Efficient Bioproduction of Human Milk Alpha-Lactalbumin in <i>Komagataella phaffii</i>

Abstract

Alpha-lactalbumin (α-LA; the most abundant whey protein in human milk) contributes to infant development, providing bioactive peptides and essential amino acids. Here, <i>Komagataella phaffii</i> (<i>K. phaffii</i>) was selected as the production host. We found that the <i>K. phaffii</i> host X33 was suitable for expressing the target protein, yielding 5.2 mg·L^-1 α-LA. Thereafter, several secretory signal peptides were applied to obtain a higher titer of α-LA. The strain with α-factor secretory signal peptide secreted the highest extracellular titer. Additionally, promoters <i>AOX1</i>, <i>GAP</i>, and <i>GAP(m)</i> were compared and applied. The strain with the promoter <i>AOX1</i> produced the highest extracellular titer. In addition, coexpressing human protein disulfide isomerase A3 (h<i>PDIA3</i>) increased the titer by 27%. Human α-LA production by the strain X33-pPICZαA-hLALBA-hPDIA3 reached 56.3 mg·L^-1 in a 3 L bioreactor. This is the first report of successful secretory human α-LA expression in <i>K. phaffii</i> and lays foundations for the simulation of human milk for infant formulas and further development of bioengineered milk.