## 2 Materials and Methods  
### Bacterial Strains, Media, and Growth Conditions  
The _E. coli_ strain XL1 was grown in an LB medium at 37 \({}^{\circ}\)C. If necessary, antibiotics were added to the medium: kanamycin--50 \(\mathrm{\SIUnitSymbolMicro g}\)/mL; and ampicillin--100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL. The _C. glutamicum_ strains were grown in 2 \(\times\) LB medium with 1% maltose at 30 \({}^{\circ}\)C. We used electroporation to transform DNA. A BHIS medium with the following composition was used for the growth of transformants (g/L): brain heart infusion (BHI) (Difco, Pinellas Park, FL, USA) 37.0; sorbitol--30.0; and agar--15.0. To select strains containing plasmids, kanamycin (10 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) was added to the medium. The list of _C. glutamicum_ strains used in this work is presented in Table 1. The _C. glutamicum_ strains ATCC 13032, VF, and L2.5 were obtained from the Russian Collection of Industrial Microorganisms (VKPM) of the National Research Center \"Kurchatov Institute\", Moscow. The _C. glutamicum_ strain ATCC 13869 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). The strains VB1, VB2, and VC2 were obtained by [PERSON]. (NCR \"Kurchatov Institute\", Kurchatov Genomic Center) from _C. glutamicum_ strains ATCC 13869 and ATCC 13032, by introducing a deletion into the _ponA_ gene to increase the efficiency of electroporation [9] and a deletion into the _ilvA_ gene (VB2, VC2) for the purpose of the further use of strains in which the synthesis of isoleucine is limited and the flow of metabolites is directed to the synthesis of valine. To determine the level of production of BCAAs by the _C. glutamicum_ strains, fermentation was carried out in test tubes in a medium with the following composition (g/L): glucose--100.0; (NH4)2 SO4--18.9; KH2 PO4--1.0; MgSO5O4 \(\times\) 7H2O--1.0; CaCO3--25.0; biotin--0.0001; and thiamine--0.0002, with the addition of 50.0 mL/L of an acid hydrolyzate of wheat gluten, which was neutralized with ammonia before being added. The hydrolysis of 320 g of wheat gluten was carried out in 1 L 3.8-4.0 N sulfuric acid at 110 \({}^{\circ}\)C for 12 h. After hydrolysis, the precipitate was separated by filtration. The content of amine nitrogen in the resulting hydrolyzate was not \begin{table}
\begin{tabular}{l l l} \hline \hline
**Strain** & **Strain Characteristics** & **Source** \\ \hline DSM 1412 (ATCC13869) & Wild-type strain & Collection of microorganisms DSMZ \\ ATCC13032 & Wild-type strain & VKPM \\ VF & Valine producer (mutagenesis) & VKPM, B5212 \\ VB1 & ATCC 13869 \(\mathrm{\SIUnitSymbolMicro g}\)_ponA_ & Received by [PERSON]. \\ VC2 & ATCC 13032 \(\mathrm{\SIUnitSymbolMicro g}\)_ponA_ \(\mathrm{\SIUnitSymbolMicro g}\)_ponA_ & Received by [PERSON]. \\ VC3 & VC2 G110A & Received by [PERSON]. \\ VB2 & VB1 \(\mathrm{\SIUnitSymbolMicro g}\)_ponA_ & Received by [PERSON]. \\ VB3 & VB2 G110A & This work \\ VB6 & VB1 G110A & This work \\ VB22 & VB1 G110T & This work \\ VB23 & VB1 G110C & This work \\ VB34 & VB1 A73G & This work \\ VB35 & VB1 G111A & This work \\ VB36 & VB1 C77T & This work \\ VB45 & VB1 C77T; G112A & This work \\ VB46 & VB1 G112A & This work \\ VB47 & VB1 G37A & This work \\ VB48 & VB1 G37A; C77T & This work \\ VB49 & VB1 C79T & This work \\ L2.5 & Leucine producer (mutagenesis) & VKPM \\ L2.5-P\({}_{\mathrm{\SIUnitSymbolMicro g}\mathrm{B}}\) & L2.5 G110A & This work \\ \hline \hline \end{tabular}
\end{table} 
Table 1: The strains of _C. glutamicum_ used in this work. lower than 15 g/L. Test tubes with cultures were incubated for 48 h with stirring (300 rpm) at 30 \({}^{\circ}\)C, and then, the content of valine, leucine, and isoleucine in the culture liquid was determined by thin-layer chromatography or high-performance liquid chromatography (HPLC). The optical density at 600 nm (OD\({}_{600}\)) was approximately 65-72 after 48 h of fermentation for all strains.