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Construction of a cellulase hyper-expression system in Trichoderma reesei by promoter and enzyme engineering.

ABSTRACT: BACKGROUND: Trichoderma reesei is the preferred organism for producing industrial cellulases. However, a more efficient heterologous expression system for enzymes from different organism is needed to further improve its cellulase mixture. The strong cbh1 promoter of T. reesei is frequently used in heterologous expression, however, the carbon catabolite repressor CREI may reduce its strength by binding to the cbh1 promoter at several binding sites. Another crucial point to enhance the production of heterologous enzymes is the stability of recombinant mRNA and the prevention of protein degradation within the endoplasmic reticulum, especially for the bacteria originated enzymes.In this study, the CREI binding sites within the cbh1 promoter were replaced with the binding sites of transcription activator ACEII and the HAP2/3/5 complex to improve the promoter efficiency. To further improve heterologous expression efficiency of bacterial genes within T. reesei, a flexible polyglycine linker and a rigid ?-helix linker were tested in the construction of fusion genes between cbh1 from T. reesei and e1, encoding an endoglucanase from Acidothermus cellulolyticus. RESULTS: The modified promoter resulted in an increased expression level of the green fluorescent protein reporter by 5.5-fold in inducing culture medium and 7.4-fold in repressing culture medium. The fusion genes of cbh1 and e1 were successfully expressed in T. reesei under the control of promoter pcbh1m2. The higher enzyme activities and thermostability of the fusion protein with rigid linker indicated that the rigid linker might be more suitable for the heterologous expression system in T. reesei. Compared to the parent strain RC30-8, the FPase and CMCase activities of the secreted enzyme mixture from the corresponding transformant R1 with the rigid linker increased by 39% and 30% at 60°C, respectively, and the reduced sugar concentration in the hydrolysate of pretreated corn stover (PCS) was dramatically increased by 40% at 55°C and 169% at 60°C when its enzyme mixture was used in the hydrolysis. CONCLUSIONS: This study shows that optimizations of the promoter and linker for hybrid genes can dramatically improve the efficiency of heterologous expression of cellulase genes in T. reesei.

SUBMITTER: Zou G

PROVIDER: S-EPMC3342899 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Construction of a cellulase hyper-expression system in Trichoderma reesei by promoter and enzyme engineering.

Zou Gen G Shi Shaohua S Jiang Yanping Y van den Brink Joost J de Vries Ronald P RP Chen Ling L Zhang Jun J Ma Liang L Wang Chengshu C Zhou Zhihua Z

Microbial cell factories 20120208

<h4>Background</h4>Trichoderma reesei is the preferred organism for producing industrial cellulases. However, a more efficient heterologous expression system for enzymes from different organism is needed to further improve its cellulase mixture. The strong cbh1 promoter of T. reesei is frequently used in heterologous expression, however, the carbon catabolite repressor CREI may reduce its strength by binding to the cbh1 promoter at several binding sites. Another crucial point to enhance the prod ...[more]

PMID: 22314137

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Project description:The filamentous fungus Trichoderma reesei is the main industrial cellulytic enzymes producer. Several strains have been developed in the past using random mutagenesis, and despite impressive performance enhancements, the pressure for low-cost cellulases has stimulated continuous research in the field. In this context, comparative study of the lower and higher producer strains obtained through random mutagenesis using systems biology tools (genome sequencing, transcriptome) can shed light on the mechanisms of cellulase production and help identify genes linked to performance. Previously, our group published comparative genome sequencing of the lower and higher producer strains NG14 and RUT C30. In this follow-up work, we examined how these mutations affect phenotype at the level of the transcriptome and cultivation behavior. We performed kinetic transcriptome analysis of the NG14 and RUT C30 strains of early cellulase production induced by lactose using bioreactor cultivations close to industrial conditions. RUT C30 exhibited both earlier onset of cellulase production and higher steady-state productivity. A rather low number of genes were regulated, most of them being specific to the NG14 strains. Clustering of these genes highlighted similar behavior for some functional categories, and allowed to distinguish between induction-related genes and productivity-related genes. The lower number of genes regulated in RUT C30 could not formally be linked to the relief in catabolic repression that is characteristic of this strain. Cross-comparison of our transcriptome data with mutations previously identified revealed that most genes from our dataset have not been mutated. Interestingly, the few mutated genes belong to the same clusters, suggesting these clusters contain genes playing a role in strain performance. This is the first kinetic transcriptome study carried out in industry-relevant conditions with two related strains of T. reesei showing distinctive performances. Our study sheds some light on some of the events occurring in these strains following induction by lactose. The fact that few regulated genes have been affected by mutagenesis suggest that the induction mechanism is essentially intact and that there is room for further improvement of T. reesei. We also provide some potential target for further genetic improvement of these strains.

Project description:BackgroundFilamentous fungus Trichoderma reesei has been widely used as a workhorse for cellulase and xylanase productions. Xylanase has been reported as the crucial accessory enzyme in the degradation of lignocellulose for higher accessibility of cellulase. In addition, the efficient hydrolysis of xylan needs the co-work of multiple xylanolytic enzymes, which rise an increasing demand for the high yield of xylanase for efficient biomass degradation.ResultsIn this study, a xylanase hyper-producing system in T. reesei was established by tailoring two transcription factors, XYR1 and ACE1, and homologous overexpression of the major endo-xylanase XYNII. The expressed xylanase cocktail contained 5256 U/mL xylanase activity and 9.25 U/mL β-xylosidase (pNPXase) activity. Meanwhile, the transcription level of the xylanolytic genes in the strain with XYR1 overexpressed was upregulated, which was well correlated with the amount of XYR1-binding sites. In addition, the higher expression of associated xylanolytic enzymes would result in more efficient xylan hydrolysis. Besides, 2310-3085 U/mL of xylanase activities were achieved using soluble carbon source, which was more efficient and economical than the traditional strategy of xylan induction. Unexpectedly, deletion of ace1 in C30OExyr1 did not give any improvement, which might be the result of the disturbed function of the complex formed between ACE1 and XYR1. The enzymatic hydrolysis of alkali pretreated corn stover using the crude xylanase cocktails as accessory enzymes resulted in a 36.64% increase in saccharification efficiency with the ratio of xylanase activity vs FPase activity at 500, compared to that using cellulase alone.ConclusionsAn efficient and economical xylanase hyper-producing platform was developed in T. reesei RUT-C30. The novel platform with outstanding ability for crude xylanase cocktail production would greatly fit in biomass degradation and give a new perspective of further engineering in T. reesei for industrial purposes.

Project description:BACKGROUND:The filamentous fungus Trichoderma reesei is the main industrial cellulolytic enzyme producer. Several strains have been developed in the past using random mutagenesis, and despite impressive performance enhancements, the pressure for low-cost cellulases has stimulated continuous research in the field. In this context, comparative study of the lower and higher producer strains obtained through random mutagenesis using systems biology tools (genome and transcriptome sequencing) can shed light on the mechanisms of cellulase production and help identify genes linked to performance. Previously, our group published comparative genome sequencing of the lower and higher producer strains NG 14 and RUT C30. In this follow-up work, we examine how these mutations affect phenotype as regards the transcriptome and cultivation behaviour. RESULTS:We performed kinetic transcriptome analysis of the NG 14 and RUT C30 strains of early enzyme production induced by lactose using bioreactor cultivations close to an industrial cultivation regime. RUT C30 exhibited both earlier onset of protein production (3 h) and higher steady-state productivity. A rather small number of genes compared to previous studies were regulated (568), most of them being specific to the NG 14 strain (319). Clustering analysis highlighted similar behaviour for some functional categories and allowed us to distinguish between induction-related genes and productivity-related genes. Cross-comparison of our transcriptome data with previously identified mutations revealed that most genes from our dataset have not been mutated. Interestingly, the few mutated genes belong to the same clusters, suggesting that these clusters contain genes playing a role in strain performance. CONCLUSIONS:This is the first kinetic analysis of a transcriptomic study carried out under conditions approaching industrial ones with two related strains of T. reesei showing distinctive cultivation behaviour. Our study sheds some light on some of the events occurring in these strains following induction by lactose. The fact that few regulated genes have been affected by mutagenesis suggests that the induction mechanism is essentially intact compared to that for the wild-type isolate QM6a and might be engineered for further improvement of T. reesei. Genes from two specific clusters might be potential targets for such genetic engineering.

Project description:The filamentous fungus Trichoderma reesei is the main industrial cellulytic enzymes producer. Several strains have been developed in the past using random mutagenesis, and despite impressive performance enhancements, the pressure for low-cost cellulases has stimulated continuous research in the field. In this context, comparative study of the lower and higher producer strains obtained through random mutagenesis using systems biology tools (genome sequencing, transcriptome) can shed light on the mechanisms of cellulase production and help identify genes linked to performance. Previously, our group published comparative genome sequencing of the lower and higher producer strains NG14 and RUTM-BM- C30. In this follow-up work, we examined how these mutations affect phenotype at the level of the transcriptome and cultivation behavior.M-BM- We performed kinetic transcriptome analysis of the NG14 and RUTM-BM- C30 strains of early cellulase production induced by lactose using bioreactor cultivations close to industrial conditions. RUTM-BM- C30 exhibited both earlier onset of cellulase production and higher steady-state productivity. A rather lowM-BM- number of genes were regulated, most of them being specific to the NG14 strains. Clustering of these genes highlighted similar behavior for some functional categories, and allowed to distinguish between induction-related genes and productivity-related genes. The lower number of genes regulated in RUTM-BM- C30 could not formally be linked to the relief in catabolic repression that is characteristic of this strain. Cross-comparison of our transcriptome data with mutations previously identified revealed that most genes from our dataset have not been mutated. Interestingly, the few mutated genes belong to the same clusters, suggesting these clusters contain genes playing a role in strain performance. This is the first kinetic transcriptome study carried out in industry-relevant conditions with two related strains of T. reesei showing distinctive performances. Our study sheds some light on some of the events occurring in these strains following induction by lactose. The fact that few regulated genes have been affected by mutagenesis suggest that the induction mechanism is essentially intact and that there is room for further improvement of T. reesei. We also provide some potential target for further genetic improvement of these strains. Two biological pool by condition in dye switch. For the two biological replicates on each four experiments we apply on the pretreated results the linear modeling approach implemented by lmFit and the empirical Bayes statistics implemented by eBayes from the limma R package (Smyth 2004). We select the list of statistically regulated genes using a 5% significance threshold.

Project description:BackgroundThe induction of cellulase production by insoluble carbon source cellulose was a common and efficient strategy, but has some drawbacks, such as difficult fermentation operation, substantial cellulase loss, long fermentation time, and high energy-consumption, resulting in high cost of cellulase production in industry. These drawbacks can be overcome if soluble carbon sources are utilized as the inducers for cellulase production. However, until now the induction efficiency of most soluble carbon sources, especially lactose and glucose, is still inferior to cellulose despite extensive efforts have been made by either optimizing the fermentation process or constructing the recombinant strains. Therefore, strain improvement by metabolic engineering for high induction efficiency of soluble carbon sources is of great interest.ResultsTrichoderma reesei mutant SEU-7 was constructed from T. reesei RUT-C30 with the overexpression of endogenous gene β-glucosidase (BGL1) by insertional mutagenesis via Agrobacterium tumefaciens-mediated transformation (AMT). Compared to RUT-C30, SEU-7 displays substantially enhanced activities of both cellulase and hemicellulase when grown on either lactose or cellulose. The induction efficiency with lactose was found to be higher than cellulose in strain SEU-7. To the best of our knowledge, we achieved the highest FPase activity in SEU-7 in both batch culture (13.0 IU/mL) and fed-batch culture (47.0 IU/mL) on lactose. Moreover, SEU-7 displayed unrivaled pNPGase activity on lactose in both batch culture (81.0 IU/mL) and fed-batch culture (144.0 IU/mL) as compared to the other reported T. reesei strains in the literature grown in batch or fed-batch experiments on cellulose or lactose. This superiority of SEU-7 over RUT-C30 improves markedly the saccharification ability of SEU-7 on pretreated corn stover. The overexpression of gene BGL1 was found either at the mRNA or at the protein level in the mutant strains with increased cellulase production in comparison with RUT-C30, but only SEU-7 displayed much higher expression of gene BGL1 on lactose than on cellulose. Two copies of gene BGL1 were inserted into the chromosome of T. reesei SEU-7 between KI911141.1:347357 and KI911141.1:347979, replacing the original 623-bp fragment that is not within any genes' coding region. The qRT-PCR analysis revealed that the mRNA levels of both cellulase and hemicellulase were upregulated significantly in SEU-7, together with the MFS transporter CRT1 and the XYR1 nuclear importer KAP8.ConclusionsRecombinant T. reesei SEU-7 displays hyper-production of both cellulase and hemicellulase on lactose with the highest FPase activity and pNPGase activity for T. reesei, enabling highly efficient saccharification of pretreated biomass. For the first time, the induction efficiency for cellulase production by lactose in T. reesei was reported to be higher than that by cellulose. This outperformance of T. reesei SEU-7, which is strain-specific, is attributed to both the overexpression of gene BGL and the collateral mutation. Moreover, the increased transcription levels of cellulase genes, the related transcription factors, and the MFS transporter CRT1 contribute to the outstanding cellulase production of SEU-7. Our research advances strain improvement to enhance the induction efficiency of soluble carbon sources to produce cost-effective cellulase and hemicellulase in industry.

Project description:BackgroundTrichoderma reesei is used for industry-scale production of plant cell wall-degrading enzymes, in particular cellulases, but also xylanases. The expression of the encoding genes was so far primarily investigated on the level of transcriptional regulation by regulatory proteins. Otherwise, the impact of chromatin remodelling on gene expression received hardly any attention. In this study we aimed to learn if the chromatin status changes in context to the applied conditions (repressing/inducing), and if the presence or absence of the essential transactivator, the Xylanase regulator 1 (Xyr1), influences the chromatin packaging.ResultsComparing the results of chromatin accessibility real-time PCR analyses and gene expression studies of the two prominent cellulase-encoding genes, cbh1 and cbh2, we found that the chromatin opens during sophorose-mediated induction compared to D-glucose-conferred repression. In the strain bearing a xyr1 deletion the sophorose mediated induction of gene expression is lost and the chromatin opening is strongly reduced. In all conditions the chromatin got denser when Xyr1 is absent. In the case of the xylanase-encoding genes, xyn1 and xyn2, the result was similar concerning the condition-specific response of the chromatin compaction. However, the difference in chromatin status provoked by the absence of Xyr1 is less pronounced. A more detailed investigation of the DNA accessibility in the cbh1 promoter showed that the deletion of xyr1 changed the in vivo footprinting pattern. In particular, we detected increased hypersensitivity on Xyr1-sites and stronger protection of Cre1-sites. Looking for the players directly causing the observed chromatin remodelling, a whole transcriptome shotgun sequencing revealed that 15 genes encoding putative chromatin remodelers are differentially expressed in response to the applied condition and two amongst them are differentially expressed in the absence of Xyr1.ConclusionsThe regulation of xylanase and cellulase expression in T. reesei is not only restricted to the action of transcription factors but is clearly related to changes in the chromatin packaging. Both the applied condition and the presence of Xyr1 influence chromatin status.

Dataset Information

Construction of a cellulase hyper-expression system in Trichoderma reesei by promoter and enzyme engineering.

Publications

Construction of a cellulase hyper-expression system in Trichoderma reesei by promoter and enzyme engineering.

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