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  • A novel aldose-aldose oxidoreductase for co-production of D-xylonate and xylitol from D-xylose with Saccharomyces cerevisiae.

A novel aldose-aldose oxidoreductase for co-production of D-xylonate and xylitol from D-xylose with Saccharomyces cerevisiae.

Applied microbiology and biotechnology (2015-08-13)
Marilyn G Wiebe, Yvonne Nygård, Merja Oja, Martina Andberg, Laura Ruohonen, Anu Koivula, Merja Penttilä, Mervi Toivari
RESUMO

An open reading frame CC1225 from the Caulobacter crescentus CB15 genome sequence belongs to the Gfo/Idh/MocA protein family and has 47 % amino acid sequence identity with the glucose-fructose oxidoreductase from Zymomonas mobilis (Zm GFOR). We expressed the ORF CC1225 in the yeast Saccharomyces cerevisiae and used a yeast strain expressing the gene coding for Zm GFOR as a reference. Cell extracts of strains overexpressing CC1225 (renamed as Cc aaor) showed some Zm GFOR type of activity, producing D-gluconate and D-sorbitol when a mixture of D-glucose and D-fructose was used as substrate. However, the activity in Cc aaor expressing strain was >100-fold lower compared to strains expressing Zm gfor. Interestingly, C. crescentus AAOR was clearly more efficient than the Zm GFOR in converting in vitro a single sugar substrate D-xylose (10 mM) to xylitol without an added cofactor, whereas this type of activity was very low with Zm GFOR. Furthermore, when cultured in the presence of D-xylose, the S. cerevisiae strain expressing Cc aaor produced nearly equal concentrations of D-xylonate and xylitol (12.5 g D-xylonate l(-1) and 11.5 g D-xylitol l(-1) from 26 g D-xylose l(-1)), whereas the control strain and strain expressing Zm gfor produced only D-xylitol (5 g l(-1)). Deletion of the gene encoding the major aldose reductase, Gre3p, did not affect xylitol production in the strain expressing Cc aaor, but decreased xylitol production in the strain expressing Zm gfor. In addition, expression of Cc aaor together with the D-xylonolactone lactonase encoding the gene xylC from C. crescentus slightly increased the final concentration and initial volumetric production rate of both D-xylonate and D-xylitol. These results suggest that C. crescentus AAOR is a novel type of oxidoreductase able to convert the single aldose substrate D-xylose to both its oxidized and reduced product.

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Sigma-Aldrich
Ethyl alcohol, Pure, 200 proof, anhydrous, ≥99.5%
Sigma-Aldrich
Ethyl alcohol, Pure, 200 proof, for molecular biology
Sigma-Aldrich
Ethyl alcohol, Pure, 190 proof, ACS spectrophotometric grade, 95.0%
Sigma-Aldrich
Glycerol, for molecular biology, ≥99.0%
Sigma-Aldrich
D-(+)-Glucose, ≥99.5% (GC)
Sigma-Aldrich
Ethyl alcohol, Pure, 200 proof, ACS reagent, ≥99.5%
Sigma-Aldrich
Glycerol, ≥99.5%
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D-(+)-Glucose, powder, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99.5%
Sigma-Aldrich
Glycerol, ACS reagent, ≥99.5%
Sigma-Aldrich
D-(−)-Fructose, ≥99%
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Glycerol, ReagentPlus®, ≥99.0% (GC)
Sigma-Aldrich
Ethyl alcohol, Pure, 200 proof, HPLC/spectrophotometric grade
Sigma-Aldrich
Dextrose, meets EP, BP, JP, USP testing specifications, anhydrous
Sigma-Aldrich
D-(+)-Glucose, ACS reagent
Sigma-Aldrich
Glycerol, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for electrophoresis, ≥99% (GC)
Sigma-Aldrich
D-(+)-Glucose, BioXtra, ≥99.5% (GC)
Sigma-Aldrich
Ethyl alcohol, Pure, 190 proof, for molecular biology
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Ethyl alcohol, Pure, 200 proof, meets USP testing specifications
Sigma-Aldrich
Xylitol, ≥99%
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Glycerin, meets USP testing specifications
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Glycerol, BioXtra, ≥99% (GC)
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Glycerol solution, 83.5-89.5% (T)
Sigma-Aldrich
D-(+)-Xylose, ≥99%
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D-(−)-Fructose, BioReagent, suitable for cell culture, suitable for insect cell culture
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Ethanol, ACS reagent, prima fine spirit, without additive, F15 o1
Sigma-Aldrich
Glycerol, FCC, FG
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D-(+)-Glucose, BioUltra, anhydrous, ≥99.5% (sum of enantiomers, HPLC)
Sigma-Aldrich
Glycerol, BioUltra, for molecular biology, anhydrous, ≥99.5% (GC)
Supelco
D-(+)-Glucose, analytical standard
Sigma-Aldrich
D-(−)-Fructose, BioUltra, ≥99.0% (HPLC)