Fig. 6. Effects of cgl0833C1439T mutation on its biological function, expression and methanol tolerance.
a Growth of C. glutamicum ATCC 13032 wild-type strain and cgl0833C1439T mutant strain on monocarboxylic acids. CGXII minimal media supplemented with different carbon sources were used for cultivation. b Effects of methanol addition and cgl0833C1439T mutation on cgl0833 expression. C. glutamicum ATCC 13032 wild-type strain and cgl0833C1439T mutant strain with gfp fused to cgl0833 were cultivated in CGXII minimal medium supplemented with 5 g/L glucose. Methanol (30 g/L) was added as required. ***P < 0.001, one-way ANOVA, N = 3; wild-type, methanol – vs. wild-type, methanol + , P = 3.2 × 10−6; wild-type, methanol – vs. C1439T mutant, methanol –, P = 5.7 × 10−8; C1439T mutant, methanol – vs. C1439T mutant, methanol + , P = 3.3 × 10−6. c Induction of cgl0833 by different C1 substrates. C. glutamicum ATCC 13032 wild-type strain with gfp fused to cgl0833 was cultivated in CGXII minimal media supplemented with 5 g/L glucose as carbon source and different inducers. ***P < 0.001, one-way ANOVA, N = 3, P = 2.5 × 10−5. d Effects of cgl0833 and cgl0833C1439T overexpression on methanol tolerance. e Effects of cgl0833 knock-down on methanol tolerance. **P < 0.01, one-way ANOVA, N = 3; CRISPRicgl0833, methanol + vs. control, methanol + , P = 0.0014. f Effects of cgl0833 knock-out on methanol tolerance. **P < 0.01, one-way ANOVA, N = 3; Δcgl0833, methanol + vs. control, methanol + , P = 0.0014. CGXII minimal medium supplemented with 5 g/L glucose and 30 g/L methanol was used to cultivate C. glutamicum ATCC 13032 and derivatives. IPTG (0.1 mM) was added at 4 h to induce cgl0833 or cgl0833C1439T overexpression and dCas9 expression. Values and error bars reflect the mean ± s.d. of three biological replicates (N = 3).