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

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, usingEscherichia colias a cell factory. We first implemented the Shemin (i.e., C4) pathway for heterologous 5-ALA biosynthesis inE. 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 represshemBexpression, 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 engineeredE. colistrains for bioreactor cultivation, we successfully demonstrated high-level 5-ALA biosynthesis from glycerol (similar to 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.

Automated summary

This study describes the development of a microbial bioprocess usingEscherichia colito produce high levels of 5-aminolevulinic acid (5-ALA). Through the implementation of the Shemin pathway and CRISPRi interference, metabolic engineering strategies were applied to direct carbon flux and enhance the biosynthesis of 5-ALA, achieving significant production yields.