Transcriptomics analysis identifies folding and secretion related genes for improving monoclonal antibody production in Thermothelomyces heterothallica C1

Background: The filamentous fungus Thermothelomyces heterothallica C1 has been developed into a highly productive protein production system for heterologous proteins like antibodies and vaccine candidates. While it is capable of secreting over 120 g/l of its native enzymes, monoclonal antibodies (mAbs) have been produced at titers exceeding 20 g/l, and strains engineered to produce human-type N-glycan structures have been developed. However, significant variability in mAb productivity and reduced production levels in glycoengineered strains limit the use of C1 as a widespread production host in the pharmaceutical industry. To address these issues, transcriptome analysis was conducted on strains producing five different mAbs with varying production efficiencies, as well as on mAb-producing strains with native and glycoengineered N-glycans. Genes related to protein folding and secretion, which are regulated in response to mAb production and glycoengineering, were over-expressed in a glycoengineered mAb producing C1 strain. In addition, genes identified based on previously described functions in the secretory pathway, including counterparts of human origin, were included in the study. Results: Transcriptome analysis revealed that the mAb heavy and light chains were among the most abundantly expressed transcripts, indicating that production bottlenecks occur after transcription. Genes associated with protein folding, quality control, glycosylation, and transport within the secretory pathway were upregulated in the mAb-producing strains. This upregulation was more pronounced in strains with low mAb yields and in glycoengineered strains. The over-expression of 10 genes (bet1, dnaj-type gene, dpm1, ero1, erv46, human calreticulin, human cypb, human mzb1, pmr1, and UDP-galactose transporter), each playing distinct roles in the secretory pathway, enhanced mAb production in glycoengineered C1 strains from 1.5- to 2.5-fold. Conclusions: Through a comprehensive analysis of transcriptome data from C1 strains producing various monoclonal antibodies (mAbs) and an extensive literature search, several factors related to protein folding and secretion were identified as potential targets for enhancing mAb production. The over-expression of some of these genes in glycoengineered C1 strains led to improvement in mAb production, with some genes enhancing mAb yields by 2.5-fold.