Production of Recombinant Hemoglobin in Tremella fuciformis Yeast-like Cells Through Selection of Hemoglobins and Secretion Elements

Hemoglobins have attracted increasing interest for their potential applications in alternative protein foods and food biotechnology, but the use of fungal yeast-like cells for recombinant hemoglobin production remains underexplored. In this study, Tremella fuciformis yeast-like cells (YLCs) were evaluated as a potential host system by screening candidate hemoglobins and secretion elements. Clover hemoglobin from Trifolium subterraneum (CHb), Vitreoscilla hemoglobin (VHb), and Vigna unguiculata leghemoglobin 2 (Lb2) were heterologously expressed in T. fuciformis YLCs. The estimated yields of CHb, VHb, and Lb2 were 32.00, 19.03, and 23.51 mg/L, respectively, with CHb showing the highest observed yield among the three tested hemoglobins. CHb was then used to evaluate the CysR signal peptide, α-mating factor prepro leader (Amf), and glucoamylase signal peptide (Gla). All three elements mediated CHb secretion, with Amf producing the highest extracellular CHb yield of 106.43 mg/L and total CHb production of 133.02 mg/L, approximately 4.16 times that of CHb without a secretion element. CysR and Gla produced extracellular CHb yields of 72.54 and 49.50 mg/L, respectively. These results identify CHb and Amf as a promising expression combination in T. fuciformis YLCs, but direct characterization of heme incorporation and functional properties is required before food applications can be evaluated.