Creating Single-Cell Protein-Producing Bacillus subtilis Mutants Using Chemical Mutagen and Amino Acid Inhibitors

Due to population growth and climate changes, there is a rising need for alternative food and protein sources to reduce protein scarcity and the environmental impact of food industries. Single-cell proteins (SCPs) have the potential to partially or fully substitute plant- and animal-derived dietary proteins. Bacillus subtilis is an appealing bacterium for SCP production because of its fast growth and ability to obtain high protein and essential amino acid (AA) content in its biomass. It is also capable of utilizing a wide range of substrates. B. subtilis attractiveness and efficiency can be further enhanced using mutagenesis. In this study, a novel approach to creating mutant strains with enhanced protein and AA content was experimentally validated. The method is based on the application of AA inhibitors for selective pressure to ensure the growth of mutants with enhanced protein and/or AA synthesis capacity. For AA inhibitors, three herbicides were used: glufosinate-ammonium (GA), L-methionine sulfoximine (MSO), and S-(2-aminoethyl)-L-cysteine (AEC). Initially, AA inhibitor doses for the complete inhibition of wild-type (WT) B. subtilis strain were determined. Then, B. subtilis was treated with EMS chemical mutagen and created mutants were cultivated on a medium containing inhibitory dose of AA inhibitors. Growing samples were selected, analyzed, and compared. The optimal inhibitory concentrations of herbicides for mutant selection were 0.05-0.4 M for GA, 0.01-0.05 M for MSO, and 0.2 M for AEC. The best-performing mutants were selected when using GA-improvement of 7.1 times higher biomass content, 1.5 times higher protein concentration, 1.2 times higher AA content, and 1.2 times higher essential AA index was achieved in comparison with WT B. subtilis. Enhanced mutants were also successfully selected when using MSO and AEC. This study demonstrates the potential of using AA inhibitors for the selection of mutants with improved protein and AA profiles.