Presentation Information
[P03-440]Translational Regulation of cell growth and cellulase production in Trichoderma reesei revealed by comparative translatome analysis
○Lan Yang1, Linjing Shen1, Xinqing Zhao1 (1. Shanghai Jiao Tong University (China))
Keywords:
Trichoderma reesei,Ribo-seq,Translational regulation,Transcription factor,Cellulase,uORF
[Purpose]
Trichoderma reesei is widely used for cellulase production and biocontrol, yet the discrepancy between mRNA levels and actual protein yield remains a bottleneck. This study aimed to decode the translatome of T. reesei to identify translational mechanisms that govern cell growth and cellulase synthesis, and discover novel regulatory targets for strain engineering.
[Methods]
A multi-omics approach was employed, integrating Ribo-seq (ribosome profiling) and RNA-seq at multiple time points using glucose and Avicel as the sole carbon source, respectively. Key regulatory candidates were identified through multi-dimensional correlation analysis. The function of a novel transcription factor was validated via homologous recombination (knockout/complementation), Western blotting, and polysome profiling. Downstream targets were identified using DAP-seq and EMSA. Finally, the regulatory role of small open reading frames (sORFs) was characterized through site-directed mutagenesis of the start codons.
[Results]
A previously uncharacterized Zn(II)2Cys6 transcription factor was identified by the omic analyses. Its deletion significantly reduced cellulase activity and delayed Cbh1 protein synthesis without altering transcript levels, confirming its role as a translational regulator. Furthermore, DAP-seq revealed that Tre03468 specifically binds to the promoter of one of the key translation initiation factors. Polysome profiling confirmed that overall translation efficiency was impaired in the mutant. We also identified carbon-source-dependent uORFs and dORFs in several key genes.
[Consideration]
These results shift the paradigm from purely transcriptional engineering to translational fine-tuning. The identification of Tre03468 suggests that T. reesei coordinates enzyme secretion and translation machinery through specific transcription factors. These non-canonical regulatory layers provide fresh targets for CRISPR-based metabolic engineering of T. reesei to enhance cellulase production.
[Conclusion]
This study provides the first comprehensive map of the T. reesei translatome. We successfully identified a novel transcription factor and specific sORFs that act as "translational switches" for cell growth and cellulase production. These findings provide basis for optimizing industrial filamentous fungi by leveraging translational control mechanisms.
Acknowledgements
The authors appreciate the help of Prof. Zhipeng Zhou with bioinformatic analysis. We also thank the master student Yajing Hou in Guangxi University for assistance in uORF functional studies. This work is supported by the National Natural Science Foundation of China (Grant No. 22578271) and the 2026 Guangxi Science and Technology Program Project "Empowerment" Action Plan Project.
Trichoderma reesei is widely used for cellulase production and biocontrol, yet the discrepancy between mRNA levels and actual protein yield remains a bottleneck. This study aimed to decode the translatome of T. reesei to identify translational mechanisms that govern cell growth and cellulase synthesis, and discover novel regulatory targets for strain engineering.
[Methods]
A multi-omics approach was employed, integrating Ribo-seq (ribosome profiling) and RNA-seq at multiple time points using glucose and Avicel as the sole carbon source, respectively. Key regulatory candidates were identified through multi-dimensional correlation analysis. The function of a novel transcription factor was validated via homologous recombination (knockout/complementation), Western blotting, and polysome profiling. Downstream targets were identified using DAP-seq and EMSA. Finally, the regulatory role of small open reading frames (sORFs) was characterized through site-directed mutagenesis of the start codons.
[Results]
A previously uncharacterized Zn(II)2Cys6 transcription factor was identified by the omic analyses. Its deletion significantly reduced cellulase activity and delayed Cbh1 protein synthesis without altering transcript levels, confirming its role as a translational regulator. Furthermore, DAP-seq revealed that Tre03468 specifically binds to the promoter of one of the key translation initiation factors. Polysome profiling confirmed that overall translation efficiency was impaired in the mutant. We also identified carbon-source-dependent uORFs and dORFs in several key genes.
[Consideration]
These results shift the paradigm from purely transcriptional engineering to translational fine-tuning. The identification of Tre03468 suggests that T. reesei coordinates enzyme secretion and translation machinery through specific transcription factors. These non-canonical regulatory layers provide fresh targets for CRISPR-based metabolic engineering of T. reesei to enhance cellulase production.
[Conclusion]
This study provides the first comprehensive map of the T. reesei translatome. We successfully identified a novel transcription factor and specific sORFs that act as "translational switches" for cell growth and cellulase production. These findings provide basis for optimizing industrial filamentous fungi by leveraging translational control mechanisms.
Acknowledgements
The authors appreciate the help of Prof. Zhipeng Zhou with bioinformatic analysis. We also thank the master student Yajing Hou in Guangxi University for assistance in uORF functional studies. This work is supported by the National Natural Science Foundation of China (Grant No. 22578271) and the 2026 Guangxi Science and Technology Program Project "Empowerment" Action Plan Project.
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