Production of recombinant human proteins in Komagataella phaffii using a sucrose-utilization-based selection marker...

Komagataella phaffii (K. phaffii) is one of the most widely used eukaryotic expression systems for recombinant protein production. However, its industrial application is constrained by reliance on traditional antibiotic-based selection and the inability to utilize sucrose. Based on the heterologous expression of the sucrose hydrolase SucF from Aspergillus flavus in K. phaffii, the strain acquired the ability to utilize sucrose. It was verified that this trait can serve as a dominant selection marker under sucrose-selective conditions, making SucF a selection marker for antibiotic-free strain construction. Under a selection pressure of 40 g/L sucrose, the gene integration efficiency reached 100 %, comparable to that of traditional antibiotic selection. Notably, this system enables the substitution of the original glycerol carbon source with sucrose for fermentation. To improve the selection efficiency of SucF, a triple mutant (T65A/R322Q/S323G) was identified through computer-aided protein engineering, exhibiting a 57 % increase in sucrose hydrolysis rate compared to the wild type. Structural analysis revealed that this mutation remodels the sucrose-binding pocket. Ultimately, the SucF mutant was used as a selection marker in combination with the histidine auxotrophic marker (HIS4) to achieve secretory expression of human lactoferrin (hLF) and human osteopontin (hOPN) in the GS115, and the resulting recombinant strain was free of antibiotic gene residues. This sucrose-based antibiotic-free platform in K. phaffii provides a promising strategy for green biomanufacturing and industrial production of recombinant proteins devoid of antibiotic resistance genes.