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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

Aerobic growth of S. cerevisiae recombinant strains carrying p426TEF-Xym1 (filled triangle), p426TEF-Xym2 (filled circle), p426TEF-XiPiromyces (filled square) or the empty plasmid p426TEF (filled diamond). All strains were cultivated at 30°C in mineral media supplemented with 50 g/L xylose.
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Figure 4: Aerobic growth of S. cerevisiae recombinant strains carrying p426TEF-Xym1 (filled triangle), p426TEF-Xym2 (filled circle), p426TEF-XiPiromyces (filled square) or the empty plasmid p426TEF (filled diamond). All strains were cultivated at 30°C in mineral media supplemented with 50 g/L xylose.

Mentions: XI is a key enzyme for the construction of recombinant S. cerevisiae strains for xylose conversion to ethanol. However, despite many efforts [19,21-24], only a few XI genes have been successfully expressed in S. cerevisiae at sufficient levels to allow growth on xylose [15,16,25]. Therefore, xym1 and xym2 were overexpressed in S. cerevisiae to assess growth in a recombinant S. cerevisiae strain lacking the initial xylose catabolic pathway. xym1, xym2 and the xylA gene from Piromyces sp. (positive control) were cloned in the multicopy vector p426TEF to generate plasmids p246TEFXiPiromyces, p426TEF-Xym1 and p426TEF-Xym2 (Table 1). The S. cerevisiae screening strain TMB3044 that has been optimised for efficient pentose utilization, but which lacks the initial conversion step from xylose to xylulose [26], was transformed with the three constructed plasmids in addition to the empty plasmid to generate strains TMB3363(empty plasmid), TMB3359(XiPiromyces), TMB3364 (xym1) and TMB3365 (xym2). No growth was observed for the negative control. In contrast, overexpression of both xym1 and xym2 enabled growth on xylose in S. cerevisiae (Figure 4). However, the strains carrying either xym1or xym2 grew at a lower growth rate (0.021 hour-1 for both strains) than the positive control harbouring Piromyces sp XI (0.069 hour-1 ± 0.006) (Figure 4).


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

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

Aerobic growth of S. cerevisiae recombinant strains carrying p426TEF-Xym1 (filled triangle), p426TEF-Xym2 (filled circle), p426TEF-XiPiromyces (filled square) or the empty plasmid p426TEF (filled diamond). All strains were cultivated at 30°C in mineral media supplemented with 50 g/L xylose.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Aerobic growth of S. cerevisiae recombinant strains carrying p426TEF-Xym1 (filled triangle), p426TEF-Xym2 (filled circle), p426TEF-XiPiromyces (filled square) or the empty plasmid p426TEF (filled diamond). All strains were cultivated at 30°C in mineral media supplemented with 50 g/L xylose.
Mentions: XI is a key enzyme for the construction of recombinant S. cerevisiae strains for xylose conversion to ethanol. However, despite many efforts [19,21-24], only a few XI genes have been successfully expressed in S. cerevisiae at sufficient levels to allow growth on xylose [15,16,25]. Therefore, xym1 and xym2 were overexpressed in S. cerevisiae to assess growth in a recombinant S. cerevisiae strain lacking the initial xylose catabolic pathway. xym1, xym2 and the xylA gene from Piromyces sp. (positive control) were cloned in the multicopy vector p426TEF to generate plasmids p246TEFXiPiromyces, p426TEF-Xym1 and p426TEF-Xym2 (Table 1). The S. cerevisiae screening strain TMB3044 that has been optimised for efficient pentose utilization, but which lacks the initial conversion step from xylose to xylulose [26], was transformed with the three constructed plasmids in addition to the empty plasmid to generate strains TMB3363(empty plasmid), TMB3359(XiPiromyces), TMB3364 (xym1) and TMB3365 (xym2). No growth was observed for the negative control. In contrast, overexpression of both xym1 and xym2 enabled growth on xylose in S. cerevisiae (Figure 4). However, the strains carrying either xym1or xym2 grew at a lower growth rate (0.021 hour-1 for both strains) than the positive control harbouring Piromyces sp XI (0.069 hour-1 ± 0.006) (Figure 4).

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