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 0.767 mg/L to 3.86 mg/L, achieving a 5.03-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.