Overexpression of an exotic thermotolerant β-glucosidase in trichoderma reesei and its significant increase in cellulolytic activity and saccharification of barley straw

Abstract

Background: Trichoderma reesei is a widely used industrial strain for cellulase production, but its low yield of β-glucosidase has prevented its industrial value. In the hydrolysis process of cellulolytic residues by T. reesei, a disaccharide known as cellobiose is produced and accumulates, which inhibits further cellulases production. This problem can be solved by adding β-glucosidase, which hydrolyzes cellobiose to glucose for fermentation. It is, therefore, of high vvalue to construct T. reesei strains which can produce sufficient β-glucosidase and other hydrolytic enzymes, especially when those enzymes are capable of tolerating extreme conditions such as high temperature and acidic or alkali pH.

Results: We successfully engineered a thermostable β-glucosidase gene from the fungus Periconia sp. into the genome of T. reesei QM9414 strain. The engineered T. reesei strain showed about 10.5-fold (23.9 IU/mg) higher β-glucosidase activity compared to the parent strain (2.2 IU/mg) after 24 h of incubation. The transformants also showed very high total cellulase activity (about 39.0 FPU/mg) at 24 h of incubation whereas the parent strain almost did not show any total cellulase activity at 24 h of incubation. The recombinant β-glucosidase showed to be thermotolerant and remains fully active after two-hour incubation at temperatures as high as 60°C. Additionally, it showed to be active at a wide pH range and maintains about 88% of its maximal activity after four-hour incubation at 25°C in a pH range from 3.0 to 9.0. Enzymatic hydrolysis assay using untreated, NaOH, or Organosolv pretreated barley straw as well as microcrystalline cellulose showed that the transformed T. reesei strains released more reducing sugars compared to the parental strain.

Conclusions: The recombinant T. reesei overexpressing Periconia sp. β-glucosidase in this study showed higher β-glucosidase and total cellulase activities within a shorter incubation time (24 h) as well as higher hydrolysis activity using biomass residues. These features suggest that the transformants can be used for β-glucosidase production as well as improving the biomass conversion using cellulases.

Figure 1

Alignment ofPericonia sp. bgl1genomic DNA and cDNA using MultAlin software. Intron regions are shown in lower case in red while the coding regions are shown in upper case and black. The 6 single-nucleotide differences between the genomic DNA (Accession No. JQ239427) and previously reported cDNA (Accession No. EU304547) (including: A660G, C678T, C851T, A903T, A914C and G921T) are shown in lower case and highlighted in boxes.

Figure 2

Construction of BGLI-overexpressionT. reeseistrain.(A) The structure of pPtef1- bgl1-cbh1 expression vector using SimVector software. Ptef1 represents tef1 promoter; bgl1 represents β-glucosidase gene from Periconia sp.; Tcbh1 indicates the cbh1 terminator; Pgpd1 represents gpd1 promoter (glyceraldehyde-3-phosphate dehydrogenase gene); hph represents hygromycin B phosphotransferase gene; Tgpd1 indicates the gpd1 terminator. (B) PCR assay ofthe bgl1 gene in the selected overexpression transformants using full-bgl1 primers. All transformants (T1-T4) showed a 2601 bp band whereas the parent strain (QM) did not amplified any specific PCR products. Periconia sp. genomic DNA (gP) as well as cDNA (cP) were used as the positive controls which amplified 2847 and 2601 bp products, respectively. (C) qPCR analysis of the isolated genomic DNA from the transformants T1-T4 using Real-Time bgl1 and tef1a primers. The transformants T1-T3 received one copy of the bgl1 gene whereas T4 obtained two copies.

Figure 3

The enzyme activities and time course study of BGLI-overexpressing transformants (T1-T4) and the parental strain (T. reeseiQM9414). The strains were pre- grown on MA-medium containing 1% glucose for 24 h and then mycelia were washed and grown for 144 h on MA-medium containing 1% microcrystalline for the induction. Enzyme activities were expressed as specific activities using international units per mg protein in the supernatant. (A) The β-glucosidase activity was measured every 24 h using the culture supernatant as the enzyme sources. The enzyme was incubated at 70°C for 10 min, pH 5.0 using 50 mM sodium citrate buffer. (B) Filter paper activity (FPA) was measured every 24 h using the culture supernatant and incubated at 50°C for 60 min, pH 4.8 using 75 mM citrate buffer. (C) Exoglucanases (Exo), and endoglucanases activities (EG) of the strains were measured after being induced with 1% microcrystalline cellulose for 120 h. No significant difference was obtained for Exo and EG activities of the four transformants compared to the parent strain usingone-way ANOVA at a confidence level of 99% (α = 0.01). Data are represented as the mean of three independent experiments and error bars denote standard error of the mean.

Figure 4

Identification of β-glucosidase in BGLI-overexpressing transformants (T1-T4) by MUG-zymogram assay. Proteins (culture supernatant) from BGLI-overexpressing transformants (T1-T4) and the parental strain (T. reesei QM9414, lane QM) were separated in 8% native PAGE. Culture supernatant of Periconia sp. (bgl1 donor strain) (lane P) grown on MA-medium containing 1% microcrystalline was used as the positive control. β-glucosidase activity was detected by MUG-zymogram assay. Lower arrow indicates the native extracellular β-glucosidase found in T. reesei strain (lanes T1-T4 and QM) whereas the upper arrow represents active β-glucosidase correlated to Periconia sp. BGLI only found in the BGLI- overexpressing transformants as well as Periconia sp. (lanes T1-T4 and P). Lane M, molecular weight marker.

Figure 5

Effect of pH and temperature on β-glucosidase activity of the selected BGLI- overexpressing transformant (T3).(A) pH profile was determined by incubating the enzyme (culture supernatant) at 70°C for 10 min at different pH, using 50 mM sodium citrate (pH 3.0- 6.0), sodium acetate (pH 4.0-6.0), MOPs (pH 6.0-8.0) and Tris buffer (pH 8.0-10.0). (B) The remaining β-glucosidase activity was determined (at 70°C in 50 mM sodium citrate buffer, pH 5.0 for 10 min) after incubating the enzyme at 25°C for 4 h at the different pHs. (C) Temperature profile was determined by incubating the enzyme (culture supernatant) in 50 mM sodium citrate buffer (pH 5.0) for 10 min at different temperature (30–90°C). (D) Thermal stability was carried out by incubating the enzyme in 50 mM sodium citrate buffer (pH 5.0) at different temperature (30–90°C) for 30, 60, 90 and 120 min before the remaining activity was assayed (at 70°C in 50 mM sodium citrate buffer, pH 5.0 for 10 min).

Figure 6

Enzymatic hydrolysis of barley straw and microcrystalline cellulose by BGLI- overexpressing transformants (T1-T4) and the parental strain (T. reeseiQM9414).(A and B) Reducing sugars and glucose yield (mg/mL) released using untreated barley straw, respectively. (C and D) Reducing sugars and glucose yield (mg/mL) released using NaOH- pretreated barley straw, respectively. (E and F) Reducing sugars and glucose yield (mg/mL) released using Organosolv-pretreated barley straw, respectively. (G and H) Reducing sugars and glucose yield (mg/mL) released using microcrystalline cellulose, respectively. 750 μl of the culture supernatants (as the enzyme source) were added to tubes containing 750 μl of 50 mM sodium citrate buffer (pH 5.0) and 0.045 g (3%) either barley straw or microcrystalline cellulose. The tubes were incubated at 50°C for 72 h and reducing sugars as well as glucose released were measured every 24 h. Data are represented as the mean of three independent experiments and error bars denote standard error of the mean. Total protein loading was as follow; T1: 0.22 mg; T2: 0.26 mg; T3: 0.24 mg; T4: 0.19 mg, and QM9414: 0.22 mg.