Abstract
Microbial synthesis of valuable hemoproteins has become a popular research topic, and Pichia pastoris is a versatile platform for the industrial production of recombinant proteins. However, the inadequate supply of heme limits the synthesis of high-active hemoproteins. Here a strategy for enhancing intracellular heme biosynthesis to improve the titers and functional activities of hemoproteins is reported. After selecting a suitable expressional strategy for globins, the efficient heme-supply P. pastoris chassis is established by removing the spatial segregation during heme biosynthesis, optimizing precursor synthesis, assembling rate-limiting enzymes using protein scaffolds, and inhibiting heme degradation. This robust chassis produces several highly active hemoproteins, including porcine myoglobin, soy hemoglobin, Vitreoscilla hemoglobin, and P450-BM3, which can be used in the development of artificial meat, high-cell-density fermentation, and whole-cell catalytic synthesis of high-value-added compounds. Furthermore, the engineered chassis strain has great potential for producing and applying other hemoproteins with high activities in various fields.