Valorization of soybean pulp for sustainable α-ketoisocaproate production using engineered Bacillus subtilis whole-cell biocatalyst

The disposal of soybean pulp (okara) (∼14 M tons annually) represents a global concern. α-ketoisocaproate (KIC) is an intrinsic L-leucine metabolite boosting mammalian muscle growth and has great potential in animal husbandry. However, the use of pure L-leucine (5000 USD/kg) for KIC (22 USD/kg) bioproduction is cost-prohibitive in practice, while okara rich in L-leucine (10%) could serve as an economical alternative. Following the concept of a circular bioeconomy, we managed to develop a cost-efficient platform to valorize okara into KIC. In this study, proteolytic Bacillus subtilis strain 168 capable of utilizing okara as a comprehensive substrate was employed as the whole-cell biocatalyst for KIC bioproduction. First, we elucidated the function of genes involved in KIC downstream metabolism in strain 168, including those encoding 2-oxoisovalerate dehydrogenase (bkdAA), 2-oxoisovalerate decarboxylase (bkdAB), enoyl-CoA hydratase (fadB), and bifunctional enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (fadN). Among those KIC downstream metabolizing mutants of strain 168, the 2-oxoisovalerate decarboxylase gene knockout strain (ΔbkdAB) was found to have a better accumulation of KIC. To further improve the KIC yield, a soluble L-amino acid deaminase (LAAD) from Proteus vulgaris was heterologously expressed in the ΔbkdAB strain and a ∼50% conversion of total L-leucine contained in okara was catalyzed into KIC, along with a ∼50% reduction of CO₂ emission compared to the wild-type cultures. Altogether, this renovated biocatalytic system provides an alternative platform to valorize okara for producing value-added chemicals in an eco-friendly manner.