Production and Purification of Soy Leghemoglobin from Pichia Pastoris Cultivated in Different Expression Media

Plant-based meat alternatives, exemplified by Impossible Foods’ Impossible Burger, offer a sustainable, ethical substitute for traditional meat, closely mimicking the taste and appearance of meat by utilizing soy leghemoglobin (LegH), a 16 kDa holoprotein found in soy plants structurally similar to heme in animal meat. Cultivation medium plays an important role in bioprocess development; however, medium development or optimization can be labor intensive, and thus the use of previously reported media can be enticing. In this study, we explored the expression of recombinant LegH in Pichia pastoris in various reported cultivation media (BSM, BMGY, FM22, D’Anjou, BSM/2, and RDM) and using different feeding approaches (µ-stat and mixed feed with sorbitol). Our findings indicate that optimization techniques tailored to the specific process did not increase LegH yields, highlighting the need to investigate strain-specific strategies. We also utilized the collected process data to create and train a novel artificial neural network-based soft sensor for estimating cell biomass, relying solely on standard bioreactor measurements (such as stirrer speed, dissolved oxygen, O2 enrichment, base feed, glycerol feed, methanol feed, and reactor volume). This soft sensor proved to be robust and exhibited a strong correlation (3.72% WCW) with experimental data.