Microbial synthesis of enantiopure (S)-2-methylbutanoic acid via L-isoleucine catabolism in Bacillus spizizenii.

Enantiopure (S)-2-methylbutanoic acid [(S)-2-MBA] is a high-value chiral compound with applications in fragrances, pharmaceuticals, and agrochemicals. However, conventional chemical synthesis lacks stereoselectivity, while existing biosynthetic methods suffer from low yield and purity. Here, we report a novel microbial process using Bacillus spizizenii ATCC 6633 for efficient (S)-2-MBA production via L-isoleucine catabolism. Through targeted screening of rhizospheric soil isolates and Bacillaceae strains, ATCC 6633 demonstrated superior performance, producing 3.67 g/L (S)-2-MBA with 99.32% enantiomeric excess (ee) under optimized conditions (45 °C, 8% inoculation, 5 g/L glucose, and 8 g/L L-isoleucine). A 58.92% conversion efficiency was achieved, and a simplified purification process recovered 63.90% product with 97.32% purity. Mechanistic studies suggested glucose depletion triggered (S)-2-MBA accumulation, aligning with starvation-induced secondary metabolism. This cost-effective, eco-friendly approach eliminates racemic separation steps and harsh reagents, positioning ATCC 6633 as a promising biocatalyst for sustainable (S)-2-MBA production.