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The impact of a single-nucleotide mutation of bgl2 on cellulase induction in a Trichoderma reesei mutant.

Shida Y, Yamaguchi K, Nitta M, Nakamura A, Takahashi M, Kidokoro S, Mori K, Tashiro K, Kuhara S, Matsuzawa T, Yaoi K, Sakamoto Y, Tanaka N, Morikawa Y, Ogasawara W - Biotechnol Biofuels (2015)

Bottom Line: The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer.The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked β-1,4, β-1,3, or a mixture of both.PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188 Japan.

ABSTRACT

Background: The filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) produces increased cellulase expression when grown on cellulose or its derivatives as a sole carbon source. It has been believed that β-glucosidases of T. reesei not only metabolize cellobiose but also contribute in the production of inducers of cellulase gene expression by their transglycosylation activity. The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer. The comparative genomics analysis of PC-3-7 and its parent revealed a single-nucleotide mutation within the bgl2 gene encoding intracellular β-glucosidase II (BGLII/Cel1a), giving rise to an amino acid substitution in PC-3-7, which could potentially account for the enhanced cellulase expression when these strains are cultivated on cellulose and cellobiose.

Results: To analyze the effects of the BGLII mutation in cellulase induction, we constructed both a bgl2 revertant and a disruptant. Enzymatic analysis of the transformant lysates showed that the strain expressing mutant BGLII exhibited weakened cellobiose hydrolytic activity, but produced some transglycosylation products, suggesting that the SNP in bgl2 strongly diminished cellobiase activity, but did not result in complete loss of function of BGLII. The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked β-1,4, β-1,3, or a mixture of both. PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates. Furthermore, the effect of this bgl2 mutation was reproduced in the common strain QM9414 in which the transformants showed cellulase production comparable to that of PC-3-7.

Conclusion: We conclude that BGLII plays an important role in cellulase induction in T. reesei and that the bgl2 mutation in PC-3-7 brought about enhanced cellulase expression on cellobiose. The results of the investigation using PC-3-7 suggested that other mutation(s) in PC-3-7 could also contribute to cellulase induction. Further investigation is essential to unravel the mechanism responsible for cellulase induction in T. reesei.

No MeSH data available.


Related in: MedlinePlus

Gene expression in PC-3-7 and QM9414 transformants grown on Avicel. Genes analyzed were cbh1 (a), egl1 (b), bgl2 (c), and xyr1 (d). PC-3-7 series are expressed in gray and the QM-series are represented in white. The relative expression of each gene is normalized that of act1 as an internal control. Values represent the mean of triplicate experiments. Error bars indicate standard deviations
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Fig9: Gene expression in PC-3-7 and QM9414 transformants grown on Avicel. Genes analyzed were cbh1 (a), egl1 (b), bgl2 (c), and xyr1 (d). PC-3-7 series are expressed in gray and the QM-series are represented in white. The relative expression of each gene is normalized that of act1 as an internal control. Values represent the mean of triplicate experiments. Error bars indicate standard deviations

Mentions: To analyze cellulase gene expression in the QM-series during Avicel cultivation, quantitative real-time PCR was performed using total cDNA obtained from mycelia grown on Avicel culture as a template (Fig. 9a–d). In the QM-series, expression of all genes was greater in QM-Mbgl2 than in the other strains at all the time points examined. In the QM-∆bgl2 strain, the expression of cbh1 and egl1 was lower at early time points than that of QM9414, but the final level of expression was comparable to what was observed in QM9414 (Fig. 9a, b). The pattern of expression of xyr1 in each strain derived from QM9414 (Fig. 9d) was nearly identical to the pattern of cellulase gene expression. In addition, the pattern of gene expression appeared to be similar to the cellulase production in each strain (Fig. 8a). In the case of PC-3-7, the final transcription level of cellulase gene was higher than that of PC-Wbgl2, although not at all time points. Interestingly, the expression of cbh1 and egl1 was the greatest in the PC-∆bgl2 strain among the PC-series. In contrast, the expression of cbh1 and egl1 in QM-∆bgl2 did not exceed that in QM-9414.Fig. 9


The impact of a single-nucleotide mutation of bgl2 on cellulase induction in a Trichoderma reesei mutant.

Shida Y, Yamaguchi K, Nitta M, Nakamura A, Takahashi M, Kidokoro S, Mori K, Tashiro K, Kuhara S, Matsuzawa T, Yaoi K, Sakamoto Y, Tanaka N, Morikawa Y, Ogasawara W - Biotechnol Biofuels (2015)

Gene expression in PC-3-7 and QM9414 transformants grown on Avicel. Genes analyzed were cbh1 (a), egl1 (b), bgl2 (c), and xyr1 (d). PC-3-7 series are expressed in gray and the QM-series are represented in white. The relative expression of each gene is normalized that of act1 as an internal control. Values represent the mean of triplicate experiments. Error bars indicate standard deviations
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4696228&req=5

Fig9: Gene expression in PC-3-7 and QM9414 transformants grown on Avicel. Genes analyzed were cbh1 (a), egl1 (b), bgl2 (c), and xyr1 (d). PC-3-7 series are expressed in gray and the QM-series are represented in white. The relative expression of each gene is normalized that of act1 as an internal control. Values represent the mean of triplicate experiments. Error bars indicate standard deviations
Mentions: To analyze cellulase gene expression in the QM-series during Avicel cultivation, quantitative real-time PCR was performed using total cDNA obtained from mycelia grown on Avicel culture as a template (Fig. 9a–d). In the QM-series, expression of all genes was greater in QM-Mbgl2 than in the other strains at all the time points examined. In the QM-∆bgl2 strain, the expression of cbh1 and egl1 was lower at early time points than that of QM9414, but the final level of expression was comparable to what was observed in QM9414 (Fig. 9a, b). The pattern of expression of xyr1 in each strain derived from QM9414 (Fig. 9d) was nearly identical to the pattern of cellulase gene expression. In addition, the pattern of gene expression appeared to be similar to the cellulase production in each strain (Fig. 8a). In the case of PC-3-7, the final transcription level of cellulase gene was higher than that of PC-Wbgl2, although not at all time points. Interestingly, the expression of cbh1 and egl1 was the greatest in the PC-∆bgl2 strain among the PC-series. In contrast, the expression of cbh1 and egl1 in QM-∆bgl2 did not exceed that in QM-9414.Fig. 9

Bottom Line: The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer.The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked β-1,4, β-1,3, or a mixture of both.PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188 Japan.

ABSTRACT

Background: The filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) produces increased cellulase expression when grown on cellulose or its derivatives as a sole carbon source. It has been believed that β-glucosidases of T. reesei not only metabolize cellobiose but also contribute in the production of inducers of cellulase gene expression by their transglycosylation activity. The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer. The comparative genomics analysis of PC-3-7 and its parent revealed a single-nucleotide mutation within the bgl2 gene encoding intracellular β-glucosidase II (BGLII/Cel1a), giving rise to an amino acid substitution in PC-3-7, which could potentially account for the enhanced cellulase expression when these strains are cultivated on cellulose and cellobiose.

Results: To analyze the effects of the BGLII mutation in cellulase induction, we constructed both a bgl2 revertant and a disruptant. Enzymatic analysis of the transformant lysates showed that the strain expressing mutant BGLII exhibited weakened cellobiose hydrolytic activity, but produced some transglycosylation products, suggesting that the SNP in bgl2 strongly diminished cellobiase activity, but did not result in complete loss of function of BGLII. The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked β-1,4, β-1,3, or a mixture of both. PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates. Furthermore, the effect of this bgl2 mutation was reproduced in the common strain QM9414 in which the transformants showed cellulase production comparable to that of PC-3-7.

Conclusion: We conclude that BGLII plays an important role in cellulase induction in T. reesei and that the bgl2 mutation in PC-3-7 brought about enhanced cellulase expression on cellobiose. The results of the investigation using PC-3-7 suggested that other mutation(s) in PC-3-7 could also contribute to cellulase induction. Further investigation is essential to unravel the mechanism responsible for cellulase induction in T. reesei.

No MeSH data available.


Related in: MedlinePlus