Enhanced heterologous gene expression in Trichoderma reesei by promoting multicopy integration

Area of Science:

• Biotechnology
• Molecular Biology
• Microbial Engineering

Background:

• Trichoderma reesei is a fungal platform for heterologous protein production.
• Previous work developed an expression cassette using the tef1 promoter and cbh1 terminator.
• Transformants showed variable fluorescence, indicating different expression levels.

Purpose of the Study:

• To investigate the genomic integration and expression of a heterologous gene in Trichoderma reesei.
• To understand the relationship between plasmid copy number and protein expression levels.
• To optimize Trichoderma reesei as a host for enhanced protein production.

Main Methods:

• Development of an expression cassette with tef1 promoter and cbh1 terminator.
• Generation of Trichoderma reesei transformants with a plasmid containing egfp transgene.
• Whole genome sequencing using Illumina and Oxford Nanopore platforms.
• Analysis of plasmid integration site and copy number.

Main Results:

• All transformants showed plasmid integration upstream of the tef1 gene via homologous recombination.
• Plasmid integration favored multicopy integration in the genome.
• Higher plasmid copy numbers correlated with increased eGFP fluorescence and protein over-production.
• The tef1 promoter and cbh1 terminator system demonstrated variable eGFP expression levels.

Conclusions:

• The tef1 promoter-based expression system facilitates multicopy integration and heterologous protein over-production in Trichoderma reesei.
• This system is a valuable tool for improving high-value protein production by increasing gene copy numbers.
• Optimized gene integration strategies can enhance the efficiency of Trichoderma reesei as a production platform.