Structural Homology Fails to Predict Secretion Efficiency in Pichia pastoris: Divergent Responses of Architecturally Similar scFvs to Multi-Parametric Genetic Engineering

Abstract

AI-driven biologics manufacturing demands an efficient protein production platform. In this study, we optimized scFv secretion in Pichia pastoris through three strategies: gene dosage optimization, expression cassette design, and endoplasmic reticulum (ER) secretory pathway reprogramming. Using two structurally homologous scFv variants-PR961 and PR953-with divergent basal secretion levels (12.35:1 ratio), we demonstrate that protein-specific thresholds govern optimization efficacy. While increasing gene copy numbers yielded limited improvements (PR961: 1.25-fold at four copies; PR953: 2.37-fold at six copies), reconfiguring the expression cassette to a V_H-linker-V_L orientation significantly enhanced secretion (11.18-fold for PR961; 5.09-fold for PR953). Twenty-one genes in three functional modules of the secretory pathway were knocked out or overexpressed. The pathway reprogramming results revealed distinct regulatory dependencies: PR961 secretion relied on ER-to-Golgi trafficking (SEC23 overexpression: 1.20-fold), whereas PR953 depended more on upstream translocation (SEC62: 1.66-fold) and oxidative folding (ERO1: 1.81-fold) enhancements. Notably, both variants exhibited a glycosylation-dependent regulation through CNE1. Our findings challenge the assumption that structural homology (63% amino acid identity; RMSD 0.47 Å) ensures consistent optimization outcomes, highlighting the imperative for protein-tailored engineering strategies in synthetic biology.

Keywords: Pichia pastoris; expression cassette design; gene dosage optimization; scFv; secretory pathway engineering.

Figure 1

Characteristic comparison of PR961 and PR953. (A) Amino acid sequences of the two scFvs were compared using ESPript 3.0. Red boxes highlight homologous regions, and arrows indicate the conserved cysteine positions. (B) Basic information for PR961 and PR953, including potential N- and O-glycosylation sites, was predicted using the DTU Health Tech website. (C) Overlay of protein crystal structures of PR961 (yellow, PDB ID: 7DET) and PR953 (orange, PDB ID: 7DEU) obtained from RCSB PDB. (D) Growth curves of PR961 and PR953-harboring yeast strains incubated for 24 h in MGY medium. (E) SDS-PAGE analysis of PR961 and PR953 (V_H-linker-V_L, 2 copies) secretion in P. pastoris. (F) PR961 and PR953 secretion levels were quantified in three independent experiments using ImageJ 1.53a gel analysis software. Statistical significance is denoted as p < 0.0001 (****).

Figure 2

Secretion of PR961 and PR953 in their respective recombinant strains with varying gene dosages. (A) Growth curves of PR961 and PR953 strains with different copy numbers incubated for 24 h in MGY medium. (B) SDS-PAGE analysis of PR961 and PR953 with different copy numbers. (C) PR961 and PR953 secretion levels were quantified in three independent experiments using ImageJ 1.53a gel analysis software. Statistical significance levels are denoted as follows: p < 0.05 (*) and p < 0.001 (***).

Figure 3

Secretion of PR961 and PR953 in their respective recombinant strains carrying constructs with both V_H-linker-V_L and V_L-linker-V_H orientations. (A) Schematic representation of V_H-linker-V_L and V_L-linker-V_H construction orientations for both PR961 and PR953. (B) Growth curves of PR961 and PR953 strains transformed with V_H-linker-V_L and V_L-linker-V_H constructs incubated for 24 h in MGY medium. (C) SDS-PAGE analysis of PR961 and PR953 in their respective expression strains, carrying constructs with different scFv domain orientations. (D) Secretion levels of PR961 and PR953 were quantified using ImageJ 1.53a software, and statistical significance was determined based on three independent experiments. Statistical significance is denoted as p < 0.0001 (****).

Figure 4

Schematic illustration of the three ER functional modules and the twenty-one specific genes targeted within these modules in this study. The illustration was generated using the BioRender mapping website (https://www.biorender.com/, accessed on 11 March 2025).

Figure 5

Effects on PR961 and PR953 secretion by disrupting ER-related genes. (A) Growth curves of nine distinct single-gene knockout strains of PR961 and PR953 incubated for 24 h in MGY medium. (B) SDS-PAGE analysis of PR961 and PR953 secretion levels. The gels were then stained with SYPRO dye. (C) Quantification of PR961 and PR953 secretion using ImageJ 1.53a software. Significant differences were determined from three independent experiments. (D) Heat map of the fold changes in PR961 and PR953 secretion relative to their respective controls. Statistical significance levels are denoted as follows: p < 0.05 (*), p < 0.001 (***), and p < 0.0001 (****).

Figure 6

Effects on PR961 and PR953 secretion by overexpressing ER-related genes. (A) Growth curves of twenty-one distinct single-gene overexpression strains of PR961 and PR953 incubated 24 h in MGY medium. (B) SDS-PAGE analysis of PR961 and PR953 secretion levels. The gels were stained using the SYPRO dye method. (C) Quantification of PR961 and PR953 secretion using the ImageJ 1.53a software. Significant differences were determined from three independent experiments. (D) Heat map of the fold changes in PR961 and PR953 secretion relative to their respective controls. Statistical significance levels are denoted as follows: p < 0.05 (*), p < 0.01 (**), and p < 0.0001 (****).