Metabolic engineering of Bacillus subtilis for enhanced free heme biosynthesis by an enzyme-chassis co-optimization strategy

Heme, an iron-incorporated porphyrin compound, serves as the prosthetic group for numerous proteins involved in diverse biological processes. The prokaryotic heme biosynthetic pathway features a complex cascade of reactions, in which glutamyl-tRNA reductase (GluTR) catalyzes the formation of 5-aminolevulinic acid (ALA) that represents a critical rate-limiting step and determines ultimate heme yield. In this study, OsGluTR A510V showed enhanced heme synthesis capacity in Oryza sativa and was used for developing microbial cell factories dedicated to free heme production. Through systematic protein engineering involving site-directed mutagenesis and N -terminal modification, OsGluTR A510V was optimized to improve the structural stability and catalytic efficiency. It yielded the recombinant enzyme GluTR A510V/S189T/KK , which achieved a maximum heme titer of 13.14 mg/L in Escherichia coli , representing a 7.6-fold improvement over that of GluTR A510V . To establish heme production in Bacillus subtilis , GluTR A510V/S189T/KK was introduced into the ΔhmoAB-hemX chassis, a modified B. subtilis host lacking key heme biosynthesis inhibitors ( hmoA , hmoB , and hemX ). This engineered system elevated the heme yield from 0.77 to 3.86 mg/L, achieving a 5.0-fold improvement. This study demonstrates a combinatory metabolic engineering strategy that reconstitutes the heme synthetic route in B. subtilis , enabling efficient production of food-grade free heme through enzyme engineering and chassis optimization.