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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

The active site and the mutation point in the three-dimensional structure of Trichoderma reesei BGLII (PDBID: 3AHY). The catalytic acid/base, Glu165, the catalytic nucleophile, Glu367, and the mutation residue in this study, Val409 are shown as CPK model. All other residues are displayed as ribbon model in a and residues from Phe323 to Tyr360 are omitted in b
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Fig10: The active site and the mutation point in the three-dimensional structure of Trichoderma reesei BGLII (PDBID: 3AHY). The catalytic acid/base, Glu165, the catalytic nucleophile, Glu367, and the mutation residue in this study, Val409 are shown as CPK model. All other residues are displayed as ribbon model in a and residues from Phe323 to Tyr360 are omitted in b

Mentions: In this study, we evaluated the effect of a single-nucleotide mutation in the bgl2 gene of the cellulase hyper-producing mutant PC-3-7 developed in Japan on cellulase production. By comparing amino acid sequences between GH1 proteins from Trichoderma species, we noted that V409 was strictly conserved. V409 is located within a loop between the 7th α-helix and the 8th β-sheet. Hence, V409 is close to the glycone or aglycone recognition site [37], and substitution of V409 to phenylalanine, which is a large amino acid, could lead to the alteration of substrate recognition and enzyme activity. Through this study, we concluded that the hydrolysis activity of BGLIIV409F was markedly low and that transglycosylation activity, although weak, remained. From a structural point of view, V409 is localized in close proximity to the surface of the BGLII protein. However, structural analysis by a calculation method of solvent-accessible surface [38] showed that the solvent accessibility of V409 was only 1 % (Fig. 10a). If the loop and the following 6th α-helix between F323 and Y360 comprising a part of the catalytic pocket of BGLII were removed, the solvent accessibility of V409 will increase to 30 % (Fig. 10b). This means that V409 interacts with the 6th α-helix and the substitution V409F is expected to affect the static and/or dynamic structure of the loop constituting a part of the substrate pocket indirectly through the 6th α-helix. Therefore, V409 is a very interesting residue from the standpoint of the protein design with a physical perturbation method [39]. We observed reduced cellulase production by reverting the bgl2 mutation of PC-3-7 and a significantly enhanced cellulase production when the mutation was introduced into the bgl2 gene of strain QM9414. These facts suggest that the single-point mutation in the gene encoding BGLIIV409F affected cellulase expression.Fig. 10


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)

The active site and the mutation point in the three-dimensional structure of Trichoderma reesei BGLII (PDBID: 3AHY). The catalytic acid/base, Glu165, the catalytic nucleophile, Glu367, and the mutation residue in this study, Val409 are shown as CPK model. All other residues are displayed as ribbon model in a and residues from Phe323 to Tyr360 are omitted in b
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig10: The active site and the mutation point in the three-dimensional structure of Trichoderma reesei BGLII (PDBID: 3AHY). The catalytic acid/base, Glu165, the catalytic nucleophile, Glu367, and the mutation residue in this study, Val409 are shown as CPK model. All other residues are displayed as ribbon model in a and residues from Phe323 to Tyr360 are omitted in b
Mentions: In this study, we evaluated the effect of a single-nucleotide mutation in the bgl2 gene of the cellulase hyper-producing mutant PC-3-7 developed in Japan on cellulase production. By comparing amino acid sequences between GH1 proteins from Trichoderma species, we noted that V409 was strictly conserved. V409 is located within a loop between the 7th α-helix and the 8th β-sheet. Hence, V409 is close to the glycone or aglycone recognition site [37], and substitution of V409 to phenylalanine, which is a large amino acid, could lead to the alteration of substrate recognition and enzyme activity. Through this study, we concluded that the hydrolysis activity of BGLIIV409F was markedly low and that transglycosylation activity, although weak, remained. From a structural point of view, V409 is localized in close proximity to the surface of the BGLII protein. However, structural analysis by a calculation method of solvent-accessible surface [38] showed that the solvent accessibility of V409 was only 1 % (Fig. 10a). If the loop and the following 6th α-helix between F323 and Y360 comprising a part of the catalytic pocket of BGLII were removed, the solvent accessibility of V409 will increase to 30 % (Fig. 10b). This means that V409 interacts with the 6th α-helix and the substitution V409F is expected to affect the static and/or dynamic structure of the loop constituting a part of the substrate pocket indirectly through the 6th α-helix. Therefore, V409 is a very interesting residue from the standpoint of the protein design with a physical perturbation method [39]. We observed reduced cellulase production by reverting the bgl2 mutation of PC-3-7 and a significantly enhanced cellulase production when the mutation was introduced into the bgl2 gene of strain QM9414. These facts suggest that the single-point mutation in the gene encoding BGLIIV409F affected cellulase expression.Fig. 10

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