Heterologous gene expression of a bacterial dipeptidyl peptidase 4 in the thermophilic filamentous fungus Humicola insolens

The thermophilic filamentous fungus Humicola insolens is a rich reservoir of enzymes with broad application potential, owing to their thermostability and neutral pH optima. H. insolens is also distinguished in secreting abundant catalase. Despite this characteristic, its potential as a host for heterologous gene expression remains largely underexploited. Herein, DsRed encoding the red fluorescence protein was established as an effective reporter gene for use in H. insolens . Integrating the DsRed expression cassette into the pks locus enabled systematical evaluation of key expression elements by screening the transformants with the albino phenotype. This strategy allowed identification of the P8.1255 promoter and sp18.4 signal peptide as superior candidates from libraries of 26 promoters and 30 signal peptides. Using a bacterial dipeptidyl peptidase 4 from Parabacteroides goldsteinii (Pg5516) as a model heterologous protein, we demonstrated that the P8.1255 promoter and sp18.4 signal peptide were 6.2-fold and 2.5-fold more efficient, respectively, than the standard strong gpd promoter and catalase signal peptide in directing heterologous protein production. Following the construction of a species-specific codon usage table and subsequent codon optimization, the combinatorial use of these optimized elements resulted in a 37.6-fold increase in recombinant production of Pg5516 (from 194.29 U/L to 7,302.94 U/L). Furthermore, overexpression of key secretory pathway genes PDI , Hsp90 , and Hac1 further enhanced the yield by 13.1%, 12.2%, and 40.6%, respectively, reaching a maximum activity of 10,265.63 U/L. Collectively, this study presents an example of enhancing secretory expression of heterologous proteins in H. insolens .