Strain engineering for high-level 5-aminolevulinic acid production in Escherichia coli

As issues surrounding depleting fossil fuels, climate change, and various other environmental impacts are becoming more prevalent, there is a growing interest in technological shifts toward a bio-based economy. Various advanced biotechnological tools have been developed to customize cell factories for the production of a wide range of complex fine chemicals from renewable feedstock. Herein, we report development of a microbial bioprocess for high-level and potentially economical 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 solely from glycerol (~30 g l-1) under both microaerobic and aerobic conditions, achieving up to 5.95 g l-1 (36.9% yield) and 6.93 g l-1 (50.9% 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.