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

Parachin NS, Gorwa-Grauslund MF - Biotechnol Biofuels (2011)

Bottom Line: Sequence and phylogenetic analyses revealed that the genes shared 67% similarity and belonged to different bacterial groups.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Applied Microbiology, Center for Chemistry and Chemical Engineering, Lund University, P,O, Box 124, SE-221 00 Lund, Sweden. Marie-Francoise.Gorwa@tmb.lth.se.

ABSTRACT

Background: 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 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.

Results: 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 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.

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.

No MeSH data available.


Related in: MedlinePlus

Conserved regions utilized for the construction of the degenerate primers. The choice of regions was based on multiple alignments of XI amino acid sequences. The microorganisms chosen, in presented order, are Piromyces sp., Streptomyces diastaticus, Escherichia coli, Pseudomonas sp., Rhizobium sp., Streptomyces coelicolor, Saccharophagus sp., Enterobacter sp., Mesorhizobium loti and Clostridium sp.
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Figure 1: Conserved regions utilized for the construction of the degenerate primers. The choice of regions was based on multiple alignments of XI amino acid sequences. The microorganisms chosen, in presented order, are Piromyces sp., Streptomyces diastaticus, Escherichia coli, Pseudomonas sp., Rhizobium sp., Streptomyces coelicolor, Saccharophagus sp., Enterobacter sp., Mesorhizobium loti and Clostridium sp.

Mentions: For the sequence-based screening, degenerate primers were designed based on the alignment of amino acid sequences of known XIs from soil microorganisms such as Streptomyces sp. and Rhizobium sp. Amino acid sequences of XI genes whose expression has been attempted in S. cerevisiae, such as the ones from Piromyces sp. [14] and E. coli [19], were also included (Figure 1). The first polymerase chain reaction (PCR) was performed with primers DF1 and DR2 (Table 2) and with the soil metagenomic library used as a template. A fragment of approximately 750 bp was amplified. The purified fragment was then utilized as the template for a nested PCR with internal primers DF2 and DR1. A single fragment of about 350 bp was obtained, purified from an agarose gel and cloned into the pGEM-T Easy Vector System (Promega, Madison, WI, USA) (Table 1). Sequence analysis revealed that this fragment belonged to a gene coding for a XI. The complete gene sequence was then obtained by PCR utilizing the metagenomic library as the template and primers designed to the sequence of the known 350-bp fragment and in plasmid pRSETB. The gene sequence encoded a 443-amino acid protein with 83% identity with Sorangium cellulosum XI and was designated xym1.


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

Parachin NS, Gorwa-Grauslund MF - Biotechnol Biofuels (2011)

Conserved regions utilized for the construction of the degenerate primers. The choice of regions was based on multiple alignments of XI amino acid sequences. The microorganisms chosen, in presented order, are Piromyces sp., Streptomyces diastaticus, Escherichia coli, Pseudomonas sp., Rhizobium sp., Streptomyces coelicolor, Saccharophagus sp., Enterobacter sp., Mesorhizobium loti and Clostridium sp.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113934&req=5

Figure 1: Conserved regions utilized for the construction of the degenerate primers. The choice of regions was based on multiple alignments of XI amino acid sequences. The microorganisms chosen, in presented order, are Piromyces sp., Streptomyces diastaticus, Escherichia coli, Pseudomonas sp., Rhizobium sp., Streptomyces coelicolor, Saccharophagus sp., Enterobacter sp., Mesorhizobium loti and Clostridium sp.
Mentions: For the sequence-based screening, degenerate primers were designed based on the alignment of amino acid sequences of known XIs from soil microorganisms such as Streptomyces sp. and Rhizobium sp. Amino acid sequences of XI genes whose expression has been attempted in S. cerevisiae, such as the ones from Piromyces sp. [14] and E. coli [19], were also included (Figure 1). The first polymerase chain reaction (PCR) was performed with primers DF1 and DR2 (Table 2) and with the soil metagenomic library used as a template. A fragment of approximately 750 bp was amplified. The purified fragment was then utilized as the template for a nested PCR with internal primers DF2 and DR1. A single fragment of about 350 bp was obtained, purified from an agarose gel and cloned into the pGEM-T Easy Vector System (Promega, Madison, WI, USA) (Table 1). Sequence analysis revealed that this fragment belonged to a gene coding for a XI. The complete gene sequence was then obtained by PCR utilizing the metagenomic library as the template and primers designed to the sequence of the known 350-bp fragment and in plasmid pRSETB. The gene sequence encoded a 443-amino acid protein with 83% identity with Sorangium cellulosum XI and was designated xym1.

Bottom Line: Sequence and phylogenetic analyses revealed that the genes shared 67% similarity and belonged to different bacterial groups.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Applied Microbiology, Center for Chemistry and Chemical Engineering, Lund University, P,O, Box 124, SE-221 00 Lund, Sweden. Marie-Francoise.Gorwa@tmb.lth.se.

ABSTRACT

Background: 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 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.

Results: 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 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.

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.

No MeSH data available.


Related in: MedlinePlus