Compartmentalization of heme biosynthetic pathways into yeast mitochondria enhances heme production

Saccharomyces cerevisiae is a generally recognized as safe (GRAS) workhorse strain widely used in the food industry for the cost-effective production of food ingredients. However, the heme production yield in yeast is significantly lower than in bacteria for two main reasons: (1) the heme biosynthetic pathway is bifurcated into the cytosol and mitochondria, and (2) yeast's heme biosynthetic protoporphyrin-dependent (PPD) pathway is thermodynamically unfavorable compared with bacteria's coproporphyrin-dependent (CPD) pathway. To overcome these limitations, the PPD and CPD pathways were compartmentalized into the mitochondria by attaching mitochondria-targeting sequences (MTSs) to the N-terminus of the enzymes. All the enzyme activities required for the CPD pathway are present in S. cerevisiae, except for copro-heme decarboxylase (HemQ); therefore, bacterial HemQ with the N-terminal MTS was introduced to complete the CPD pathway. The resulting S. cerevisiae H4+_MTS9HemQ^Cg strain with mitochondrial PPD and CPD pathways showed 65% higher heme concentration than the engineered strain with only the mitochondrial PPD pathway. Furthermore, the functional expression level of HemQ from Corynebacterium glutamicum was significantly enhanced in vitro and in vivo by the co-expression of Group-I HSP60 chaperonins (GroEL and GroES) derived from Escherichia coli. The engineered S. cerevisiae H4+_MTS9HemQ^Cg+GroELS strain containing the mitochondrial PPD and CPD pathways and the Group-I HSP60 chaperonins produced the highest heme concentration (4.6 mg/L), which was 17% higher than that produced by the H4+_MTS9HemQ^Cg strain.