Characterizing the role of an endogenous serine protease KpSub2 in recombinant collagen degradation in Komagataella phaffii

Abstract

The yeast Komagataella phaffii is an emerging microbial host for the production of functional recombinant proteins. However, proteolytic degradation during fermentation often compromises product yield and stability, posing a major hurdle for industrial-scale applications. This study presents a strategy to enhance the production of recombinant humanized type I collagen (rhColI) by engineering a host strain with reduced protease activity. An initial production strain, CL1, was engineered using post-transformational vector amplification (PTVA), achieving a titer of 558.86 ± 20.05 mg/L in flask culture. Subsequent scale-up fermentation, however, revealed significant rhColI degradation. To address this, we systematically deleted 11 candidate endogenous protease genes. The knockout of a serine protease gene, KpSub2, resulted in the most pronounced improvement, elevating the rhColI titer to 1039.06 ± 34.08 mg/L, a 23.47% increase over the parent strain CL1. Furthermore, the purified KpSub2 protein, obtained from inclusion bodies in Escherichia coli, demonstrated broad proteolytic activity against various types of recombinant humanized collagens. This broad substrate specificity was consistent with the observation that KpSub2 deletion also mitigated the degradation of recombinant humanized type III collagen (rhColIII). Our findings establish KpSub2 as a key mediator of collagen degradation in K. phaffii and provide an effective engineering strategy for optimizing the production of collagen and other degradation-susceptible functional proteins in this host.

Graphical abstract

Keywords

Recombinant humanized collagen / Serine protease / Proteolytic degradation / Komagataella phaffii

References

[1] Carmona-Gutiérrez D, Bauer MA, Ring Jet al. . The propeptide of yeast cathepsin D inhibits programmed necrosis. Cell Death Dis. 2011, 2: e161-e161.

[2] Cheng J-H, Zhang X-Y, Wang Zet al. . Potential of thermolysin-like protease A69 in preparation of bovine collagen peptides with moisture-retention ability and antioxidative activity. Mar Drugs. 2021, 19: 676.

[3] Cheng J-H, Zhao W-X, Cao H-Yet al. . Mechanistic insight into the production of collagen oligopeptides by the S8 family protease A4095. J Agric Food Chem. 2023, 71603-614.

[4] Daly R, Hearn MT. Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit: Interdiscip J. 2005, 18: 119-138.

[5] Fidler AL, Boudko SP, Rokas A, Hudson BG. The triple helix of collagens—an ancient protein structure that enabled animal multicellularity and tissue evolution. J Cell Sci. 2018, 131: jcs203950.

[6] Gutierrez-Canul CD, Can-Herrera LA, de Ramírez-Rivera Eet al. . A review of classical and rising approaches the extraction and utilization of marine collagen. BioTech. 2025, 1426.

[7] Hu J, Yu L, Shu Q, Chen Q. Identification of down-regulated proteome in Saccharomyces cerevisiae with the deletion of yeast cathepsin D in response to nitrogen stress. Microorganisms. 2019, 7214.

[8] Jiao L, Zhou Q, Liu W, Yan Y. New insight into the method of posttransformational vector amplification (PTVA) in Pichia pastoris. J Microbiol Methods. 2018.

[9] Kurt I, Kulhan M, AlAshqar A, Borahay MA. Uterine collagen cross-linking: biology, role in disorders, and therapeutic implications. Reproductive Sci. 2024, 31645-660.

[10] Liu Q, Shi X, Song Let al. . CRISPR–Cas9-mediated genomic multiloci integration in Pichia pastoris. Microb Cell Fact. 2019, 18: 1-11.

[11] Ma X, Liu Y, Li Qet al. . Expression, purification and identification of a thermolysin-like protease, neutral protease I, from Aspergillus oryzae with the Pichia pastoris expression system. Protein Exp Purif. 2016, 128: 52-59.

[12] Majeed T, Lee CC, Orts WJet al. . Characterization of a thermostable protease from Bacillus subtilis BSP strain. BMC Biotechnol. 2024, 24: 49.

[13] Myers LK, Myllyharju J, Nokelainen Met al. . Relevance of posttranslational modifications for the arthritogenicity of type II collagen. J Immunol. 2004, 1722970-2975.

[14] O’Donoghue AJ, Knudsen GM, Beekman Cet al. . Destructin-1 is a collagen-degrading endopeptidase secreted by Pseudogymnoascus destructans, the causative agent of white-nose syndrome. Proc Natl Acad Sci USA. 2015, 112: 7478-7483.

[15] Patel A, Singh N, Mehta A, Shah R. Production and characterization of neutral and alkaline protease from different Bacillus subtilis strains. J Microb Biochem Technol. 2023, 15: 172-181.

[16] Pathak J, Singh SP, Pathak Met al. . Enhanced post-thaw functional characteristics of cryopreserved caprine dermal fibroblasts through adhesion to extracellular matrix proteins. Mol Biol Rep. 2025, 52: 116.

[17] Ran L-Y, Su H-N, Zhao G-Yet al. . Structural and mechanistic insights into collagen degradation by a bacterial collagenolytic serine protease in the subtilisin family. Mol Microbiol. 2013, 90997-1010.

[18] Ran LY, Su HN, Zhou MYet al. . Characterization of a novel subtilisin-like protease myroicolsin from deep sea bacterium Myroides profundi D25 and molecular insight into its collagenolytic mechanism. J Biol Chem. 2014, 289: 6041-6053.

[19] Raschmanová H, Weninger A, Knejzlík Zet al. . Engineering of the unfolded protein response pathway in Pichia pastoris: enhancing production of secreted recombinant proteins. Appl Microbiol Biotechnol. 2021, 105: 4397-4414.

[20] Raza A, Pothula R, Abdelgaffar Het al. . Identification and functional characterization of a β-glucosidase from Bacillus tequelensis BD69 expressed in bacterial and yeast heterologous systems. PeerJ. 2020, 8: e8792.

[21] Reichard U, Cole G, Rüchel R, Monod M. Molecular cloning and targeted deletion of PEP2 which encodes a novel aspartic proteinase from Aspergillus fumigatus. Int J Med Microbiol. 2000, 29085-96.

[22] Salamin K, Sriranganadane D, Lechenne Bet al. . Secretion of an endogenous subtilisin by Pichia pastoris strains GS115 and KM71. Appl Environ Microbiol. 2010, 76: 4269-4276.

[23] Sazonova EA, Zobnina AE, Padkina MV. Effect of disruption of Pichia pastoris YPS1 gene on viability and production of recombinant proteins. Russian J Genet. 2013, 49: 602-608.

[24] Scorer CA, Buckholz RG, Clare JJ, Romanes MA. The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris. Gene. 1993, 136: 111-119.

[25] Shemesh P, Fishman A. Optimal fermentation conditions for growth and recombinant protein production in Pichia pastoris: strain selection, ploidy level and carbon source. Curr Res Food Sci. 2024, 9: 100840.

[26] Song X, Chu T, Shi W, He J. Expression, characterization, and application of human-like recombinant gelatin. Bioresources Bioprocess. 2024, 111-13.

[27] Sorushanova A, Delgado LM, Wu Zet al. . The collagen suprafamily: from biosynthesis to advanced biomaterial development. Adv Mater. 2019, 31: 1801651.

[28] Tarallo M, Hayhurst M, Loo TS et al (2024) Heterologous expression of secreted proteins from phytophthora in Pichia pastoris followed by protein purification by immobilized metal ion affinity (IMAC). In: Phytophthora: methods and protocols. Springer, pp 233–248. https://doi.org/10.1007/978-1-0716-4330-3_16

[29] Vidal-Montiel A, Clark-Flores D, Valentín-Gómez Eet al. . The GPI-anchored aspartyl proteases encoded by the YPS1 and YPS7 Genes of Candidozyma auris and their role under stress conditions. J Fungi. 2025, 11573.

[30] Vijayakumar VE, Venkataraman K. A systematic review of the potential of Pichia pastoris (Komagataella phaffii) as an alternative host for biologics production. Mol Biotechnol. 2024, 661497.

[31] Wang X, Wang P, Li Wet al. . Effect and mechanism of signal peptide and maltose on recombinant type III collagen production in Pichia pastoris. Appl Microbiol Biotechnol. 2023, 1074369-4380.

[32] Wang K, Yu S, Sun Ret al. . Biosynthesis of a functional fragment of human collagen II in Pichia pastoris. ACS Synth Biol. 2024, 13: 2567-2576.

[33] Wang J, Xiang Z-X, Luan M-Fet al. . High-level secretory expression of recombinant type III human-like collagen α1 in Pichia pastoris via multilevel systematic optimization. Int J Biol Macromol. 2025.

[34] Werten MW, de Wolf FA. Reduced proteolysis of secreted gelatin and Yps1-mediated α-factor leader processing in a Pichia pastoris kex2 disruptant. Appl Environ Microbiol. 2005, 71: 2310-2317.

[35] Wu M, Shen Q, Yang Yet al. . Disruption of YPS1 and PEP4 genes reduces proteolytic degradation of secreted HSA/PTH in Pichia pastoris GS115. J Ind Microbiol Biotechnol. 2013, 40: 589-599.

[36] Yan W, Huang C, Yan Yet al. . Expression, characterization and antivascular activity of amino acid sequence repeating collagen hexadecapeptide. Int J Biol Macromol. 2024, 260: 131886.

[37] Zhang Y, Liu R, Wu X. The proteolytic systems and heterologous proteins degradation in the methylotrophic yeast Pichia pastoris. Ann Microbiol. 2007, 57553.

[38] Zhao Z, Deng J, Fan D. Green biomanufacturing in recombinant collagen biosynthesis: trends and selection in various expression systems. Biomater Sci. 2023, 115439-5461.

Funding

National Key Research and Development Program of China (CN)(2022YFC2403105)

RIGHTS & PERMISSIONS

The Author(s)