Efficient Secretory Expression and Purification of Food-Grade Porcine Myoglobin in Komagataella phaffii

Abstract

Myoglobin (MG) is one of the eukaryotic heme-binding proteins that is closely associated with the real color and metallic taste of meat and can be used as a color additive in artificial meat alternatives. However, the traditional extraction methods are expensive and time-consuming and the heterologous biosynthesis of MG has never been reported. Herein, we achieved the secretory expression of porcine MG by engineered Komagataella phaffii using the suitable host (X33), signal peptide (α-factor signal peptide), and modified constitutive promoter (G1 promoter). In addition, the fermentation conditions for MG production were optimized at shaking-flask level (BMGY medium with 40 mg/L of hemin, 30 °C) and at fermenter level (30% DO, feeding 150 mg/L of hemin), resulting in the highest titer of 285.42 mg/L MG in fed-batch fermentations. Furthermore, a purification method for food-grade MG was developed, which can obtain 0.22 mol of heme/mol of MG with 88.0% purity and 66.1% recovery rate.

Whole‐Cell P450 Biocatalysis Using Engineered Escherichia coli with Fine‐Tuned Heme Biosynthesis

“…In addition to P450s, the insufficient supply of heme is a common bottleneck for synthesizing other active hemoproteins because heme plays vital roles in their folding, assembly, and function. [51] These hemoproteins have been widely applied in the fields of food and medicine, including food processing (soy hemoglobin and porcine myoglobin as coloring and flavoring agents), [52,53] food preservation (lactoperoxidase as a bioavailable bacteriostatic agent), [54] emergency medicine (human hemoglobin as an acellular oxygen carrier), [55] and medical diagnosis (horseradish peroxidase as a chemiluminescent sensor). [56] Importantly, hemoglobin and myoglobin have become indispensable components in plant-based and cell-based alternative meats.…”

Whole-cell P450 biocatalysts using engineered Escherichia coli with fine-tuned heme supply

“…Aside from P450s, the insu cient supply of heme is a common bottleneck for the synthesis of other active hemoproteins because heme plays vital roles in their folding, assembly, and function 44 . These hemoproteins have been widely applied in the elds of food and medicine, including food processing (soy hemoglobin and porcine myoglobin as coloring and avoring agents) 45,46 , food preservation (lactoperoxidase as a bioavailable bacteriostatic agent) 47 , emergency medicine (human hemoglobin as an acellular oxygen carrier) 48 , and medical diagnosis (horseradish peroxidase as a chemiluminescent sensor) 49 . Especially, hemoglobin and myoglobin have become the indispensable components in plantbased and cell-based arti cial meat alternatives 50 .…”

1Progress, applications, challenges and prospects of protein purification technology

“…Methanol is flammable and toxic, which is detrimental to cell growth (Santoso et al, 2012) It can express membrane proteins very well (Bill and Hedfalk, 2021). The production of anticancer drug (Liu et al, 2015), viral surface antigens (Gupta et al, 2020), antiviral proteins, important enzymes as well as viral subunit recombinant protein vaccines (Zhang et al, 2015;Chen et al, 2017), synthetic food-grade proteins and food additives (Bhataya et al, 2009;Zhang et al, 2021a)…”