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Development of a powerful synthetic hybrid promoter to improve the cellulase system of Trichoderma reesei for efficient saccharification of corncob residues.

ABSTRACT:

Background

The filamentous fungus Trichoderma reesei is a widely used workhorse for cellulase production in industry due to its prominent secretion capacity of extracellular cellulolytic enzymes. However, some key components are not always sufficient in this cellulase cocktail, making the conversion of cellulose-based biomass costly on the industrial scale. Development of strong and efficient promoters would enable cellulase cocktail to be optimized for bioconversion of biomass.

Results

In this study, a synthetic hybrid promoter was constructed and applied to optimize the cellulolytic system of T. reesei for efficient saccharification towards corncob residues. Firstly, a series of 5' truncated promoters in different lengths were established based on the strong constitutive promoter Pcdna1. The strongest promoter amongst them was Pcdna1-3 (- 640 to - 1 bp upstream of the translation initiation codon ATG), exhibiting a 1.4-fold higher activity than that of the native cdna1 promoter. Meanwhile, the activation region (- 821 to - 622 bp upstream of the translation initiation codon ATG and devoid of the Cre1-binding sites) of the strong inducible promoter Pcbh1 was cloned and identified to be an amplifier in initiating gene expression. Finally, this activation region was fused to the strongest promoter Pcdna1-3, generating the novel synthetic hybrid promoter Pcc. This engineered promoter Pcc drove strong gene expression by displaying 1.6- and 1.8-fold stronger fluorescence intensity than Pcbh1 and Pcdna1 under the inducible condition using egfp as the reporter gene, respectively. Furthermore, Pcc was applied to overexpress the Aspergillus niger β-glucosidase BGLA coding gene bglA and the native endoglucanase EG2 coding gene eg2, achieving 43.5-fold BGL activity and 1.2-fold EG activity increase, respectively. Ultimately, to overcome the defects of the native cellulase system in T. reesei, the bglA and eg2 were co-overexpressed under the control of Pcc promoter. The bglA-eg2 double expression strain QPEB70 exhibited a 178% increase in total cellulase activity, whose cellulase system displayed 2.3- and 2.4-fold higher saccharification efficiency towards acid-pretreated and delignified corncob residues than the parental strain, respectively.

Conclusions

The synthetic hybrid promoter Pcc was generated and employed to improve the cellulase system of T. reesei by expressing specific components. Therefore, construction of synthetic hybrid promoters would allow particular cellulase genes to be expressed at desired levels, which is a viable strategy to optimize the cellulolytic enzyme system for efficient biomass bioconversion.

SUBMITTER: Wang Y

PROVIDER: S-EPMC8725555 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe filamentous fungus Trichoderma reesei is extensively used for the industrial-scale cellulase production. It has been well known that the transcription factor Xyr1 plays an important role in the regulatory network controlling cellulase gene expression. However, the role of Xyr1 in the regulation of cellulase expression has not been comprehensively elucidated, which hinders further improvement of lignocellulolytic enzyme production.ResultsHere, the expression dosage of xyr1 was tailored in T. reesei by differentially overexpressing the xyr1 gene under the control of three strong promoters (Pegl2, Pcbh1, and Pcdna1), and the transcript abundance of xyr1 was elevated 5.8-, 12.6-, and 47.2-fold, respectively. We found expression of cellulase genes was significantly increased in the Pegl2-driven xyr1 overexpression strain QE2X, whereas relatively low in the Pcbh1- and Pcdna1-driven overexpression strains. We also found that the Pegl2-driven overexpression of xyr1 caused a more significant opening of chromatin in the core promoter region of the prominent cellulase genes. Furthermore, the cellulase activity showed a 3.2-fold increase in the strain QE2X, while insignificant improvement in the Pcbh1- and Pcdna1-driven strains. Finally, the saccharification efficiency toward acid-pretreated corncob residues containing high-content lignin by the crude enzyme from QE2X was increased by 57.2% compared to that from the parental strain. Moreover, LC-MS/MS and RT-qPCR analysis revealed that expression of accessory proteins (Cip1, Cip2, Swo1, and LPMOs) was greatly improved in QE2X, which partly explained the promoting effect of the Pegl2-driven overexpression on enzymatic hydrolysis of lignocellulose biomass.ConclusionsOur results underpin that the precise tailoring expression of xyr1 is essential for highly efficient cellulase synthesis, which provide new insights into the role of Xyr1 in regulating cellulase expression in T. reesei. Moreover, these results also provides a prospective strategy for strain improvement to enhance the lignocellulolytic enzyme production for use in biorefinery applications.

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Project description:Background:Trichoderma reesei is widely used for cellulase production and accepted as an example for cellulase research. Cre1-mediated carbon catabolite repression (CCR) can significantly inhibit the transcription of cellulase genes during cellulase fermentation in T. reesei. Early efforts have been undertaken to modify Cre1 for the release of CCR; however, this approach leads to arrested hyphal growth and decreased biomass accumulation, which negatively affects cellulase production. Results:In this study, novel fusion transcription factors (fTFs) were designed to release or attenuate CCR inhibition in cellulase transcription, while Cre1 was left intact to maintain normal hyphal growth. Four designed fTFs were introduced into the T. reesei genome, which generated several transformants, named Kuace3, Kuclr2, Kuace2, and Kuxyr1. No obvious differences in growth were observed between the parent and transformant strains. However, the transcription levels of cel7a, a major cellulase gene, were significantly elevated in all the transformants, particularly in Kuace2 and Kuxyr1, when grown on lactose as a carbon source. This suggested that CCR inhibition was released or attenuated in the transformant strains. The growth of Kuace2 and Kuxyr1 was approximately equivalent to that of the parent strain in fed-batch fermentation process. However, we observed a 3.2- and 2.1-fold increase in the pNPCase titers of the Kuace2 and Kuxyr1 strains, respectively, compared with that of the parent strain. Moreover, we observed a 6.1- and 3.9-fold increase in the pNPCase titers of the Kuace2 and Kuxyr1 strains, respectively, compared with that of ?cre1 strain. Conclusions:A new strategy based on fTFs was successfully established in T. reesei to improve cellulase titers without impairing fungal growth. This study will be valuable for lignocellulosic biorefining and for guiding the development of engineering strategies for producing other important biochemical compounds in fungal species.