Figure 4.

Effects of CRISPRi‐mediated suppression on lactate and acetate production and impact on the fermentation profile. For lactate and acetate production, positive transformants referred as ldh_P, ldh _S, pta_P, pta_S and control ldh_non‐targeting (NT) were analysed in HPLC at OD₆₀₀ – 0.5–0.7 (T1). The silencing transformants for ldh gene referred as ldh_P – targeting the promoter, ldh _S – targeting the start of the gene; positive silencing transformants for pta gene referred as pta_P – targeting the promoter, pta_S – targeting the start of the gene. H. thermocellum DSM 1313 cells were transformed with both non‐targeting pThermoCas9i_NT and targeting pThermoCas9i_ldh and pta plasmids. The Tm^R colonies of both the non‐targeting and targeting plasmids were transferred to 50 ml CP medium (1 g l⁻¹ yeast extract) containing 6 µg ml⁻¹ Tm^R and cultured to OD₆₀₀ ~ 0.5–0.7 (T1) and > 1.0 (T2), and 1 ml cells were sampled for 2 days for HPLC analysis. Data represent the mean values of three biological replicates and the standard deviation (SD). The level of significance of the differences when results were compared was estimated by means of analysis of variance (ANOVA), with α = 0.05.