Enhancing heme import to synthesize active myoglobin and hemoglobin in Pichia pastoris.

Myoglobin and hemoglobin are both heme-binding proteins that have widespread applications in food processing and medical treatments. Pichia pastoris is often chosen to produce heterologous hemoproteins. However, due to the insufficient heme supply, the inactive hemoproteins take a large proportion of synthesized products. To overcome this problem, the intracellular heme supply was increased by improving heme uptake efficiency. At first, an efficient method was created for the screen of potential heme importers from various microorganisms by knocking out HEM1 gene to obtain a deficient P. pastoris strain X33-ΔKu70-ΔHEM1 (HEMEX) in heme synthesis. Based on the significantly improved cell growth, two effective heterogenous heme importers (Caflc1 and Shu1) were selected when the exogenous heme was supplemented. Finally, the titers of porcine myoglobin (PMB) and soybean hemoglobin (SHB) increased by 112.28% and 5.78-fold, respectively, through the overexpression of heme importers. The applied strategy provides a promising approach to synthesize other valuable hemoproteins in P. pastoris. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-025-04286-6.