A Growth-Coupled Evolutionary Strategy Enhances Heme Biosynthesis in Saccharomyces cerevisiae

Enhancing the nutritional and sensory qualities of microbial single-cell proteins (SCPs) requires strategies to increase heme content in edible microorganisms. We adapted the Growth-Acceleration Targeting Evolution (GATE) platform, initially developed in Corynebacterium glutamicum, for use in Saccharomyces cerevisiae. By engineering a plasmid that connects the heme-responsive CYC1 promoter to the growth-promoting PTH1 gene, we established a feedback loop that links intracellular heme levels to accelerated cell proliferation. After 100 h of continuous culture under growth-selective pressure, we cured out the plasmid to isolate an Evol-GATE strain. Compared to the parental type, Evol-GATE displayed a five-fold increase in intracellular heme, a slight reduction in biomass, and coordinated upregulation of the heme biosynthetic pathway. Transcriptome analysis confirmed increased expression of heme biosynthesis and associated respiratory genes in Evol-GATE. Whole-genome sequencing revealed only a small number of dispersed variants, and no residual plasmid sequences, supporting its classification as a non-GMO mutant. Our results demonstrate that GATE can effectively select yeast mutants with significantly improved heme productivity, providing a promising approach to develop non-GMO SCPs enriched in heme for next-generation meat analogues.