Cytokinetic engineering enhances the secretory production of recombinant human lysozyme in Komagataella phaffii

Abstract

Background: Human lysozyme (hLYZ) is a natural antibacterial protein with broad applications in food and pharmaceutical industries. Recombinant production of hLYZ in Komagataella phaffii (K. phaffii) has attracted considerable attention, but there are very limited strategies for its hyper-production in yeast.

Results: Here through Atmospheric and Room Temperature Plasma (ARTP)-based mutagenesis and transcriptomic analysis, the expression of two genes MYO1 and IQG1 encoding the cytokinesis core proteins was identified downregulated along with higher hLYZ production. Deletion of either gene caused severe cytokinesis defects, but significantly enhanced hLYZ production. The highest hLYZ yield of 1,052,444 ± 23,667 U/mL bioactivity and 4.12 ± 0.11 g/L total protein concentration were obtained after high-density fed-batch fermentation in the Δmyo1 mutant, representing the best production of hLYZ in yeast. Furthermore, O-linked mannose glycans were characterized on this recombinant hLYZ.

Conclusions: Our work suggests that cytokinesis-based morphology engineering is an effective way to enhance the production of hLYZ in K. phaffii.

Keywords: Komagataella phaffii; Cytokinesis; Genetic engineering; Human lysozyme; Secretory production.

Fig. 1

ARTP mutagenesis screening and transcriptomic analysis. (a) Schematic of ARTP mutagenesis, high-yield mutant screening, and analysis workflow. ARTP mutagenesis was applied to the initial strain to generate a mutant library. These mutants underwent a high-throughput bioactivity-guided screening against Micrococcus lysodeikticus on agar plates, followed by a secondary screening in the 48 deep-well plates and hLYZ activity assay. Transcriptomic analysis was conducted to identify potential genes related to the high-yield phenotype. (b) hLYZ bioactivity of the ARTP mutant strain CH2 and the initial strain LYZ-C1. The strain LYZ-C1 served as the control. Data are presented as the means ± standard deviation. ***p < 0.001. (c) Volcano plot of differential expressed genes in the strain CH2 compared to the reference strain LYZ-C1. Red dots, significantly upregulated genes. blue dots, significantly downregulated genes. Light grey dots, genes with no significant differences

Fig. 2

The effect of cytokinetic engineering on hLYZ production. The growth curves (a) and hLYZ bioactivity (b) of strains CH2, Δiqg1-CH2 and Δmyo1-CH2 by shaking flask fermentation. (c, d) Western blotting detection of intracellular hLYZ. Total cell lysates prepared from cells as depicted in Fig. 2b were subjected to Western blotting using an anti-hLYZ antibody, with β-actin used as the loading control. RL, the relative level of intracellular hLYZ. *p < 0.05, **p < 0.01, *** p < 0.001

Fig. 3

Characterization of Δmyo1-CH2 in high-density fermentation. (a) Fed-batch fermentation. The fermentation of the strain Δmyo1-CH2 was conducted in a 5 L bioreactor using BSM medium. The hLZY bioactivity, total protein concentration in the supernatant, and wet cell weight were assessed. (b) SDS-PAGE analysis of the fermentation supernatant. An equal volume of 1.6 µL of the supernatant was sampled, separated using a 15% SDS-PAGE gel, and then stained with Coomassie brilliant blue

Fig. 4

Morphological changes upon deletion of MYO1 and IQG1. (a) Microscopic examination of yeast cell morphology. Chitin was stained with Calcofluor White. BF, Bright field. Scale bar, 10 μm. (b) Flow cytometry analysis. For each quadrant, the relative distribution of cells is indicated in % from total, with quadrant Q4 representing normal shaped cells with one or no bud, and quadrant Q2 representing morphology changed cells with more than one bud. Cells (> 10⁵) were counted in each case