## 2 Materials and Methods
### Bacterial Strains, Media, and Growth Conditions
The Construction of a Recombinant Integrating Plasmids and Strains of C. glutamicum with Mutations in the Regulatory Region of the ilvBNC Operon  
Oligonucleotides for DNA-specific primers were synthesized by Evrogen, Moscow. The sequences of the primers are presented in Supplementary Table S1. The targeted introduction of a mutation into the regulatory region of the _ilvBNC_ operon of the _C. glutamicum_ chromosome was carried out by a substitution method based on homologous recombination [10]. A DNA fragment of the regulatory region of the _ilvBNC_ operon, limited by primers 944--923, containing a certain mutation, was obtained using the PCR method [11] and cloned into the plasmid pIKA-sac13, a derivative of pUC19, with the _sacB_ gene from _Bacillus subtilis_ 168 and the kanamycin resistance gene [12]. The plasmid pIKA-sac13 is not capable of autonomous maintenance in the host cell but can be integrated into the chromosome along homologous regions and excluded from it during growth on a medium with sucrose, leaving a mutant copy of the gene in the chromosome. The list of the plasmids carrying fragments of the regulatory region of the _ilvBNC_ operon with various mutations is presented in Supplementary Table S2. _C. glutamicum_ strains with mutations in the regulatory region of the _ilvBNC_ operon were constructed based on _C. glutamicum_ strains VB1, VB2, VC2, and L2.5 by replacing the native sequence of the regulatory region of the _ilvBNC_ operon with a mutant copy. The preparation of a competent culture of _C. glutamicum_ and electroporation were carried out in accordance with the method described in [13]. The electroporation conditions were as follows: 2500 V, 2525 \(\mu\)F, 200 ohm. The duration of the electrical pulse was 4.5-5.5 ms, depending on the quality of the cells and the purity of the DNA. DNA sequencing was performed using an automatic ABI PRISM3500 sequencer (ThermoFisher Scientific, Waltham, MA, USA) at the Resource Centers of the NRC \"Kurchatov Institute\". Km\({}^{\text{R}}\) transformants were plated on a BH medium with 10% sucrose. Among the grown colonies, Km\({}^{\text{S}}\) clones that lost the plasmid were selected based on the lack of growth on the BH medium with kanamycin at 10 \(\mu\)g/mL. Chromosomal DNA was isolated from Km\({}^{\text{S}}\) colonies, and PCR was performed using primers selected to detect a particular mutation (Supplementary Table S1). \"Wild\" and \"mutant\" primers, differing in their last nucleotide, paired with primer 923, should give signals of different intensity after PCR, depending on the template sequence in the region of the replacement. If there is a mutation in the regulatory region of the _ilvB_ gene, the PCR signal using the mutant primer will be more intense than that using the wild-type primer. PCR was performed with the chromosomal DNA of strain ATCC 13032 as a negative control, and with plasmid DNA used to introduce the mutation as a positive control. As a result of screening, the strains VB3, VB6, VC3, VB34, VB35, VB36, VB45, VB46, VB47, VB48, VB49, and L2.5-P\({}_{ilvB}\) (Table 1) with mutations in the regulatory region of the _ilvBNC_ operon were selected. The strains VB22 and VB23 were selected among clones that did not synthesize a PCR fragment with the pair of primers 926 and 923. In all cases, the presence of mutations was confirmed by sequencing fragments amplified from the chromosomal DNA of strains using specific primers.