Cloning and heterologous expression of xylanase from Pichia stipitis in Escherichia coli

Basaran, Pervin; Hang, Y. D.; Basaran, Nese; Worobo, Randy W.

doi:10.1046/j.1365-2672.2001.01237.x

Cited by 24 publications

(8 citation statements)

References 42 publications

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“…lanases produced by these microbial symbionts are only distantly related to known xylanases further supports the view that the insect gut is a closed ecosystem in which glycosyl hydrolases have evolved independently. Although from the same glycosyl hydrolase family, the enzymes discovered in this work are only distantly similar to the xylanase from the beetlegut yeast, Pichia stipitis (2,36). Interestingly, xylanases most closely related to three of the four enzymes described here are found in species of anaerobic bacteria and fungi that normally inhabit the gastrointestinal tract of ruminant and nonruminant herbivores, another closed ecosystem (although very different from the insect gut) inhabited by commensal and symbiotic species of obligately anaerobic microorganisms, typically not found elsewhere in nature.…”

Section: Discussionmentioning

confidence: 88%

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Unusual Microbial Xylanases from Insect Guts

Brennan

Callen

Christoffersen

et al. 2004

Appl Environ Microbiol

158

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Recombinant DNA technologies enable the direct isolation and expression of novel genes from biotopes containing complex consortia of uncultured microorganisms. In this study, genomic libraries were constructed from microbial DNA isolated from insect intestinal tracts from the orders Isoptera (termites) and Lepidoptera (moths). Using a targeted functional assay, these environmental DNA libraries were screened for genes that encode proteins with xylanase activity. Several novel xylanase enzymes with unusual primary sequences and novel domains of unknown function were discovered. Phylogenetic analysis demonstrated remarkable distance between the sequences of these enzymes and other known xylanases. Biochemical analysis confirmed that these enzymes are true xylanases, which catalyze the hydrolysis of a variety of substituted ␤-1,4-linked xylose oligomeric and polymeric substrates and produce unique hydrolysis products. From detailed polyacrylamide carbohydrate electrophoresis analysis of substrate cleavage patterns, the xylan polymer binding sites of these enzymes are proposed.The arthropod gut is a differentiated organ harboring a complex biotope comprising both resident and transient members from protozoal, bacterial, and archaeal genera. Many of these organisms are symbionts that contribute in a concerted way to a complex chemical cycle sustaining the metabolic competence of the host. These symbiotic relationships help to define the metabolic traits of the insect, contributing to efficient sharing of the derived nutrients. In addition to carbon metabolism, data have demonstrated that the microbial consortia contribute to nitrogen cycling, methano-and acetogenesis, and prevention of foreign microbial pathogenesis (29).A complete understanding of the complexities of the gut biotopes is lacking, however, due to the difficulties encountered in culturing the myriad of contributing microbes and in describing the physiologies of the individual species. Studies aimed at a description of microbial complexity have recently been undertaken in termites by using 16S ribosomal DNA analysis and have shown that a number of unique lineages of microorganism inhabit the hindgut (28). These data support the idea that the insect gut represents a contained biome wherein unique species and chemistries evolve.Polysaccharide hydrolysis is a key element in insect nutrition. Since the diet of most arthropods comprises primarily plant matter, digestion of the structural polysaccharides cellulose and hemicellulose is essential for energy metabolism and the ability to obtain carbon from these sources contributes materially to the success of the order. These polysaccharides are resistant to degradation, and the insects themselves do not secrete all of the digestive enzymes to hydrolyze ␤-linkages in the polymer. Rather, much of the hydrolysis of these polysaccharides is carried out by enzymes produced by the microbial symbionts (4,5,36,41).Hemicellulose consists primarily of xylan and it is the second most abundant polymer type in plant materi...

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Section: Discussionmentioning

confidence: 88%

“…5B). Xylan was hydrolyzed into xylose and (Xyl) 2 , with additional oligosaccharides that migrated between the oligoxylose standards. Oligosaccharides have a specific migration in PACE based on degree of polymerization and composition (15,19).…”

Section: Vol 70 2004 Xylanases From Insect Guts 3611mentioning

confidence: 99%

Unusual Microbial Xylanases from Insect Guts

Brennan

Callen

Christoffersen

et al. 2004

Appl Environ Microbiol

158

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“…Pichia stipitis possesses b-xylosidase (Manzanares et al, 1999;Basaran & Ozcan, 2008) and native Family 11 xylanase activities. The latter has been cloned and characterized from P. stipitis NRRL Y-11543 (Basaran et al, 2001). The published xylanases sequence does not match with any identified ORF in the sequenced genome of P. stipitis CBS 6054 (= NRRL Y-11545, ATCC 58785), but the sequenced genome does include Family 10 endo-1,4-b-xylanase, and endoglucanase activities that might also act on xylan.…”

Section: Physiological Features Of P Stipitismentioning

confidence: 99%

Pichia stipitisgenomics, transcriptomics, and gene clusters

Jeffries

Vleet

2009

FEMS Yeast Research

100

119

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Genome sequencing and subsequent global gene expression studies have advanced our understanding of the lignocellulose-fermenting yeast Pichia stipitis. These studies have provided an insight into its central carbon metabolism, and analysis of its genome has revealed numerous functional gene clusters and tandem repeats. Specialized physiological traits are often the result of several gene products acting together. When coinheritance is necessary for the overall physiological function, recombination and selection favor colocation of these genes in a cluster. These are particularly evident in strongly conserved and idiomatic traits. In some cases, the functional clusters consist of multiple gene families. Phylogenetic analyses of the members in each family show that once formed, functional clusters undergo duplication and differentiation. Genome-wide expression analysis reveals that regulatory patterns of clusters are similar after they have duplicated and that the expression profiles evolve along with functional differentiation of the clusters. Orthologous gene families appear to arise through tandem gene duplication, followed by differentiation in the regulatory and coding regions of the gene. Genome-wide expression analysis combined with cross-species comparisons of functional gene clusters should reveal many more aspects of eukaryotic physiology.

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“…Bifidobacteria have been considered as useful probiotics, as they have been shown to lower incidence of food allergies [16] and delay proliferation of several tumor types [17]. Many xylanases have been purified and characterized from various microbial sources such as Bacillus species, fungi, Streptomyces species, and yeasts [18][19][20][21][22]. Generally, higher thermostability and broader pH stability of the enzyme are being demanded by the industrial requests.…”

Section: Introductionmentioning

confidence: 99%