Background:
Xylose isomerase (XI) catalyses the isomerization 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 the DNA into suitable vectors, was undertaken.
Results:
A soil metagenomic library was constructed and two screening methods based on protein sequence similarity (1) and enzyme activity (2) were investigated to isolate novel XI encoding genes. These 2 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 were obtained as when Piromyces XI gene was expressed. However, expression in S. cerevisiae resulted in only one fourth the growth rate of that obtained for the strain expressing Piromyces XI gene.
Conclusions:
For 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.