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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

Cellulase productivity of QM9414, QM-Mbgl2, QM-∆bgl2. Cellulase activity of transformants grown on Avicel (a) or cellobiose (b), measured as CMCase FPase, and pNPCase activity. Activities of QM9414 (filled squares), QM-Mbgl2 (filled circles), and QM-∆bgl2 (filled triangles) are shown. Values represent the mean of triplicate experiments. Error bars indicate standard deviation. X-axis shows the enzyme activity per 1 mL culture medium
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Fig8: Cellulase productivity of QM9414, QM-Mbgl2, QM-∆bgl2. Cellulase activity of transformants grown on Avicel (a) or cellobiose (b), measured as CMCase FPase, and pNPCase activity. Activities of QM9414 (filled squares), QM-Mbgl2 (filled circles), and QM-∆bgl2 (filled triangles) are shown. Values represent the mean of triplicate experiments. Error bars indicate standard deviation. X-axis shows the enzyme activity per 1 mL culture medium

Mentions: To investigate cellulase production in the transformants, they were cultivated on Avicel or cellobiose as the sole carbon source. When Avicel was used as the carbon source (Fig. 8a), the rate of cellulase production by QM-∆bgl2 was initially slower than that observed by QM9414, but toward the end of the culture period, the rate picked up and there was virtually no difference between the two strains with regards to cellulase production. Surprisingly, cellulase production by QM-Mbgl2 was significantly greater than QM9414 and was comparable to that observed by PC-3-7. This observation was supported by the data obtained from SDS-PAGE analysis of the culture filtrate (Additional file 1: Figure S1). Generally, cellobiose is regarded as a poor inducer of cellulase production in QM9414, and we indeed observed significantly less cellulase activity when QM9414 was cultured on cellobiose. However, when cellobiose was used as the poor cellulase inducer for QM9414, QM-Mbgl2 showed significantly more robust protein production than did QM9414 (Fig. 8b). QM-∆bgl2 exhibited significantly lower cellulase activity compared to the other two strains. In the case of FPase from QM9414 transformant, activity of each strain was under the detection limit. The cell biomass of these strains was comparable when cultivated on cellobiose, indicating the absence of any influence of the bgl2 mutation or disruption (data not shown).Fig. 8


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)

Cellulase productivity of QM9414, QM-Mbgl2, QM-∆bgl2. Cellulase activity of transformants grown on Avicel (a) or cellobiose (b), measured as CMCase FPase, and pNPCase activity. Activities of QM9414 (filled squares), QM-Mbgl2 (filled circles), and QM-∆bgl2 (filled triangles) are shown. Values represent the mean of triplicate experiments. Error bars indicate standard deviation. X-axis shows the enzyme activity per 1 mL culture medium
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig8: Cellulase productivity of QM9414, QM-Mbgl2, QM-∆bgl2. Cellulase activity of transformants grown on Avicel (a) or cellobiose (b), measured as CMCase FPase, and pNPCase activity. Activities of QM9414 (filled squares), QM-Mbgl2 (filled circles), and QM-∆bgl2 (filled triangles) are shown. Values represent the mean of triplicate experiments. Error bars indicate standard deviation. X-axis shows the enzyme activity per 1 mL culture medium
Mentions: To investigate cellulase production in the transformants, they were cultivated on Avicel or cellobiose as the sole carbon source. When Avicel was used as the carbon source (Fig. 8a), the rate of cellulase production by QM-∆bgl2 was initially slower than that observed by QM9414, but toward the end of the culture period, the rate picked up and there was virtually no difference between the two strains with regards to cellulase production. Surprisingly, cellulase production by QM-Mbgl2 was significantly greater than QM9414 and was comparable to that observed by PC-3-7. This observation was supported by the data obtained from SDS-PAGE analysis of the culture filtrate (Additional file 1: Figure S1). Generally, cellobiose is regarded as a poor inducer of cellulase production in QM9414, and we indeed observed significantly less cellulase activity when QM9414 was cultured on cellobiose. However, when cellobiose was used as the poor cellulase inducer for QM9414, QM-Mbgl2 showed significantly more robust protein production than did QM9414 (Fig. 8b). QM-∆bgl2 exhibited significantly lower cellulase activity compared to the other two strains. In the case of FPase from QM9414 transformant, activity of each strain was under the detection limit. The cell biomass of these strains was comparable when cultivated on cellobiose, indicating the absence of any influence of the bgl2 mutation or disruption (data not shown).Fig. 8

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