Systematic engineering of Escherichia coli for biosynthesis of 3-hydroxypropionic acid from glucose and malonate

Abstract Background 3-Hydroxypropionic acid (3-HP) is a promising C3 platform chemical with wide industrial applications. However, its microbial production remains limited by insufficient intracellular malonyl-CoA availability and metabolic imbalance. Result In this study, we systematically engineered Escherichia coli for enhanced 3-HP biosynthesis. The malonate assimilation genes (matB, smatPQM) and 3-HP biosynthesis gene (mcr) were chromosomally integrated using CRISPR/Cas9, resulting in a plasmid-free, antibiotic-free strain (WYY04) that produced 21.97 mM 3-HP, 0.51-fold higher than the plasmid-based system. Further improvement was achieved by CRISPRi-mediated repression of fatty acid biosynthesis genes (fabD, fabF), increasing 3-HP titer by 66%. Introduction of a malonyl-CoA-responsive FapR/fapO biosensor enabled dynamic regulation of mcr expression, enhancing 3-HP production by 59%. Through all these above engineering, the 3-HP production of the strain WYY19 increased by 2.29 times compared to that of the plasmid-expressing system. Under optimized fermentation conditions, the final engineered strain WYY19 produced 42.22 g/L 3-HP with the specific productivity of 0.69 g/g and 0.46 g/L/h from glucose and malonate in fed-batch bioreactor. Conclusions This study demonstrates a robust, genetically stable, and scalable microbial platform for 3-HP biosynthesis.