Reprogrammed Komagataella phaffii for enhanced secretory expression of human lactoferrin

Area of Science:

• Biotechnology
• Microbial Engineering
• Protein Expression

Background:

• Human lactoferrin (hLF) is a vital glycoprotein with antibacterial and immunomodulatory properties, crucial for infant nutrition.
• Current methods for hLF production face challenges in protein synthesis efficiency and host stability.
• Microbial hosts often struggle with the proper folding and secretion of complex glycoproteins like hLF.

Purpose of the Study:

• To develop a robust yeast expression system for high-yield secretory production of human lactoferrin (hLF).
• To enhance hLF production by optimizing the host cell's protein processing machinery and stability.
• To validate the functional activity of the produced hLF.

Main Methods:

• Engineered a Komagataella phaffii yeast strain using CRISPR/Cas9 for endoplasmic reticulum (ER) and vacuole reprogramming.
• Integrated a dual-expression cassette for hLF under the AOX1 promoter into multiple genomic sites.
• Implemented strategies including ER expansion, protease knockout, and FeCl₃ supplementation for enhanced production.

Main Results:

• Achieved a secretory intact hLF titer of 2214mg/L, a 76.3-fold increase over baseline.
• Optimized cultivation conditions in shake flasks and a 5L bioreactor for high-density growth.
• Demonstrated significant antibacterial activity of the produced hLF against Escherichia coli, Staphylococcus aureus, and yeast.

Conclusions:

• The engineered K. phaffii platform enables highly efficient secretory production of human lactoferrin.
• This system shows potential for the cost-effective manufacturing of lactoferrin and other lactoproteins.
• The developed strain and methods overcome previous limitations in heterologous protein expression in yeast.