The disposal of soybean waste (~14M tons annually) represents a global concern. Following the concept of a circular bioeconomy, we managed to develop a cost-efficient platform for α-ketoisocaproic acids (KIC) bioproduction using a big portion of wasted biomass. The use of pure L-leucine (5 USD/g) for KIC (22 USD/kg) bioproduction is cost-prohibitive in practice. Hence, soybean pulp rich in L-leucine (10%) was used as the representative of protein waste. KIC is an intrinsic compound in mammals responsible for muscle growth and has been used for treating Sarcopenia. This study aims to harness okara as the source of L-leucine for KIC bioproduction and as the substrate for bacteria. Proteolytic Bacillus subtilis strain 168 capable of directly utilizing okara as a comprehensive substrate was selected as the model organism for KIC bioproduction. To produce KIC, we elucidated the function of genes involved in the leucine degradation 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). We observed KIC production in the cultures of bkdAB-disrupted strain 168 (ΔbkdAB) showed a dominant KIC production. To further enhance the KIC production, we heterologously expressed a truncated L-amino acid deaminase from proteus vulgaris in the ΔbkdAB and observed a stoichiometric conversion of L-leucine in okara to KIC in the cultures of the ΔbkdAB expressing L-amino acid deaminase. In addition to the KIC production, our engineered B. subtilis generated less carbon dioxide (~50%) than the wild type, which provide a plausible biotransformation process for value-added chemicals production in an eco-friendly manner. Altogether, this renovated system provides an alternative platform to valorize soybean offal for producing value-added chemicals.    

Keywords: Bacillus subtilis; resource recovery; ɑ-ketoisocaproic acids; whole-cell biocatalyst; branched-chain amino acid metabolism; reduced carbon dioxide emission