Animal-free heme production for artificial meat in Corynebacterium glutamicum via systems metabolic and membrane engineering

Recently, heme has attracted much attention as a main ingredient that mimics meat flavor in artificial meat in the food industry. Here, we developed Corynebacterium glutamicum capable of high-yield production of heme with systems metabolic engineering and modification of membrane surface. The combination of two precursor pathways based on thermodynamic information increased carbon flux toward heme and porphyrin intermediate biosynthesis. The co-overexpression of genes involved in a noncanonical downstream pathway and the gene encoding the transcriptional regulator DtxR significantly enhanced heme production. The overexpression of the putative heme exporters, knockout of heme-binding proteins, modification of the cell wall by chemical treatment, and reduction of intermediate UP III substantially improved heme secretion. The fed-batch fermentation showed a maximum heme titer of 309.18 ± 16.43 mg l−1, including secreted heme of 242.95 ± 11.45 mg l−1, a yield on glucose of 0.61 mmol mol−1, and productivity of 6.44 mg l−1h−1, which are the highest values reported to date. These results demonstrate that engineered C. glutamicum can be an attractive cell factory for animal-free heme production. •The rational design and systematic consolidation of the complex heme biosynthesis pathway based on thermodynamic information substantially enhanced heme production in Corynebacterium glutamicum.•The global transcriptional regulator DtxR prevented the heme biosynthesis from being abolished by the disruption of heme metabolism.•Membrane engineering to reduce the binding of heme to the cell membrane was first attempted and successfully enhanced heme secretion.•Fed-batch fermentation of engineered C. glutamicum achieved the highest production titer, yield and productivity reported to date.