Trichoderma reesei, a filamentous fungus, is known to secrete proteins at a level of more than 100 grams per liter, making it an attractive platform to express heterologous genes. Now researchers at the French Institute of Petroleum (IFP Energies Nouvelles) have developed a way to further enhance its production capabilities.
In a paper, Frederique Bidard, PhD, research engineer, and colleagues at the IFP report that integrating “a circular plasmid with the green fluorescence protein (eGFP) transgene under the control of [the] tef1 promoter favors multicopy [gene] integration and allows over-production of this heterologous protein on glucose.”
In a prior study involving engineering fluorescent spores of T reesei, first author and IFP research scientist Hugues Mathis, PhD found a correlation between high eGFP expression levels and the presence of the tef1 promoter, noting that that promoter also was compatible with flow cytometry.
The current study by Mathis, Bidard, and colleagues evaluates transgene integration sites to identify those best able to modulate target gene expression levels.
The team used the hyper-productive T. reesei mutant C1847 as the reference strain, and also assessed nine of its transformants.
tef1 promoter supports multicopy insertions
After 25 hours, analysis showed differences in the ratio levels of fluorescence to spore size ranging between 2.8 and 6.9, depending on the strain. The differences in ratios are related to the number of expression cassette copies at the insertion site.
Rather than finding multiple eGFP integration sites among the transformants and the C1847 strain, the scientists found one localization site in the genome of the transformed strains. It is “localized upstream of the tef1 locus with a copy number ranging from 5 to 11,” they report, and supports multicopy gene insertions.
Long-read sequencing showed the eGFP-containing plasmid also had the ref1 promoter and the cbh1 terminator in the genome of eight transformed strains. Importantly, the eGFP expression cassette “consistently integrated upstream of the tef1 promoter, even without two homologous flanks,” the team reported.
“This result suggests that the use of the tef1 promoter may favor multicopy insertion and, therefore, promote a higher expression for heterologous genes.”
Mathis and his team suggest that using an expression cassette with a circular plasmid that includes the tef1 promotor but does not include the tef1 terminator could be an important step in producing heterologous proteins on an industrial scale for many industries, including the biopharmaceutical sector.
