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

• Main Authors: Parachin Nádia, Gorwa-Grauslund Marie F
• Format: Article
• Language: English
• Published: BMC 2011-05-01
• Series: Biotechnology for Biofuels
• Online Access: http://www.biotechnologyforbiofuels.com/content/4/1/9

<p>Abstract</p> <p>Background</p> <p>Xylose 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 <it>Saccharomyces cerevisiae</it>, 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.</p> <p>Results</p> <p>A 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 <it>xym1 </it>and <it>xym2 </it>genes, respectively. Sequence and phylogenetic analyses revealed that the genes shared 67% similarity and belonged to different bacterial groups. When <it>xym1 </it>and <it>xym2 </it>were overexpressed in a <it>xylA</it>-deficient <it>Escherichia coli </it>strain, similar growth rates to those in which the <it>Piromyces </it>XI gene was expressed were obtained. However, expression in <it>S. cerevisiae </it>resulted in only one-fourth the growth rate of that obtained for the strain expressing the <it>Piromyces </it>XI gene.</p> <p>Conclusions</p> <p>For the first time, the screening of a soil metagenomic library in <it>E. coli </it>resulted in the successful isolation of two active XIs. However, the discrepancy between XI enzyme performance in <it>E. coli </it>and <it>S. cerevisiae </it>suggests that future screening for XI activity from soil should be pursued directly using yeast as a host.</p>