Isolation of xylose isomerases by sequence- and function-based screening from a soil metagenomic library

Abstract

BackgroundXylose isomerase (XI) catalyses the isomerisation of xylose to xylulose in bacteria and some fungi. Currently, only a limited number of XI genes have been functionally expressed in Saccharomyces cerevisiae, the microorganism of choice for lignocellulosic ethanol production. The objective of the present study was to search for novel XI genes in the vastly diverse microbial habitat present in soil. As the exploitation of microbial diversity is impaired by the ability to cultivate soil microorganisms under standard laboratory conditions, a metagenomic approach, consisting of total DNA extraction from a given environment followed by cloning of DNA into suitable vectors, was undertaken.ResultsA soil metagenomic library was constructed and two screening methods based on protein sequence similarity and enzyme activity were investigated to isolate novel XI encoding genes. These two screening approaches identified the xym1 and xym2 genes, respectively. Sequence and phylogenetic analyses revealed that the genes shared 67% similarity and belonged to different bacterial groups. When xym1 and xym2 were overexpressed in a xylA-deficient Escherichia coli strain, similar growth rates to those in which the Piromyces XI gene was expressed were obtained. However, expression in S. cerevisiae resulted in only one-fourth the growth rate of that obtained for the strain expressing the Piromyces XI gene.ConclusionsFor the first time, the screening of a soil metagenomic library in E. coli resulted in the successful isolation of two active XIs. However, the discrepancy between XI enzyme performance in E. coli and S. cerevisiae suggests that future screening for XI activity from soil should be pursued directly using yeast as a host.

Growth and fermentation of D-xylose by Saccharomyces cerevisiae expressing a novel D-xylose isomerase originating from the bacterium Prevotella ruminicola TC2-24

“…Prior studies have reported V max for the Piromyces XI at 0.05 μmole/min/mg protein (Table 3). Higher enzyme activity than 0.05 μmole/min/mg for the Piromyces sp E2 XI has been observed, but V max was not reported in these studies [12,25,26]. Following strain adaptation, V max for the P .…”

“…The specific growth rate for YRH1114 was 0.23 h -1 , an increase of 3.8-fold compared to the unadapted strain, is among the highest reported growth rates which range from 0.01 h -1 to 0.22 h -1 for a S . cerevisiae strain expressing D-xylose isomerase [6,13,14,16,17,24-26]. …”

Applying functional metagenomics to search for novel lignocellulosic enzymes in a microbial consortium derived from a thermophilic composting phase of sugarcane bagasse and cow manure

“…The use of function-based metagenomic approaches to search for novel lignocellulosic enzymes have led to the discovery of novel b-galactosidases/a-arabinopyranosidases (Beloqui et al 2010), cellulases Kim et al 2008;Lopes et al 2015;Pandey et al 2016), xylanases (Hu et al 2008;Kanokratana et al 2015), xylose isomerases (Parachin and Gorwa-Grauslund 2011), b-xylosidase/a-arabinofuranosidase (Matsuzawa et al 2015) and b-glucosidases (Del Pozo et al 2012;Biver et al 2014), supporting this strategy as a powerful activity-based screening tool to identify entirely new classes of gene sequences encoding new or known functions (Handelsman 2004;Parachin and Gorwa-Grauslund 2011). Several sources of potential cellullose-and hemicellulosedepleting microbial communities are being used for metagenomic analysis.…”

Directed Evolution of Xylose Isomerase for Improved Xylose Catabolism and Fermentation in the Yeast Saccharomyces cerevisiae

“…(28), and Clostridium phytofermentans (8) in S. cerevisiae raise the prospect of efficient xylose fermentation. Furthermore, researchers have applied adaptive evolutionary engineering (21), optimized metabolic flux by introducing/overexpressing xylose transporter and/or overexpressing the downstream pathway (20,27), and employed bioprospecting to identify other putative xylose isomerase enzymes (8,34). In all of these cases, extensive downstream overexpression and/or evolutionary engineering is required to improve cell growth and xylose consumption.…”