[Enhancing heterologous expression of bovine casein macropeptide via promoter engineering]

Bovine casein macropeptide (bCMP) is a glycosylated polypeptide generated by the chymosin hydrolysis of κ-casein. It exhibits various bioactivities, including antioxidant, anti-inflammatory, and gut microbiota-regulating effects. This study established a heterologous expression system for bCMP in Pichia pastoris and systematically optimized the promoter, signal peptide, and fermentation conditions to enhance its expression level. Initially, on the basis of the successful construction and expression verification of the bCMP-expressing strain, a fusion expression system of bCMP with green fluorescent protein (GFP) was developed, enabling real-time monitoring of target protein expression via fluorescence intensity. After identifying the optimal promoter pAOX1, the effects of the signal peptides α-factor, SCW, PHO1, SUC, and Cel5A on the secretion level of the fusion protein bCMP-GFP were analyzed and compared, with α-factor identified as the most effective signal peptide for promoting bCMP secretion. Furthermore, a synthetic promoter mutant library of pAOX1 was generated via error-prone PCR. After fluorescence-activated cell sorting (FACS), six mutants (pAOX1-1 to pAOX1-6) were isolated, which exhibited increases of 59%-160% in expression strength compared with the wild type. Finally, the fermentation parameters were optimized as 1.5% methanol, pH 7.5, initial OD₆₀₀ of 4.0, and 26 ℃, which led to a 240% increase in expression compared with the initial conditions. Ultimately, following the integration of the aforementioned optimization strategies, the production titer of bCMP by P. pastoris reached 1.02 g/L. This study successfully developed and optimized a heterologous expression system for bCMP in P. pastoris, yielding novel pAOX1 promoter variants with application potential and providing effective strategies and key regulatory elements for the efficient heterologous expression of other mammalian-derived functional proteins in P. pastoris.