Metabolic Engineering of Escherichia coli for Bioproduction of (R)-3-Hydroxybutyric Acid through a Three-Pronged Approach

( R )-3-Hydroxybutyric acid ( R -3HB) is an important chiral chemical with extensive applications in the agricultural, food, and chemical industries. The synthesis of R -3HB by microbial fermentation is of interest due to its remarkable stereoselectivity and economy. However, the low production of R -3HB failed to meet the needs of large-scale industrial production. In this study, an engineered strain for the efficient biosynthesis of R -3HB was constructed through a three-pronged approach encompassing biosynthetic pathway optimization, engineering of NADPH regenerators, and central metabolism regulation. The engineered strain Q5081 produced 75.7 g/L R -3HB, with a productivity of 1.26 g/L/h and a yield of 0.34 g/g glucose in fed-batch fermentation, showing the highest reported titer and productivity of R -3HB to date. We also performed transcriptome sequencing and annotation to illustrate the mechanism underlying the enhanced R -3HB production. The systematic metabolic engineering by a three-pronged approach demonstrated the feasibility of improving the biosynthesis, and the engineered strain Q5081 has the potential for widespread applications in the industrial production of R -3HB.