Combining Xylose Reductase from Spathaspora arborariae with Xylitol Dehydrogenase from Spathaspora passalidarum to Promote Xylose Consumption and Fermentation into Xylitol by Saccharomyces cerevisiae

Adriane Mouro,Boris U Stambuk,Carlos A Rosa,Gabriela F Gubert,César Fonseca,Denis D Agnolo,Angela A Dos Santos,Elba P S Bon

doi:10.3390/fermentation6030072

Abstract

In recent years, many novel xylose-fermenting yeasts belonging to the new genus Spathaspora have been isolated from the gut of wood-feeding insects and/or wood-decaying substrates. We have cloned and expressed, in Saccharomyces cerevisiae, a Spathaspora arborariae xylose reductase gene (SaXYL1) that accepts both NADH and NADPH as co-substrates, as well as a Spathaspora passalidarum NADPH-dependent xylose reductase (SpXYL1.1 gene) and the SpXYL2.2 gene encoding for a NAD+-dependent xylitol dehydrogenase. These enzymes were co-expressed in a S. cerevisiae strain over-expressing the native XKS1 gene encoding xylulokinase, as well as being deleted in the alkaline phosphatase encoded by the PHO13 gene. The S. cerevisiae strains expressing the Spathaspora enzymes consumed xylose, and xylitol was the major fermentation product. Higher specific growth rates, xylose consumption and xylitol volumetric productivities were obtained by the co-expression of the SaXYL1 and SpXYL2.2 genes, when compared with the co-expression of the NADPH-dependent SpXYL1.1 xylose reductase. During glucose-xylose co-fermentation by the strain with co-expression of the SaXYL1 and SpXYL2.2 genes, both ethanol and xylitol were produced efficiently. Our results open up the possibility of using the advantageous Saccharomyces yeasts for xylitol production, a commodity with wide commercial applications in pharmaceuticals, nutraceuticals, food and beverage industries.

Highlights

Industrial biotechnology will play an increasing role in creating a more sustainable global economy.Lignocellulosic biomass, an abundant and renewable feedstock, is an attractive raw material for fuels and chemicals production, since it does not compete with food and feed production [1,2]
Considering that Spathaspora species may constitute a suitable platform for new genes encoding enzymes involved in xylose consumption to engineer industrial S. cerevisiae yeasts [24], in the present work, we extended our knowledge regarding the Spathaspora xylose-utilizing enzymes by cloning and expressing in S. cerevisiae the Sp. arborariae SaXYL1 xylose reductase that accepts both NADH and NADPH as cofactors, as well as the Sp. passalidarum
Yeasts were grown on rich YP medium (1% yeast extract, 2% Bacto peptone) or synthetic complete (YNB) medium (0.67% yeast nitrogen base without amino acids, supplemented with adequate auxotrophic requirements), containing 2% glucose or xylose

Summary

Introduction

Industrial biotechnology will play an increasing role in creating a more sustainable global economy.Lignocellulosic biomass, an abundant and renewable feedstock, is an attractive raw material for fuels and chemicals production, since it does not compete with food and feed production [1,2]. To develop an economically feasible industrial process, it is necessary to efficiently consume and metabolize both sugars [3,4], in a scenario where xylose is not as readily consumed as glucose by the Saccharomyces yeasts [5]. The efficient consumption of pentose sugars is, important in the overall bioconversion of plant biomass for the production of liquid fuels and chemicals. While several yeast species have been shown to be able to consume this sugar (including recombinant S. cerevisiae strains), the efficiency and rates of xylose utilization are slow and/or inefficient, challenging the industrial production of lignocellulosic valuable chemical compounds [6]. There is still a need for the development of new yeasts capable of efficient xylose consumption for fuels and chemicals production

Methods

Results

Conclusion