High-Level Expression of β-Glucosidase in Aspergillus niger ATCC 20611 Using the Trichoderma reesei Promoter Pcdna1 to Enhance Cellulose Degradation
<p>Construction of the <span class="html-italic">gpd1</span> promoter-driven EGFP expression strains of <span class="html-italic">A. niger</span> ATCC 20611: (<b>A</b>) Schematic representation of the <span class="html-italic">egfp</span> expression under the control of <span class="html-italic">gpd1</span> promoter. The resistance gene <span class="html-italic">ptrA</span> was used as the reporter gene. (<b>B</b>) The transformant strains expressing EGFP under the control of <span class="html-italic">gpd1</span> promoter were selected on an MM plate with 0.3% PT (the left). The plate diagram on the right showed the growth of <span class="html-italic">A. niger</span> ATCC 20611 on a MM plate with 0.3% PT as the control. (<b>C</b>) The transcript levels of <span class="html-italic">egfp</span> in the transformant AGE9 and the parental strain <span class="html-italic">A. niger</span> ATCC 20611 were detected by RT–qPCR at 24 h. The relative expression was the level of transcripts normalized to that of <span class="html-italic">actin</span> gene. (<b>D</b>) Detection of the fluorescence in the mycelium of <span class="html-italic">A. niger</span> AGF9-1 and the parental strain ATCC 20611 at 48 h by light and fluorescent microscopy. Error bars indicate the standard deviation (SD) of three biological replicates. Student’s <span class="html-italic">t</span>-test: ** <span class="html-italic">p</span> < 0.01. ND, not detected.</p> "> Figure 2
<p>Construction of the <span class="html-italic">gpd1</span> promoter-driven β-glucosidase (BGLA) expression strains of <span class="html-italic">A. niger</span> ATCC 20611: (<b>A</b>) Schematic representation of the <span class="html-italic">bglA</span> expression cassette, which contained the promotor of <span class="html-italic">gpd1</span> (P<span class="html-italic">gpd1</span>), the signal peptides of <span class="html-italic">cbh1</span> (SP), the β-glucosidase-encoding gene (<span class="html-italic">bglA</span>) and the terminator of <span class="html-italic">cbh1</span> (T<span class="html-italic">cbh1</span>). (<b>B</b>) Detection of the β-glucosidase activities of transformants on the CMC-esculin plates. The parental strain ATCC 20611 was used as control. (<b>C</b>) The ratios of halo diameter to colony diameter of the transformants in (<b>B</b>). (<b>D</b>) The transcript levels of <span class="html-italic">bglA</span> in transformants AGB1, AGB33 and the parental strain ATCC 20611 at 24 h by RT–qPCR. The relative expression was the level of transcripts normalized to that of <span class="html-italic">actin</span> gene. (<b>E</b>) Detection of β-glucosidase activity using p-Nitrophenyl-β-d-glucopyranoside (pNPG) as the substrate of the <span class="html-italic">cdna1</span> promoter-driven β-glucosidase (BGLA) expression strains and the parental strain ATCC 20611 after 4-day fermentation. Error bars indicate the standard deviation (SD) of three biological replicates. Student’s <span class="html-italic">t</span>-test: ** <span class="html-italic">p</span> < 0.01. ND, not detected.</p> "> Figure 3
<p>Transcript analysis of endoplasmic reticulum-associated genes (<span class="html-italic">bip1</span>, <span class="html-italic">pdi1 yos9</span> and <span class="html-italic">der1</span>) by RT-qPCR. The transcript levels of UPR—(<b>A</b>) and ERAD—(<b>B</b>) related genes of transformants AGB1, AGB33 and the parental strain ATCC 20611. The relative expression was the level of transcripts normalized to that of the <span class="html-italic">actin</span> gene. Error bars indicate the standard deviation (SD) of three biological replicates. Student’s <span class="html-italic">t</span>-test: ** <span class="html-italic">p</span> < 0.01, * <span class="html-italic">p</span> < 0.05. n.s., no significant differences.</p> "> Figure 4
<p>Construction of the <span class="html-italic">cdna1</span> promoter-driven β-glucosidase (BGLA) expression strains of <span class="html-italic">A. niger</span> ATCC 20611: (<b>A</b>) Schematic representation of the <span class="html-italic">bglA</span> expression under the control of <span class="html-italic">gpd1</span> promoter, which contained the promotor <span class="html-italic">gpd1</span> (P<span class="html-italic">cdna1</span>), the signal peptides of <span class="html-italic">cbh1</span> (SP), the β-glucosidase-encoding gene (<span class="html-italic">bglA</span>) and the terminator of <span class="html-italic">cbh1</span> (T<span class="html-italic">cbh1</span>). (<b>B</b>) Detection of β-glucosidase activities of transformant on the CMC-esculin plate. The parental strain ATCC 20611 as control. (<b>C</b>) The ratios of halo diameter to colony diameter. The data assessed the β-glucosidase production capacity. Analysis of the expression level of <span class="html-italic">bglA</span>. (<b>D</b>) The transcript levels of <span class="html-italic">bglA</span> in transformants ACB8, ACB11 and the parental strain ATCC 20611 at 24 h by RT–qPCR. The relative expression was the level of transcripts normalized to that of the <span class="html-italic">actin</span> gene. Error bars indicate the standard deviation (SD) of three biological replicates. Student’s <span class="html-italic">t</span>-test: ** <span class="html-italic">p</span> < 0.01. ND, not detected.</p> "> Figure 5
<p>Analysis of the expression level of <span class="html-italic">bglA</span>: (<b>A</b>) Detection of β-glucosidase activity using p-Nitrophenyl-β-d-glucopyranoside (pNPG) as the substrate of the <span class="html-italic">cdna1</span> promoter-driven β-glucosidase (BGLA) expression strains and the parental strain ATCC 20611 after 4-day fermentation. (<b>B</b>) SDS PAGE showing secreted BGLA expression. (<b>C</b>) Detection of extracellular total protein concentration by Bradford’s method. Error bars indicate the standard deviation (SD) of three biological replicates. Student’s <span class="html-italic">t</span>-test: ** <span class="html-italic">p</span> < 0.01, * <span class="html-italic">p</span> < 0.05.</p> "> Figure 6
<p>Transcript analysis of endoplasmic reticulum-associated genes (<span class="html-italic">bip1</span>, <span class="html-italic">pdi1 yos9</span> and <span class="html-italic">der1</span>) by RT-qPCR: The transcript levels of UPR—(<b>A</b>) and ERAD—(<b>B</b>) related genes of transformants ACB8, ACB11 and the parental strain ATCC 20611. The relative expression was the level of transcripts normalized to that of actin. Error bars indicate the standard deviation (SD) of three biological replicates. Student’s <span class="html-italic">t</span>-test: ** <span class="html-italic">p</span> < 0.01, * <span class="html-italic">p</span> < 0.05.</p> "> Figure 7
<p>Enzymatic characterization of BGLA in <span class="html-italic">A. niger</span> ACB 8. The BGLA in <span class="html-italic">T. reesei</span> QVB-1 was the control: (<b>A</b>) Optimal temperature. The activity of BGLA was detected in the temperature range of 30–80 °C at pH 4.8. (<b>B</b>) and (C) Thermostability. After preincubation of the fermentation broths of the ACB8 and QVB-1 at 50, 60 and 70 °C for 0.5 h to 10 h, respectively, the residual activities were determined at pH 4.8 and 50 °C and the initial activity at pH 4.8 and 50 °C was defined as 100%. (<b>D</b>) Optimal pH. The activity of BGLA was detected at 50 °C in the pH range 3 to 8. (<b>E</b>) pH stability. After preincubation of the fermentation broths of the ACB8 and QVB-1 in citric acid buffer of different pH for 24 h in 4 °C, respectively, the residual activities were determined at pH 4.8 and 50 °C and the initial activity at pH 4.8 and 50 °C was defined as 100%.</p> "> Figure 8
<p>Saccharification of different pretreated corncob: (<b>A</b>) Glucose released from saccharification of acid-pretreated corncob residues by <span class="html-italic">T. reesei</span> SN1 with the addition of the fermentation broth of <span class="html-italic">A. niger</span> ACB8 for 48 h. (<b>B</b>) Glucose released from the saccharification of delignified corncob residues by <span class="html-italic">T. reesei</span> SN1 adding the fermentation broth of ACB8 for 48 h. Saccharification by adding the fermentation broth of the parental strain <span class="html-italic">A. niger</span> ATCC 20611 was used as the control. Error bars indicate the standard deviation (SD) of three biological replicates. Student’s <span class="html-italic">t</span>-test: * <span class="html-italic">p</span> < 0.05. n.s., no significant differences.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains and Culture Conditions
2.2. Construction of Expression Strains
2.3. Fluorescence and Light Microscopy
2.4. RNA Extraction and RT-qPCR Analysis
2.5. Enzyme Assay and SDS PAGE
2.6. Enzymatic Characterization
2.7. Saccharification of Different Pretreated Corncob Residues
3. Results
3.1. The T. reesei gpd1 Promoter-Driven EGFP Expression in A. niger ATCC 20611
3.2. The gpd1 Promoter-Driven β-Glucosidase Expression in A. niger ATCC 20611
3.3. Detection of the ER Pressure in the gpd1 Promoter-Driven β-glucosidase Expression Strains
3.4. The cdna1 Promoter-Driven β-Glucosidase Expression in A. niger ATCC 20611
3.5. Enzymatic Property of β-Glucosidase in A. niger ACB8
3.6. Saccharification of the Corncob Residues by Supplementing the β-glucosidase to the Cellulase Mixture of T. reesei
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Chang, J.; Wang, J.; Li, Z.; Wang, L.; Lu, P.; Zhong, Y.; Liu, H. High-Level Expression of β-Glucosidase in Aspergillus niger ATCC 20611 Using the Trichoderma reesei Promoter Pcdna1 to Enhance Cellulose Degradation. Fermentation 2024, 10, 461. https://doi.org/10.3390/fermentation10090461
Chang J, Wang J, Li Z, Wang L, Lu P, Zhong Y, Liu H. High-Level Expression of β-Glucosidase in Aspergillus niger ATCC 20611 Using the Trichoderma reesei Promoter Pcdna1 to Enhance Cellulose Degradation. Fermentation. 2024; 10(9):461. https://doi.org/10.3390/fermentation10090461
Chicago/Turabian StyleChang, Jingjing, Juan Wang, Zhihong Li, Lu Wang, Peng Lu, Yaohua Zhong, and Hong Liu. 2024. "High-Level Expression of β-Glucosidase in Aspergillus niger ATCC 20611 Using the Trichoderma reesei Promoter Pcdna1 to Enhance Cellulose Degradation" Fermentation 10, no. 9: 461. https://doi.org/10.3390/fermentation10090461