Systematic Engineering of <i>Komagataella phaffii</i> for Efficient Kestose Biosynthesis: Integrating Expression Optimization with Secretory Pathway Engineering

was engineered for enhanced secretion of β-Ffase as an efficient biocatalyst. Initial optimization of transcriptional elements increased extracellular enzyme titer by 8.1-fold, but significant intracellular accumulation persisted. Targeted engineering of the secretory pathway, including modulation of endoplasmic reticulum-associated degradation (ERAD) and vesicle trafficking, further improved secretion efficiency. The highest-yielding strain, BA2-2, achieved a 15.2-fold increase in extracellular activity (80.3 ± 5.6 U/mL) and reduced intracellular retention to 12.3% in shake-flask cultures. Application of highly secreted β-Ffase from this strain in a one-pot bioconversion strategy enabled sucrose conversion to 248.9 ± 20.6 g/L kestose with a productivity of 4.08 ± 0.28 g/L/h. Collectively, these results validate a systematic engineering strategy that combines expression optimization with secretory pathway engineering, enabling the development of an efficient microbial platform for industrial kestose production.