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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

Gene expression profile of PC-3-7 transformants following cellobiose induction. Genes analyzed were cbh1 (a), egl1 (b), bgl2 (c), and xyr1 (d). Stippled gray bar represents PC-3-7, solid gray bar represents PC-Wbgl2, and the shaded gray barindicates PC-∆bgl2. Values represent the means of triplicate experiments. Error bars indicate standard deviations
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Fig7: Gene expression profile of PC-3-7 transformants following cellobiose induction. Genes analyzed were cbh1 (a), egl1 (b), bgl2 (c), and xyr1 (d). Stippled gray bar represents PC-3-7, solid gray bar represents PC-Wbgl2, and the shaded gray barindicates PC-∆bgl2. Values represent the means of triplicate experiments. Error bars indicate standard deviations

Mentions: To investigate the transcriptional response of cellulase gene toward several carbon sources without taking cell growth into account, induction experiments were carried out by using the mycelia transfer method. Each transformant was pregrown on glucose and mycelia were transferred to an induction solution containing α-sophorose, cellobiose, or Avicel as an inducer. For the target gene of expression analysis, cbh1 and egl1 were selected as the major cellulase genes among T. reesei cellulase genes that were coordinately expressed with other cellulase genes in T. reesei. xyr1, the gene encoding Xyr1 that is the key activator of T. reesei cellulase genes, was also chosen as the target gene. Total cDNA derived from induced mycelia was used as a template and expression amounts of cbh1, egl1, bgl2, and xyr1 were determined by quantitative real-time PCR. When α-sophorose, a strong cellulase inducer for T. reesei, was used, the expression amounts of cbh1 and xyr1 were nearly the same between all three strains, although cbh1 expression at time point of 180 min was higher in PC-Wbgl2 compared to the other two strains (Fig. 6a, d). In the case of bgl2 expression, the level of bgl2 of PC-3-7 was higher than that of PC-Wbgl2 at all time points (Fig. 6c). This suggests that bgl2 expression was partly different from cbh1 and egl1 (Fig. 6b). As compared with α-sophorose induction, however, significant difference in gene expression pattern was observed when cellobiose or Avicel was used as an inducer. Compared to PC-3-7, the transcription of cbh1, egl1, bgl2, and xyr1 of PC-Wbgl2 was higher than that of PC-3-7 at the early stages of induction (after 30–60 min) on cellobiose (Fig. 7), but the pattern was completely reversed after 180 min and PC-3-7 showed significantly higher expression than PC-Wbgl2. Similar expression pattern was observed on Avicel as well (Additional file 1: Figure S2). Contrary to cellulase production, a significant delay in transcription of all the other genes was observed in PC-∆bgl2 as compared with the other two strains in cellobiose induction (Fig. 7). Furthermore, transcription was not detected from mycelia induced on Avicel (Additional file 1: Figure S2).Fig. 6


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)

Gene expression profile of PC-3-7 transformants following cellobiose induction. Genes analyzed were cbh1 (a), egl1 (b), bgl2 (c), and xyr1 (d). Stippled gray bar represents PC-3-7, solid gray bar represents PC-Wbgl2, and the shaded gray barindicates PC-∆bgl2. Values represent the means of triplicate experiments. Error bars indicate standard deviations
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig7: Gene expression profile of PC-3-7 transformants following cellobiose induction. Genes analyzed were cbh1 (a), egl1 (b), bgl2 (c), and xyr1 (d). Stippled gray bar represents PC-3-7, solid gray bar represents PC-Wbgl2, and the shaded gray barindicates PC-∆bgl2. Values represent the means of triplicate experiments. Error bars indicate standard deviations
Mentions: To investigate the transcriptional response of cellulase gene toward several carbon sources without taking cell growth into account, induction experiments were carried out by using the mycelia transfer method. Each transformant was pregrown on glucose and mycelia were transferred to an induction solution containing α-sophorose, cellobiose, or Avicel as an inducer. For the target gene of expression analysis, cbh1 and egl1 were selected as the major cellulase genes among T. reesei cellulase genes that were coordinately expressed with other cellulase genes in T. reesei. xyr1, the gene encoding Xyr1 that is the key activator of T. reesei cellulase genes, was also chosen as the target gene. Total cDNA derived from induced mycelia was used as a template and expression amounts of cbh1, egl1, bgl2, and xyr1 were determined by quantitative real-time PCR. When α-sophorose, a strong cellulase inducer for T. reesei, was used, the expression amounts of cbh1 and xyr1 were nearly the same between all three strains, although cbh1 expression at time point of 180 min was higher in PC-Wbgl2 compared to the other two strains (Fig. 6a, d). In the case of bgl2 expression, the level of bgl2 of PC-3-7 was higher than that of PC-Wbgl2 at all time points (Fig. 6c). This suggests that bgl2 expression was partly different from cbh1 and egl1 (Fig. 6b). As compared with α-sophorose induction, however, significant difference in gene expression pattern was observed when cellobiose or Avicel was used as an inducer. Compared to PC-3-7, the transcription of cbh1, egl1, bgl2, and xyr1 of PC-Wbgl2 was higher than that of PC-3-7 at the early stages of induction (after 30–60 min) on cellobiose (Fig. 7), but the pattern was completely reversed after 180 min and PC-3-7 showed significantly higher expression than PC-Wbgl2. Similar expression pattern was observed on Avicel as well (Additional file 1: Figure S2). Contrary to cellulase production, a significant delay in transcription of all the other genes was observed in PC-∆bgl2 as compared with the other two strains in cellobiose induction (Fig. 7). Furthermore, transcription was not detected from mycelia induced on Avicel (Additional file 1: Figure S2).Fig. 6

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