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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 PC-3-7, PC-Wbgl2, PC-∆bgl2. Cellulase activity of transformants grown on Avicel (a) or cellobiose (b), measured as CMCase, FPase, and pNPCase activity. As displayed in the panel, the open circle represents PC-3-7. The open square and the open triangle represent PC-Wbgl2 and PC-∆bgl2, respectively. 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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Fig5: Cellulase productivity of PC-3-7, PC-Wbgl2, PC-∆bgl2. Cellulase activity of transformants grown on Avicel (a) or cellobiose (b), measured as CMCase, FPase, and pNPCase activity. As displayed in the panel, the open circle represents PC-3-7. The open square and the open triangle represent PC-Wbgl2 and PC-∆bgl2, respectively. Values represent the mean of triplicate experiments. Error bars indicate standard deviation. X-axis shows the enzyme activity per 1 mL culture medium

Mentions: From the enzymatic analysis, it was evident that the SNP of bgl2 reduced the cellobiase activity of BGLIIV409F, which appeared to be responsible for three quarters of intracellular cellobiose degrading activity. PC-3-7 shows enhanced cellulase production on cellobiose as the sole carbon source. When T. reesei is cultivated on cellulose, it has been inferred that cellobiose is supplied continuously as cellulose is degraded by cellulase. Therefore, we attempted to determine whether mutation of bgl2 affects cellulase production on cellulose or cellobiose. PC-3-7, PC-Wbgl2, and PC-∆bgl2 were grown on media containing Avicel or cellobiose and a time-course of CMCase activities was analyzed. In the case of Avicel culture, the rate of cellulase production by PC-3-7 was faster than that observed in the other two strains at the early stage of cultivation (Fig. 5a). Nevertheless, the other two strains caught up and a comparable final level of production was achieved by all three strains. This comparable cellulase activity could also be observed in the protein patterns seen on SDS-PAGE (Additional file 1: Figure S1). When transformants were grown on cellobiose, however, differences between the three strains began to emerge. PC-3-7 showed a faster production rate at the early cultivation stage similar to what we observed on the Avicel medium, but cellulase production rate of PC-Wbgl2 was significantly slower and the final activity was lower than PC-3-7, whereas PC-∆bgl2 showed slower production rate initially, but the final activity was higher than that of PC-3-7 (Fig. 5b). SDS-PAGE analysis also showed that cellulase production of PC-Wbgl2 was lower than that of PC-3-7 or PC-∆bgl2 (Additional file 1: Figure S1). These data suggest not only that SNP of bgl2 and the resulting BGLIIV409F mutation affected cellulase production but that another factor(s) responsible for cellobiose signaling may also exist in PC-3-7.Fig. 5


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 PC-3-7, PC-Wbgl2, PC-∆bgl2. Cellulase activity of transformants grown on Avicel (a) or cellobiose (b), measured as CMCase, FPase, and pNPCase activity. As displayed in the panel, the open circle represents PC-3-7. The open square and the open triangle represent PC-Wbgl2 and PC-∆bgl2, respectively. 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

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Show All Figures
getmorefigures.php?uid=PMC4696228&req=5

Fig5: Cellulase productivity of PC-3-7, PC-Wbgl2, PC-∆bgl2. Cellulase activity of transformants grown on Avicel (a) or cellobiose (b), measured as CMCase, FPase, and pNPCase activity. As displayed in the panel, the open circle represents PC-3-7. The open square and the open triangle represent PC-Wbgl2 and PC-∆bgl2, respectively. Values represent the mean of triplicate experiments. Error bars indicate standard deviation. X-axis shows the enzyme activity per 1 mL culture medium
Mentions: From the enzymatic analysis, it was evident that the SNP of bgl2 reduced the cellobiase activity of BGLIIV409F, which appeared to be responsible for three quarters of intracellular cellobiose degrading activity. PC-3-7 shows enhanced cellulase production on cellobiose as the sole carbon source. When T. reesei is cultivated on cellulose, it has been inferred that cellobiose is supplied continuously as cellulose is degraded by cellulase. Therefore, we attempted to determine whether mutation of bgl2 affects cellulase production on cellulose or cellobiose. PC-3-7, PC-Wbgl2, and PC-∆bgl2 were grown on media containing Avicel or cellobiose and a time-course of CMCase activities was analyzed. In the case of Avicel culture, the rate of cellulase production by PC-3-7 was faster than that observed in the other two strains at the early stage of cultivation (Fig. 5a). Nevertheless, the other two strains caught up and a comparable final level of production was achieved by all three strains. This comparable cellulase activity could also be observed in the protein patterns seen on SDS-PAGE (Additional file 1: Figure S1). When transformants were grown on cellobiose, however, differences between the three strains began to emerge. PC-3-7 showed a faster production rate at the early cultivation stage similar to what we observed on the Avicel medium, but cellulase production rate of PC-Wbgl2 was significantly slower and the final activity was lower than PC-3-7, whereas PC-∆bgl2 showed slower production rate initially, but the final activity was higher than that of PC-3-7 (Fig. 5b). SDS-PAGE analysis also showed that cellulase production of PC-Wbgl2 was lower than that of PC-3-7 or PC-∆bgl2 (Additional file 1: Figure S1). These data suggest not only that SNP of bgl2 and the resulting BGLIIV409F mutation affected cellulase production but that another factor(s) responsible for cellobiose signaling may also exist in PC-3-7.Fig. 5

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