starch and cane sugar, carbohydrates predominantly occur as dimers or polymers of hexose sugars. The different redox cofactor preferences of XR and XDH represent a challenge in their use for constructing d-xylose-fermenting S. cerevisiae strains This redox problem causes the production of substantial amounts of xylitol by anaerobic cultures of such engineered strains[5,6,7]. Kuyper et al (2005a)[19] reported that expression of xylA combined with constitutive overexpression of the genes encoding the native S. cerevisiae xylulokinase (XKS1, EC 2.7.1.17), ribulose 5-phosphate epimerase (RPE1, EC 5.3.1.1), ribulose 5-phosphate isomerase (RKI1, EC 5.3.1.6), transketolase (TKL1, EC 2.2.1.1) and transaldolase (TAL1, EC 2.2.1.2) was sufficient to enable anaerobic growth on d-xylose, at a specific growth rate of 0.07 h−1. Additional mutations that improve pentose-fermentation kinetics, mainly identified by resequencing of laboratory-evolved strains, affected structural genes encoding native yeast hexose transporters[26,27,28,29,30] and in the ‘secondary’ transaldolase and transketolase isoenzymes NQM1 and TKL220,31

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