| 1. |
- Verhoeven, Maarten D., et al.
(författare)
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Laboratory evolution of a glucose-phosphorylation-deficient, arabinose-fermenting S. cerevisiae strain reveals mutations in GAL2 that enable glucose-insensitive L-arabinose uptake
- 2018
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Ingår i: FEMS yeast research (Print). - : Oxford University Press. - 1567-1356 .- 1567-1364. ; 18:6
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Tidskriftsartikel (refereegranskat)abstract
- Cas9-assisted genome editing was used to construct an engineered glucose-phosphorylation-negative S. cerevisiae strain, expressing the Lactobacillus plantarum L-arabinose pathway and the Penicillium chrysogenum transporter PcAraT. This strain, which showed a growth rate of 0.26 h(-1) on L-arabinose in aerobic batch cultures, was subsequently evolved for anaerobic growth on L-arabinose in the presence of D-glucose and D-xylose. In four strains isolated from two independent evolution experiments the galactose-transporter gene GAL2 had been duplicated, with all alleles encoding Gal2(N376T) or Gal(2N376I) substitutions. In one strain, a single GAL2 allele additionally encoded a Gal2(T89I) substitution, which was subsequently also detected in the independently evolved strain IMS0010. In C-14-sugar-transport assays, Gal2(N376S), Gal2(N376T) and Gal(2N376I) substitutions showed a much lower glucose sensitivity of L-arabinose transport and a much higher Km for D-glucose transport than wild-type Gal2. Introduction of the Gal2(N376I) substitution in a non-evolved strain enabled growth on L-arabinose in the presence of D-glucose. Gal2(N376T), T89I and Gal2(T89I) variants showed a lower K-m for L-arabinose and a higher K-m for D-glucose than wild-type Gal2, while reverting Gal2(N376T), T89I to Gal2(N376) in an evolved strain negatively affected anaerobic growth on L-arabinose. This study indicates that optimal conversion of mixed-sugar feedstocks may require complex 'transporter landscapes', consisting of sugar transporters with complementary kinetic and regulatory properties.
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| 2. |
- Marques, Wesley Leoricy, et al.
(författare)
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Elimination of sucrose transport and hydrolysis in Saccharomyces cerevisiae : a platform strain for engineering sucrose metabolism
- 2017
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Ingår i: FEMS yeast research (Print). - : Oxford University Press. - 1567-1356 .- 1567-1364. ; 17:1
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Tidskriftsartikel (refereegranskat)abstract
- Many relevant options to improve efficacy and kinetics of sucrose metabolism in Saccharomyces cerevisiae and, thereby, the economics of sucrose-based processes remain to be investigated. An essential first step is to identify all native sucrose-hydrolysing enzymes and sucrose transporters in this yeast, including those that can be activated by suppressor mutations in sucrose-negative strains. A strain in which all known sucrose-transporter genes (MAL11, MAL21, MAL31, MPH2, MPH3) were deleted did not grow on sucrose after 2 months of incubation. In contrast, a strain with deletions in genes encoding sucrose-hydrolysing enzymes (SUC2, MAL12, MAL22, MAL32) still grew on sucrose. Its specific growth rate increased from 0.08 to 0.25 h(-1) after sequential batch cultivation. This increase was accompanied by a 3-fold increase of in vitro sucrose-hydrolysis and isomaltase activities, as well as by a 3- to 5-fold upregulation of the isomaltase-encoding genes IMA1 and IMA5. One-step Cas9-mediated deletion of all isomaltase-encoding genes (IMA1-5) completely abolished sucrose hydrolysis. Even after 2 months of incubation, the resulting strain did not grow on sucrose. This sucrose-negative strain can be used as a platform to test metabolic engineering strategies and for fundamental studies into sucrose hydrolysis or transport.
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| 3. |
- Verhoeven, Maarten D, et al.
(författare)
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Fermentation of glucose-xylose-arabinose mixtures by a synthetic consortium of single-sugar-fermenting Saccharomyces cerevisiae strains
- 2018
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Ingår i: FEMS yeast research (Print). - : Oxford University Press. - 1567-1356 .- 1567-1364. ; 18:8
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Tidskriftsartikel (refereegranskat)abstract
- D-glucose, D-xylose and L-arabinose are major sugars in lignocellulosic hydrolysates. This study explores fermentation of glucose-xylose-arabinose mixtures by a consortium of three 'specialist' Saccharomyces cerevisiae strains. A D-glucose- and L-arabinose-tolerant xylose specialist was constructed by eliminating hexose phosphorylation in an engineered xylose-fermenting strain and subsequent laboratory evolution. A resulting strain anaerobically grew and fermented D-xylose in the presence of 20 g L-1 of D-glucose and L-arabinose. A synthetic consortium that additionally comprised a similarly obtained arabinose specialist and a pentose non-fermenting laboratory strain, rapidly and simultaneously converted D-glucose and L-arabinose in anaerobic batch cultures on three-sugar mixtures. However, performance of the xylose specialist was strongly impaired in these mixed cultures. After prolonged cultivation of the consortium on three-sugar mixtures, the time required for complete sugar conversion approached that of a previously constructed and evolved 'generalist' strain. In contrast to the generalist strain, whose fermentation kinetics deteriorated during prolonged repeated-batch cultivation on a mixture of 20 g L-1 D-glucose, 10 g L-1 D-xylose and 5 g L-1 L-arabinose, the evolved consortium showed stable fermentation kinetics. Understanding the interactions between specialist strains is a key challenge in further exploring the applicability of this synthetic consortium approach for industrial fermentation of lignocellulosic hydrolysates.
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| 4. |
- Verhoeven, Maarten D., et al.
(författare)
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Mutations in PMR1 stimulate xylose isomerase activity and anaerobic growth on xylose of engineered Saccharomyces cerevisiae by influencing manganese homeostasis
- 2017
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)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.
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