Multistep engineering of the secretory pathway for enhanced leghemoglobin expression in Kluyveromyces marxianus

Leghemoglobin (LegH) is a heme-binding protein and a key ingredient in plant-based meat products, as it imparts the characteristic color and flavor of animal meat. However, achieving high-level secretory expression of LegH in food-grade microbial hosts remains challenging. Here, we applied multistep engineering of the secretory pathway in Kluyveromyces marxianus, a food-safe yeast, to enhance secretion of leghemoglobin A (LBA). A chimeric signal peptide combining the Ost1 signal peptide with the α-factor pro-region enabled efficient secretion of LBA. Further improvements were achieved by disrupting late-stage N-glycosylation genes (MNS1 or MNL1), overexpressing COPII vesicle assembly genes (SEC23 or SAR1), cargo receptor genes (ERV29 or EMP47) and transport factor gene (BET1), and deleting the vacuolar protease gene PRB1, while individual modifications increased LBA secretion by 16%-74%. Combining key modifications (mns1Δ, prb1Δ, and SEC23, SAR1 overexpression) with attenuation of the heme-degrading enzyme Hmx1 culminated in a secretory titer of 1.02 g/L LBA in a 5-L bioreactor, representing a 2.3-fold increase over the parental strain. The secreted LBA preserved heme-binding capacity (73.1% ratio), oxygen-binding spectral features, and peroxidase activity (956.77 U mg^-1), confirming proper folding and functionality. Our findings establish K. marxianus as an efficient platform for producing functional, food-safe LegH, with broad implications for meat-analog development and medical applications.