Engineered Folding and Secretion for Expression Improvement of α‐Amylase in <i>Komagataella Phaffii</i>

ABSTRACT α‐Amylase is the second most widely produced enzyme globally, with diverse applications in the fields of food, pharmaceutical, bioenergy, papermaking, etc. However, natural α‐amylase often fails to withstand the extreme conditions encountered in industrial processes, such as low pH and high temperatures. Previous studies identified an α‐amylase derived from deep‐sea sources with resistance to low pH, and subsequent amino acid mutations well enhanced its thermal stability. Nevertheless, the advantageous enzyme mutant exhibited low expression levels in Escherichia coli , highlighting the need for a more suitable expression host. In this study, an engineered industrial host, Komagataella phaffii , was involved for heterologous production of α‐amylase. High‐efficiency signal peptides were screened and multi‐copy integrant strains were constructed to achieve a high‐yield strain. A total of 31 key chaperones and 11 vesicle transport factors were further investigated to facilitate protein folding and secretion, which resulted in a 3.4‐fold increase in α‐amylase production. Finally, batch fermentation in a 3‐L bioreactor achieved a maximum α‐amylase activity of 2.5 × 10 4 U/mL. This study demonstrates the development of a high‐yield α‐amylase strain for potential industrial applications, offering valuable insights and strategies for engineering high‐producing strains of other industrial enzymes.