Enhanced heterologous gene expression in Trichoderma reesei by promoting multicopy integration

Abstract

Trichoderma reesei displays a high capability to produce extracellular proteins and therefore is used as a platform for the expression of heterologous genes. In a previous study, an expression cassette with the constitutive tef1 promoter and the cbh1 terminator compatible with flow cytometry analysis was developed. Independent transformants obtained by a random integration into the genome of a circular plasmid containing the expression cassette showed a wide range of fluorescence levels. Whole genome sequencing was conducted on eight of the transformed strains using two next-generation sequencing (NGS) platforms: Illumina paired-end sequencing and Oxford Nanopore. In all strains, the expression plasmid was inserted at the same position in the genome, i.e., upstream of the tef1 gene, indicating an integration by homologous recombination. The different levels of fluorescence observed correspond to different copy numbers of the plasmid. Overall, the integration of a circular plasmid with the green fluorescence protein (egfp) transgene under the control of tef1 promoter favors multicopy integration and allows over-production of this heterologous protein on glucose. In conclusion, an expression system based on using the tef1 promotor could be one of the building blocks for improving high-value heterologous protein production by increasing the copy number of the encoding genes into the genome of the platform strain. KEY POINTS: • Varied eGFP levels from tef1 promoter and cbh1 terminator expression. • Whole genome sequencing on short and long reads platforms reveals various plasmid copy numbers in strains. • Plasmids integrate at the same genomic site by homologous recombination in all strains.

Keywords: Trichoderma reesei; tef1; Heterologous expression; Multicopy integration.

Fig. 1

Evolution of ratio of fluorescence to spore size over time for pTEF-EGFP transformants E1 to E9 (excluding E7). Comparison with CL847 controls. Events in the SF gating region were used for measurement. Averages obtained based on triplicates

Fig. 2

Strategy for identifying the integration sites of the plasmid. A Classification of paired-end reads localization according to their localization in the transformant genomes. PR1, reads mapping to the genome; PR2, reads mapping to the plasmid; PR3 or hybrid reads, one read mapping to the plasmid and the other one to the genome; PR4, reads with one mapping to either the plasmid or the genome whereas the second one mapped nowhere. B Pipeline to recover the hybrid paired reads and localize plasmid integration site

Fig. 3

Annotation of different expression plasmid elements inserted into the genome of transformed strains. A Plasmid vector for transformation and native chromosome. B Multicopy integrations as observed for 4 transformants. Abbreviations: P promoter, T terminator, E egfp, H hygromycin; green arrows indicate functional copies of egfp; black arrows indicate dynamics of insertion of the circular plasmid into the genome