Sequence Analysis, Overexpression, and Antisense Inhibition of a β-Xylosidase Gene,
            <i>xylA</i>
            , from
            <i>Aspergillus oryzae</i>
            KBN616

Kitamoto, Noriyuki; Yoshino, Shoko; Ohmiya, Kunio; Tsukagoshi, Norihiro

doi:10.1128/aem.65.1.20-24.1999

Cited by 75 publications

(16 citation statements)

References 18 publications

(16 reference statements)

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“…2011), have been isolated. There was no intron interrupting the coding region of TlXyl43, similar to that observed for some other β‐xylosidase genes from filamentous fungi (Kitamoto et al. 1999; Ito et al.…”

Section: Discussionsupporting

confidence: 78%

“…2011). In comparison, some purified fungal β‐xylosidases have low specific activities (Kitamoto et al. 1999; Suzuki et al.…”

Section: Discussionmentioning

confidence: 99%

“…2008). Although many β‐xylosidases have been purified and biochemically characterized from mesophilic fungi (Kitamoto et al. 1999; Rizzatti et al.…”

Section: Introductionmentioning

confidence: 99%

“…2008). In the past few decades, some β‐xylosidase genes from filamentous fungi have been cloned (Kitamoto et al. 1999; Ito et al.…”

Section: Introductionmentioning

confidence: 99%

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Secretory expression of a β-xylosidase gene fromThermomyces lanuginosusinEscherichia coliand characterization of its recombinant enzyme

Chen

Jia

Yang

et al. 2012

Letters in Applied Microbiology

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Aims: To characterize a β‐xylosidase from the thermophilic fungus Thermomyces lanuginosus and to investigate its potential in saccharification of hemicellulosic xylans. Methods and Results: A gene (designated TlXyl43) encoding β‐xylosidase was cloned from T. lanuginosus CAU44 and expressed in Escherichia coli. The gene consists of a 1017‐bp open reading frame without introns. It encodes a mature protein of 338 residues with no predicted signal peptide, belonging to glycoside hydrolase (GH) family 43. Over 60% of the recombinant β‐xylosidase (TlXyl43) was secreted into the culture medium. TlXyl43 was purified 2·6‐fold to homogeneity with an estimated mass of 51·6 kDa by SDS‐PAGE. The purified enzyme exhibited optimal activity at pH 6·5 and 55°C and was stable at 50°C. It was competitively inhibited by xylose with a Ki value of 63 mmol l−1. Conclusions: In this study, a GH family 43 β‐xylosidase gene (TlXyl43) from T. lanuginosus CAU44 was cloned and functionally expressed in E. coli, and over 60% of recombinant protein was secreted into the culture. Significance and Impact of the Study: This is the first report of the cloning and functional expression of a β‐xylosidase gene from Thermomyces species. TlXyl43 holds great potential for variety of industries.

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Section: Discussionsupporting

confidence: 78%

“…2011). In comparison, some purified fungal β‐xylosidases have low specific activities (Kitamoto et al. 1999; Suzuki et al.…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Secretory expression of a β-xylosidase gene fromThermomyces lanuginosusinEscherichia coliand characterization of its recombinant enzyme

Chen

Jia

Yang

et al. 2012

Letters in Applied Microbiology

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“…Silencing of gene expression by transcription of an antisense construct has been successfully employed with fungi such as Aspergillus oryzae and Aspergillus nidulans (Kitamoto et al 1999;Bautista et al 2000). It was also explored in T. reesei and gained some effective results (Carle-Urioste et al 1997).…”

Section: Introductionmentioning

confidence: 99%

Antisense inhibition of xylitol dehydrogenase gene, xdh1 from Trichoderma reesei

Wang

Zhong

Huang

et al. 2005

Lett Appl Microbiol

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Aims: To inhibit xylitol dehydrogenase (XDH) in Trichoderma reesei by antisense inhibition strategy and construct novel strains capable of accumulating xylitol. Methods and Results: The xdh1 antisense expression plasmid pGTA-xdh was constructed by inserting xdh1 DNA fragment inversely between the gpdA promoter and the trpC terminator from Aspergillus nidulans into a pUC19 plasmid backbone. Trichoderma reesei protoplasts were co-transformated with pGTA-xdh and hygromycin B resistance plasmid pAN7-1. Of 20 transformants screened from the selective medium, one transformant with the highest xylitol accumulation, designated ZY15, showed a distinct reduction (c. 52%) in XDH activity compared with the original strain Rut-C30. The results of Southern hybridization and PCR assay showed that the antisense expression cassette of xdh1 was integrated into the genome of T. reesei. The RT-PCR analysis proved that antisense RNA effectively inhibited XDH expression (c. 65%). Xylitol accumulation (2AE37 mg ml )1 ) of ZY15 was five times higher than that (0AE46 mg ml )1 ) of the original strain Rut-C30.Conclusions: Strain ZY15 successfully downregulated XDH production and exhibited xylitol accumulation in xylose liquid medium. Significance and Impact of the Study: This study contributed to the budding field of fungal genetics in two points. First, it confirmed that antisense RNA strategy could be used as a means of reducing gene expression in the filamentous fungus T. reesei. Secondly, it verified that the strategy appears most promising for creating novel filamentous fungi strains capable of accumulating intermediary metabolites.

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Characterization of theN-linked glycosylation site of recombinant pectate lyase

Colangelo

Licon

Benen³

et al. 1999

Rapid Commun. Mass Spectrom.

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Recombinant pectate lyase from Aspergillus niger was overexpressed in Aspergillus nidulans. The two recombinant proteins produced differed in molecular mass by 1200 Da, which suggested that the larger molecular weight protein was glycosylated. The deduced amino acid sequence was searched for potential N-linked glycosylation sites, and one potential site was identified at residue 64. The proteins were analyzed for their ability to bind various lectins as an assay for the presence of carbohydrates. The proteins were then digested with trypsin to facilitate the isolation of the potential glycosylation site. The resulting digestion products were subsequently analyzed by liquid chromatography/mass spectrometry using in-source collision induced dissociation to detect glycopeptides. Once the glycopeptide had been identified, treatment with an endoglycosidase both verified the location of glycosylation and identified the mass of the glycan. The Complex Carbohydrate Structural Database was searched for possible N-linked structures with the same mass, and the suggested primary sequence was confirmed by an exoglycosidase digestion. The data demonstrated that the larger recombinant protein contained a high mannose N-linked structure (Man(5)GlcNAc(2)) attached to N-64, while this site was not occupied in the smaller protein.

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Sequence Analysis, Overexpression, and Antisense Inhibition of a β-Xylosidase Gene, xylA , from Aspergillus oryzae KBN616

Cited by 75 publications

References 18 publications

Secretory expression of a β-xylosidase gene fromThermomyces lanuginosusinEscherichia coliand characterization of its recombinant enzyme

Secretory expression of a β-xylosidase gene fromThermomyces lanuginosusinEscherichia coliand characterization of its recombinant enzyme

Antisense inhibition of xylitol dehydrogenase gene, xdh1 from Trichoderma reesei

Characterization of theN-linked glycosylation site of recombinant pectate lyase

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