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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

Chromatogram of transglycosylation products by rBGLIIwt. HPLC analysis of transglycosylation products using purified recombinant wild-type BGLII in E. coli. a The chromatogram generated by the Prominence HPLC system. In the standard chromatogram, G4, G3, G2, and G1 represent cellotetraose, cellotriose, cellobiose, and glucose peaks, respectively. Other β-disaccharides, α-sophorose, laminaribiose, and gentiobiose are represented by Sp, Lm, and Ge, respectively. b The chromatogram of size exclusion chromatography. Cello-oligosaccharides were used as the standard substance. Putative transglycosylation products are indicated by arrows. Asterisk refers to the peak of a buffer component in the reactant
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Fig3: Chromatogram of transglycosylation products by rBGLIIwt. HPLC analysis of transglycosylation products using purified recombinant wild-type BGLII in E. coli. a The chromatogram generated by the Prominence HPLC system. In the standard chromatogram, G4, G3, G2, and G1 represent cellotetraose, cellotriose, cellobiose, and glucose peaks, respectively. Other β-disaccharides, α-sophorose, laminaribiose, and gentiobiose are represented by Sp, Lm, and Ge, respectively. b The chromatogram of size exclusion chromatography. Cello-oligosaccharides were used as the standard substance. Putative transglycosylation products are indicated by arrows. Asterisk refers to the peak of a buffer component in the reactant

Mentions: The results from PC-3-7 and its transformants revealed that the formation of transglycosylation products from cellobiose was likely due to BGLII or BGLIIV409F. However, there is also the possibility of contamination from other enzymes because we used a cell-free extract for the analysis of enzyme activity. To rule out this possibility and to measure the transglycosylation activity of BGLII exclusively, wild-type BGLII was expressed in E. coli. cDNA of bgl2 was cloned into the expression vector of E. coli, the His-tagged recombinant wild-type BGLII, rBGLIIwt, was expressed by the induction strategy, and rBGLIIwt was purified by affinity chromatography. For the HPLC analysis of transglycosylation products of rBGLIIwt, not only glucose, cellobiose, cellotriose, and α-sophorose, but also cellotetraose, laminaribiose, and gentiobiose were used as the standard substance. HPLC data (Fig. 3a) showed that the pattern of transglycosylation products was almost the same as that from the cell-free extract of PC-3-7. In addition, the peak around 50 min of retention time was consistent with the peak of laminaribiose. To investigate the degree of polymerization of transglycosylation products, the sample was applied to a ligand exchange and size exclusion column. The analysis revealed that some kinds of oligosaccharides were present in the reactant and the larger molecules seemed to be at least tetrasaccharides (Fig. 3b). In order to analyze the transglycosylation products further, the samples were subjected to TLC together with cello-oligosaccharides, laminari-oligosaccharides, and α-sophorose as the standard material. Figure 4 shows the results of TLC in which cellobiose as the substrate was reduced and glucose as the hydrolysis product was increased according to the increase in the amount of protein in the reaction. In addition, spots having the same migration patterns as that of laminaribiose, laminaritetraose, and cellotetraose were observed. However, few spots that were not consistent with those of the standard substances were also noted.Fig. 3


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)

Chromatogram of transglycosylation products by rBGLIIwt. HPLC analysis of transglycosylation products using purified recombinant wild-type BGLII in E. coli. a The chromatogram generated by the Prominence HPLC system. In the standard chromatogram, G4, G3, G2, and G1 represent cellotetraose, cellotriose, cellobiose, and glucose peaks, respectively. Other β-disaccharides, α-sophorose, laminaribiose, and gentiobiose are represented by Sp, Lm, and Ge, respectively. b The chromatogram of size exclusion chromatography. Cello-oligosaccharides were used as the standard substance. Putative transglycosylation products are indicated by arrows. Asterisk refers to the peak of a buffer component in the reactant
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Chromatogram of transglycosylation products by rBGLIIwt. HPLC analysis of transglycosylation products using purified recombinant wild-type BGLII in E. coli. a The chromatogram generated by the Prominence HPLC system. In the standard chromatogram, G4, G3, G2, and G1 represent cellotetraose, cellotriose, cellobiose, and glucose peaks, respectively. Other β-disaccharides, α-sophorose, laminaribiose, and gentiobiose are represented by Sp, Lm, and Ge, respectively. b The chromatogram of size exclusion chromatography. Cello-oligosaccharides were used as the standard substance. Putative transglycosylation products are indicated by arrows. Asterisk refers to the peak of a buffer component in the reactant
Mentions: The results from PC-3-7 and its transformants revealed that the formation of transglycosylation products from cellobiose was likely due to BGLII or BGLIIV409F. However, there is also the possibility of contamination from other enzymes because we used a cell-free extract for the analysis of enzyme activity. To rule out this possibility and to measure the transglycosylation activity of BGLII exclusively, wild-type BGLII was expressed in E. coli. cDNA of bgl2 was cloned into the expression vector of E. coli, the His-tagged recombinant wild-type BGLII, rBGLIIwt, was expressed by the induction strategy, and rBGLIIwt was purified by affinity chromatography. For the HPLC analysis of transglycosylation products of rBGLIIwt, not only glucose, cellobiose, cellotriose, and α-sophorose, but also cellotetraose, laminaribiose, and gentiobiose were used as the standard substance. HPLC data (Fig. 3a) showed that the pattern of transglycosylation products was almost the same as that from the cell-free extract of PC-3-7. In addition, the peak around 50 min of retention time was consistent with the peak of laminaribiose. To investigate the degree of polymerization of transglycosylation products, the sample was applied to a ligand exchange and size exclusion column. The analysis revealed that some kinds of oligosaccharides were present in the reactant and the larger molecules seemed to be at least tetrasaccharides (Fig. 3b). In order to analyze the transglycosylation products further, the samples were subjected to TLC together with cello-oligosaccharides, laminari-oligosaccharides, and α-sophorose as the standard material. Figure 4 shows the results of TLC in which cellobiose as the substrate was reduced and glucose as the hydrolysis product was increased according to the increase in the amount of protein in the reaction. In addition, spots having the same migration patterns as that of laminaribiose, laminaritetraose, and cellotetraose were observed. However, few spots that were not consistent with those of the standard substances were also noted.Fig. 3

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