Systematic Engineering of Komagataella phaffii for Efficient Kestose Biosynthesis: Integrating Expression...

Area of Science:

• Biotechnology and Synthetic Biology
• Enzyme Engineering
• Microbial Fermentation

Background:

• Kestose, the smallest fructooligosaccharide (FOS), offers superior bioactivity and sweetness.
• Enzymatic synthesis of kestose requires β-fructofuranosidase (β-Ffase), but its efficient heterologous production is difficult.
• Current methods face challenges in achieving high extracellular enzyme titers for industrial applications.

Purpose of the Study:

• To engineer Komagataella phaffii for enhanced secretion of β-Ffase.
• To develop an efficient microbial platform for industrial kestose production.
• To optimize enzyme secretion and bioconversion processes.

Main Methods:

• Genetic engineering of Komagataella phaffii to improve β-Ffase secretion.
• Optimization of transcriptional elements and secretory pathway components.
• Shake-flask cultivation and one-pot bioconversion assays.

Main Results:

• Achieved an 8.1-fold increase in extracellular β-Ffase activity through initial optimization.
• Further improved secretion efficiency by modulating ERAD and vesicle trafficking pathways.
• Developed strain BA2-2 with a 15.2-fold increase in extracellular activity (80.3 U/mL) and reduced intracellular retention to 12.3%.
• Enabled sucrose conversion to 248.9 g/L kestose with a productivity of 4.08 g/L/h using the engineered strain.

Conclusions:

• A systematic engineering strategy combining expression and secretory pathway optimization is effective.
• The engineered yeast provides an efficient microbial platform for industrial kestose production.
• This work facilitates the scalable and cost-effective synthesis of valuable fructooligosaccharides.