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Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

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Published on: December 15, 2017

Building a Versatile Protein Production Platform Using Engineered Trichoderma reesei.

Shunxing Chai^1,2, Zhihua Zhu^1,2, Ernuo Tian¹

¹CAS-Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 300 Fenglin Rd, Shanghai 200032, China.

ACS Synthetic Biology

|December 20, 2021

Summary

This summary is machine-generated.

Engineered Trichoderma reesei strains significantly reduce native protein secretion and protease activity. This optimized fungal expression platform enhances the production of valuable heterologous proteins like xylanase, LZ8, and human serum albumin.

Keywords:

Trichoderma reesei chassis heterologous protein expression iterative gene deletion

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A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

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Area of Science:

Biotechnology
Molecular Biology
Industrial Microbiology

Background:

* Trichoderma reesei is a highly efficient protein producer, making it a promising host for heterologous protein expression.
* High secretion of native proteins and extracellular protease activity in T. reesei limit its utility for producing foreign proteins.

Purpose of the Study:

* To develop improved Trichoderma reesei expression chassis by reducing native protein secretion and protease activity.
* To enhance the yield of heterologous proteins using the engineered T. reesei strains.

Main Methods:

* Iterative gene deletion using an efficient genome editing system to create modified T. reesei strains.
* Development of specialized donor DNAs for streamlined screening of deletion strains without ectopic insertion.
* Construction of marker-free T. reesei chassis through 11 consecutive gene deletion rounds.

Main Results:

* Successfully constructed marker-free T. reesei chassis with significantly reduced native protein secretion.
* Achieved low levels of extracellular protease activity in the engineered strains.
* Demonstrated higher production yields for three distinct heterologous proteins (bacterial xylanase XYL7, fungal LZ8, and human serum albumin HSA) using the cbh1 promoter.

Conclusions:

* The developed T. reesei chassis offers a robust platform for high-yield production of heterologous proteins.
* This engineered system has the potential for the cost-effective manufacturing of diverse high-value proteins.