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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

Effect of bgl2 complementation and disruption in T. reesei PC-3-7. a Specific activity of the intracellular cellobiase from transformants PC-3-7, PC-Wbgl2, and PC-∆bgl2. Cellobiase activity represents the mean of triplicate experiments. Error bars indicate standard deviation. b HPLC analysis of transglycosylation products using cell-free extracts from transformants. In the standard chromatogram, G3, G2, and G1 represent cellotriose, cellobiose, and glucose peaks, respectively. Sp represents α-sophorose. Putative transglycosylation products are indicated by arrows
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Fig2: Effect of bgl2 complementation and disruption in T. reesei PC-3-7. a Specific activity of the intracellular cellobiase from transformants PC-3-7, PC-Wbgl2, and PC-∆bgl2. Cellobiase activity represents the mean of triplicate experiments. Error bars indicate standard deviation. b HPLC analysis of transglycosylation products using cell-free extracts from transformants. In the standard chromatogram, G3, G2, and G1 represent cellotriose, cellobiose, and glucose peaks, respectively. Sp represents α-sophorose. Putative transglycosylation products are indicated by arrows

Mentions: The strains PC-3-7, PC-Wbgl2, and PC-∆bgl2 were cultivated on medium containing Avicel for 3 days and the cellobiase activity was measured in cell-free extracts prepared from each strain. PC-Wbgl2 showed cellobiase activity that was four times that of PC-3-7, whereas PC-∆bgl2 exhibited lower activity than PC-3-7 (Fig. 2a). These results indicate that BGLIIV409F did not lead to a complete loss of β-glucosidase activity. To determine whether PC-Wbgl2 retained transglycosylation activity, cell-free extracts prepared from each strain were incubated under high cellobiose concentrations. Consequently, formation of transglycosylation products was observed in the presence of the PC-Wbgl2 cell-free extract. PC-3-7 extracts showed reduced activity, but some transglycosylation products were observed, whereas no such compounds were detected in the presence of PC-∆bgl2 extracts (Fig. 2b).Fig. 2


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)

Effect of bgl2 complementation and disruption in T. reesei PC-3-7. a Specific activity of the intracellular cellobiase from transformants PC-3-7, PC-Wbgl2, and PC-∆bgl2. Cellobiase activity represents the mean of triplicate experiments. Error bars indicate standard deviation. b HPLC analysis of transglycosylation products using cell-free extracts from transformants. In the standard chromatogram, G3, G2, and G1 represent cellotriose, cellobiose, and glucose peaks, respectively. Sp represents α-sophorose. Putative transglycosylation products are indicated by arrows
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Effect of bgl2 complementation and disruption in T. reesei PC-3-7. a Specific activity of the intracellular cellobiase from transformants PC-3-7, PC-Wbgl2, and PC-∆bgl2. Cellobiase activity represents the mean of triplicate experiments. Error bars indicate standard deviation. b HPLC analysis of transglycosylation products using cell-free extracts from transformants. In the standard chromatogram, G3, G2, and G1 represent cellotriose, cellobiose, and glucose peaks, respectively. Sp represents α-sophorose. Putative transglycosylation products are indicated by arrows
Mentions: The strains PC-3-7, PC-Wbgl2, and PC-∆bgl2 were cultivated on medium containing Avicel for 3 days and the cellobiase activity was measured in cell-free extracts prepared from each strain. PC-Wbgl2 showed cellobiase activity that was four times that of PC-3-7, whereas PC-∆bgl2 exhibited lower activity than PC-3-7 (Fig. 2a). These results indicate that BGLIIV409F did not lead to a complete loss of β-glucosidase activity. To determine whether PC-Wbgl2 retained transglycosylation activity, cell-free extracts prepared from each strain were incubated under high cellobiose concentrations. Consequently, formation of transglycosylation products was observed in the presence of the PC-Wbgl2 cell-free extract. PC-3-7 extracts showed reduced activity, but some transglycosylation products were observed, whereas no such compounds were detected in the presence of PC-∆bgl2 extracts (Fig. 2b).Fig. 2

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