Point mutation of the xylose reductase (XR) gene reduces xylitol accumulation and increases citric acid production in Aspergillus carbonarius.

István Weyda,Mette Lübeck,Birgitte K Ahring,Peter S Lübeck

doi:10.1007/s10295-014-1415-6

István Weyda, Mette Lübeck + Show 2 more

Open Access

https://doi.org/10.1007/s10295-014-1415-6

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Abstract

Aspergillus carbonarius accumulates xylitol when it grows on d-xylose. In fungi, d-xylose is reduced to xylitol by the NAD(P)H-dependent xylose reductase (XR). Xylitol is then further oxidized by the NAD+-dependent xylitol dehydrogenase (XDH). The cofactor impairment between the XR and XDH can lead to the accumulation of xylitol under oxygen-limiting conditions. Most of the XRs are NADPH dependent and contain a conserved Ile-Pro-Lys-Ser motif. The only known naturally occurring NADH-dependent XR (from Candida parapsilosis) carries an arginine residue instead of the lysine in this motif. In order to overcome xylitol accumulation in A. carbonarius a Lys-274 to Arg point mutation was introduced into the XR with the aim of changing the specificity toward NADH. The effect of the genetic engineering was examined in fermentation for citric acid production and xylitol accumulation by using d-xylose as the sole carbon source. Fermentation with the mutant strain showed a 2.8-fold reduction in xylitol accumulation and 4.5-fold increase in citric acid production compared to the wild-type strain. The fact that the mutant strain shows decreased xylitol levels is assumed to be associated with the capability of the mutated XR to use the NADH generated by the XDH, thus preventing the inhibition of XDH by the high levels of NADH and ensuring the flux of xylose through the pathway. This work shows that enhanced production of citric acid can be achieved using xylose as the sole carbon source by reducing accumulation of other by-products, such as xylitol.

Highlights

Pentose sugars are among the major components of plantbased lignocellulosic biomass [35]
Aspergillus carbonarius, a fungus closely related to Aspergillus niger [1], is able to consume a large variety of carbohydrates, including the two most abundant pentose sugars found in lignocellulosic biomass, d-xylose and l-arabinose ([42], unpublished data)
The conversion of d-xylose and l-arabinose occurs via the pentose catabolic pathway (PCP) [18]. This pathway involves a series of reversible reduction– oxidation steps followed by a phosphorylation step resulting in xylulose-5-phosphate, which is channeled into the glycolysis via the pentose phosphate pathway (PPP) as described for A. niger [41] (Fig. 1a)

Summary

Introduction

Pentose sugars are among the major components of plantbased lignocellulosic biomass [35]. Leitgeb et al [24] described the effect of a single point mutation in the conserved Ile-Pro-Lys-Ser motif [22, 30] of the NADPH/NADH-dependent XR in C. tenuis This point mutation consists of the replacement of Lys-274 with an Arg residue and results in conformational changes in the coenzyme binding pocket of the XR, altering the enzyme’s cofactor preference from NADPH towards NADH. In contrast to the eukaryotic pathway, the bacterial PCP does not include the two-step conversion of d-xylose to d-xylulose by the cofactor-dependent XR and XDH. Instead, this conversion is catalyzed by the xylose isomerase (XI) in a single-step reaction.

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