Table 1 Bacteria strains and plasmids used in this study
Strain or plasmid | Relevant characteristics | References |
|---|---|---|
Strains | ||
 E. coli DH5α | F−(80d lacZ M15) (lacZYA-argF) U169 hsdR17(r− m+) recA1 endA1 relA1 deoR96 | [26] |
 C. glutamicum ATCC 13032 | Wild-type strain, biotin auxotroph | ATCC |
 C. glutamicum Wt derivatives | Wild-type strain containing pCoryne-dCas9 and pCoryne-sgRNA-target_gene-r | This study |
 C. glutamicum DM1919 | pyc(P458S), hom(V59A), 2 copies of lysC(T311I), Δpck, l-lysine producer | Evonik Laboratory |
Plasmids | ||
 pBbEB2c | ColE1(Ec), pBL1(Cg), Cmr, P trc , BglBrick sites, CoryneBrick vector, TetR-P tetA | [11] |
 pdCas9-bacteria | p15A(Ec), Cmr, inactive bacterial Cas9 (Streptococcus pyogenes), addgene#44249 | [1] |
 pCoryne-dCas9 | pBbEB2c derivative containing the dCas9 gene | This study |
 pZ8-1 | p15A(Ec), pHM1519(Cg), Kmr, P tac , E. coli–C. glutamicum shuttle vector | [27] |
 pgRNA-bacteria | ColE1 (Ec), Ampr, customizable guide RNA (gRNA), addgene#44251 | [1] |
 pCoryne-sgRNA | pZ8-1 derivative, EcoR1, BglII and BamHI compatible | This study |
 psgRNA-pyc-r | pCoryne-sgRNA containing sgRNA-pyc-r targeting transcriptional repression of the pyc gene | This study |
 psgRNA-gltA-r1 | pCoryne-sgRNA containing sgRNA-gltA-r1 targeting transcriptional repression of the gltA gene | This study |
 psgRNA-gltA-r2 | pCoryne-sgRNA containing sgRNA-gltA-r2 targeting transcriptional repression of the gltA gene | This study |
 psgRNA-gltA-r3 | pCoryne-sgRNA containing sgRNA-gltA-r3 targeting transcriptional repression of the gltA gene | This study |
 psgRNA-idsA-r1 | pCoryne-sgRNA containing sgRNA-idsA-r1 targeting transcriptional repression of the idsA gene | This study |
 psgRNA-idsA-r2 | pCoryne-sgRNA containing sgRNA-idsA-r2 targeting transcriptional repression of the idsA gene | This study |
 psgRNA-idsA-r3 | pCoryne-sgRNA containing sgRNA-idsA-r3 targeting transcriptional repression of the idsA gene | This study |
 psgRNA-glgC-r1 | pCoryne-sgRNA containing sgRNA-glgC-r1 targeting transcriptional repression of the glgC gen | This study |
 psgRNA-glgC-r2 | pCoryne-sgRNA containing sgRNA-glgC-r2 targeting transcriptional repression of the glgC gene | This study |
 psgRNA-glgC-r2-idsA-r1 | pCoryne-sgRNA containing sgRNA-glgC-r2, sgRNA-idsA-r1 targeting transcriptional repression of the glgC and idsA gene simultaneously | This study |
 psgRNA-idsA-r1-glgC-r2 | pCoryne-sgRNA containing sgRNA-idsA-r1, sgRNA-glgC-r2 targeting transcriptional repression of the idsA and glgC gene simultaneously | This study |