Fig. 4: A. oryzae mycelium bioengineered for ergothioneine and heme content.

A Fungal biosynthesis of ergothioneine, a powerful antioxidant associated with several health benefits in humans. The characterized biosynthetic pathway from the fungus Neurospora crassa involves the enzymes Egt1 and Egt2. B A. oryzae homologs of N. crassa Egt1 and Egt2 were identified bioinformatically (see Supplementary Table 7 and Supplementary Fig. 12 for details) and expressed from neutral loci using a bidirectional promoter (strain VMR-Eg1-2) or as two separate genes at two different genomic locations, with each gene under the control of its own promoter (strain VMR-Eg1_2). The strategy is described in Supplementary Fig. 12. Oyster mushroom, the dietary mushroom with the highest ergothioneine content, was included for comparison. Biomass was analyzed by LC–MS. Results are average and SEM of three biological replicates. C Engineering of heme biosynthesis in A. oryzae biomass. The strategy is described in Supplementary Fig. 15. Heme was quantified using LC–MS in the biomass. The intracellular heme levels in the engineered strain were 4-fold higher than in the background strain, RIB40, and 40% of those found in IMPOSSIBLE™ burger made from plants, a leading plant-based meat product incorporating heme for flavor and color, was included for comparison. Results are average and SEM of three biological replicates. D Color of harvested background and engineered heme strain after culturing. The engineered strain overproducing heme (VMR-HEM_v1) was distinctly red in color, while RIB40 was off-white. The harvested fungal biomass could be readily formulated into an imitation meat patty with minimal processing. The color difference remained upon cooking, further enhancing the meat-like appearance of the naturally textured fibrous biomass.