Efficient extracellular expression and rapid screening of human-like recombinant gelatin in Komagataella phaffii

To obtain a strain that efficiently secretes and expresses hlrGEL (recombinant human-like gelatin), and to establish a rapid method for detecting this expression, this study began with the original strain GS115/pPICZα-gel6 (4# clone). High-copy transformants were obtained through Zeocin resistance screening, and positive expression strains were rapidly screened using single-cell laser Raman spectroscopy (SCLRS) technology. The results revealed that the Raman spectral features of positive expressing strains were located at 1447 cm^{- 1}, 1658 cm^{- 1}, and 2929 ~ 2943 cm^{- 1}, and their peak intensities were correlated with the expression level of hlrGEL while being consistent with SDS-PAGE, Western blotting and BCA methods analysis results. Additionally, real-time fluorescence quantitative PCR (qPCR) was employed to detect the copy numbers of gel6 in positive transformants subjected to various antibiotic treatments. The results demonstrated that gel6 copy numbers were 4.29 × 10³, 5.66 × 10³, 6.01 × 10³, and 6.29 × 10³/DNA (ng), corresponding to protein expression levels of 0.21, 0.31, 0.36, and 0.19 mg/mL, respectively. The research shows that as a non-invasive detection method, SCLRS can quickly screen positive transformants. Combined with biological experiments, highly expressing strains can be efficiently obtained.