Abstract

Systematic engineering enabled high-level rhCSF2 production in P. pastoris. • Multi-copy integration and combinatorial signal peptide boosted rhCSF2 titers. • The strategy applied to SuperMan5, yielding rhCSF2 with human-like N -glycans. • The engineered strains yielded the highest rhCSF2 titers ever reported in yeast. Human granulocyte–macrophage colony-stimulating factor (hCSF2) is a clinically important therapeutic glycoprotein with growing market demand, underscoring the need for efficient recombinant production platforms. To enable high-level production of recombinant hCSF2 (rhCSF2), the Pichia pastoris GS115 strain was systematically engineered through multi-copy integration of the expression cassette, screening for suitable secretory signal peptides, and enhancement of protein folding and transport pathways. The combination of multi-copy integration at multiple genomic loci with the combinatorial use of endogenous and exogenous signal peptides proved critical for improving rhCSF2 titers, while reinforcement of the folding and transport machinery further alleviated the bottleneck of protein throughput. Following high-density fermentation in a 3‑L bioreactor, the optimally engineered strain achieved an rhCSF2 titer of 3.4 g/L—markedly exceeding the highest titer previously reported in yeast hosts by an order of magnitude. Importantly, this engineering strategy was successfully extended to a glycoengineered strain SuperMan5, where it enabled production of rhCSF2 with more homogeneous and humanized N ‑glycans and a titer of 4.3 g/L under high‑density fermentation condition. Our work demonstrates that the high-yield production of recombinant human therapeutic glycoproteins bearing homogeneous, humanized N -glycans in engineered yeast offers promising prospects. Download: Download high-res image (169KB) Download: Download full-size image