Biosynthesis of 3-Hydroxy-3-methylbutyrate by Probiotic Escherichia coli Nissle 1917.

3-Hydroxy-3-methylbutyrate (HMB) is an important nutritional supplement for managing sarcopenia. This study engineered an HMB biosynthetic pathway in the probiotic Escherichia coli Nissle 1917 (EcN). The initial engineered strain exhibited low HMB titers, as carbon flux was primarily diverted toward acetate formation. Deleting the pyruvate oxidase gene (poxB) reduced acetate but caused significant pyruvate accumulation, revealing a severe kinetic bottleneck in endogenous pyruvate dehydrogenase (PDH) activity. Supplementing pantothenic acid (Vitamin B5) profoundly expanded the intracellular coenzyme A (CoA) pool, providing the essential backbone for pathway intermediates. This strategy enhanced PDH flux and improved HMB production by 5.5-fold. Through fed-batch fermentation, the engineered strain achieved an HMB titer of 8.6 g/L with a yield of 0.14 g/g glucose. The results of this study provided a probiotic strain that can synthesize HMB de novo and demonstrated the importance of pantothenic acid availability for EcN metabolic engineering.