Efficient biosynthesis of active hemoglobins through enhancing the import of heme in Saccharomyces cerevisiae

Hemoglobins, with heme as a cofactor, are functional proteins that have extensive applications in the fields of artificial oxygen carriers and foods. Although Saccharomyces cerevisiae is an ideal host for hemoglobin synthesis, it lacks a suitable transport system to utilize additional heme for active expression of hemoglobins, resulting in the cellular aggregation and degradation of the latter. Here, an effective heme importer, heme‐responsive gene 4 (Hrg‐4), was selected from six candidates through the comparison of effects on the growth rates of Δhem1 S. cerevisiae strain and the activities of various hemoglobins when supplemented with 5 mg·L−1 exogenous heme. Additionally, to counter the instability of plasmid‐based expression and the metabolic burden introduced from overexpressing Hrg‐4, a series of hrg‐4 integrated strains were constructed and the best engineered strain with five copies of hrg‐4 was chosen. We found that this engineered strain was associated with an increased binding rate of heme in monomeric leghemoglobin and multimeric human hemoglobin (76.3% and 16.5%, respectively), as well as an enhanced expression of both hemoglobins (52.8% and 17.0%, respectively). Thus, the engineered strain with improved heme uptake can be used to efficiently synthesize other heme‐binding proteins and enzymes in S. cerevisiae.