Xylose metabolism in the anaerobic fungus Piromyces sp. strain E2 follows the bacterial pathway

Abstract

The anaerobic fungus Piromyces sp. strain E2 metabolizes xylose via xylose isomerase and d-xylulokinase as was shown by enzymatic and molecular analyses. This resembles the situation in bacteria. The clones encoding the two enzymes were obtained from a cDNA library. The xylose isomerase gene sequence is the first gene of this type reported for a fungus. Northern blot analysis revealed a correlation between mRNA and enzyme activity levels on different growth substrates. Furthermore, the molecular mass calculated from the gene sequence was confirmed by gel permeation chromatography of crude extracts followed by activity measurements. Deduced amino acid sequences of both genes were used for phylogenetic analysis. The xylose isomerases can be divided into two distinct clusters. The Piromyces sp. strain E2 enzyme falls into the cluster comprising plant enzymes and enzymes from bacteria with a low G+C content in their DNA. The d-xylulokinase of Piromyces sp. strain E2 clusters with the bacterial d-xylulokinases. The xylose isomerase gene was expressed in the yeast Saccharomyces cerevisiae, resulting in a low activity (25+/-13 nmol min(-1)mg protein(-1)). These two fungal genes may be applicable to metabolic engineering of Saccharomyces cerevisiae for the alcoholic fermentation of hemicellulosic materials.

Development of Efficient Xylose Fermentation in Saccharomyces cerevisiae: Xylose Isomerase as a Key Component

“…The phylogenetic tree contains only one other eukaryotic XI sequence, namely that of the anaerobic fungus Piromyces sp. E2 [28]. Interestingly, this eukaryotic XI sequence clusters with those of the prokaryotic phylum Bacteroidetes, which has led to the suggestion that the fungus may have acquired XI via horizontal gene transfer [28], as previously suggested for other enzymes in anaerobic fungi [20].…”

“…E2 [28]. Expression of this Piromyces xylA gene in S. cerevisiae resulted in high enzyme activities (up to 1.1 µmol(mg protein) -1 min -1 at 30 • C [42].…”

Genetic Analysis of a Novel Pathway for d -Xylose Metabolism in Caulobacter crescentus

“…Numerous bacteria, including Escherichia coli (15), Bacillus species (24,25), and Lactobacillus species (16), use xylose isomerase to convert D-xylose to xylulose, which is then phosphorylated to enter the pentose phosphate pathway. Although some fungi have recently been shown to use this "bacterial" pathway (11), fungi more commonly transform Dxylose into xylitol by using xylose reductase and xylitol dehydrogenase (13). The freshwater bacterium Caulobacter crescentus, which readily uses D-xylose as a carbon and energy source, expresses an NAD-dependent xylose dehydrogenase (XDH) activity, suggesting that xylose metabolism occurs through a distinct pathway (21).…”

Bioethanol a Microbial Biofuel Metabolite; New Insights of Yeasts Metabolic Engineering

“…Adaptive evolution and metabolic engineering of S. cerevisiae for fermentation of xylose were successful to certain degrees [103][104][105]. Engineering S. cerevisiae with the basic xylose assimilatory machinery with expressing xylose reductases, xylose isomerase [13,106] and xylitol dehydrogenases [107] can theoretically enhance the ethanol yield.…”

“…Recently using direct evolution approach, the activity of XI could be enhanced more nine-fold than previous studies at a higher temperature of 60 • C and for xylitol much higher inhibition constants [133]. Recently, eukaryotic XI has been identified, followed by the introduction of XI from anaerobic fungus (AraA) into S. cerevisiae [103]. The transformation of S. cerevisiae with AraA enhanced slowly the utilization rate of xylose assimilation [134].…”