LAUSR

Herein, we report the development of a microbial bioprocess for high‐level production of 5‐aminolevulinic acid (5‐ALA), a valuable non‐proteinogenic amino acid with multiple applications in medical, agricultural, and food industries, using Escherichia coli as a cell factory. We first implemented the Shemin (i.e., C4) pathway for heterologous 5‐ALA biosynthesis in E. coli. To reduce, but not to abolish, the carbon flux toward essential tetrapyrrole/porphyrin biosynthesis, we applied clustered regularly interspersed short palindromic repeats interference (CRISPRi) to repress hemB expression, leading to extracellular 5‐ALA accumulation. We then applied metabolic engineering strategies to direct more dissimilated carbon flux toward the key precursor of succinyl‐CoA for enhanced 5‐ALA biosynthesis. Using these engineered E. coli strains for bioreactor cultivation, we successfully demonstrated high‐level 5‐ALA biosynthesis from glycerol (~30 g L−1) under both microaerobic and aerobic conditions, achieving up to 5.95 g L−1 (36.9% of the theoretical maximum yield) and 6.93 g L−1 (50.9% of the theoretical maximum yield) 5‐ALA, respectively. This study represents one of the most effective bio‐based production of 5‐ALA from a structurally unrelated carbon to date, highlighting the importance of integrated strain engineering and bioprocessing strategies to enhance bio‐based production.