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

Marilyn G Wiebe,Mervi Toivari,Anu Koivula,Yvonne Nygård,Martina Andberg,Laura Ruohonen,Merja Penttilä,Merja Oja

doi:10.1007/s00253-015-6878-5

Marilyn G Wiebe, Mervi Toivari + Show 6 more

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https://doi.org/10.1007/s00253-015-6878-5

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Abstract

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.

Highlights

D-Xylose, the main component of hemicellulose, is the second most abundant sugar in nature after cellulose and starchderived D-glucose
The putative glucose-fructose oxidoreductase encoding gene from C. crescentus CB15 (CC1225, AAK23207.1, NCBI), hereafter called Cc aaor or Cc AAOR when referring to the enzyme), was obtained as a synthetic gene, codon optimized for S. cerevisiae, deposited sequence KR269738 (Gene Art, Germany)
A DNA sequence coding for a similar region of amino acids is found upstream of the predicted start codon of the CC1225 open reading frame (ORF) in the CB15 strain, and in C. segnis ATCC 21756, suggesting the possibility that the translation start codon is not correctly predicted in the ORFs of C. segnis and C. crescentus CB15

Summary

Introduction

D-Xylose, the main component of hemicellulose, is the second most abundant sugar in nature after cellulose and starchderived D-glucose. Various studies have shown possibilities for biotechnical conversion of D-xylose to ethanol, butanol, lactic acid, succinic acid, xylonic acid, xylitol, hydrogen, modified sugars (transglycosylation) or compounds derived from fatty acid metabolism (Dumon et al 2012; Peng et al 2012) These products are formed via metabolism by natural or engineered microbes, e.g. of species like Escherichia coli, Corynebacterium glutamicum, Zymomonas mobilis, Saccharomyces cerevisiae and Scheffersomyces stipitis. It is well known that introduction of a redox reaction affects the cellular cofactor pool and that optimal production may necessitate further engineering of redox metabolism, use of a cosubstrate or increased aeration Some enzymes, such as the glucose-fructose oxidoreductase (EC 1.1.99.28), have a bound cofactor and can perform both oxidation and reduction reactions without interfering with the cellular redox balance (Piersma et al 1997; Zachariou and Scopes 1986). Glucosefructose oxidoreductase activity generates two products, an acid and an alcohol, which could be applicable, e.g. in polymer synthesis

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