Fig. 4

Fermentation profile of constructed strains. Mutants generated in this study are validated using PCR and Sanger Sequencing (a) before characterization (b–d). To achieve a clean in-frame deletion, typically homology arms (left and right) are designed to include only the start and stop codons of the desired knockout-target gene. Primers flanking just outside of the homology arms (1, 2) are used for PCR screening. Lactate dehydrogenase (ldh) locus is screened with primers FC_LDH and RC_LDH (lane 1–3), with the expected size of 2031 bp for wild type (WT), 1130 bp for LS001 (Δldh) and 1981 bp for TM89 (ldh ⁻). Pyruvate dehydrogenase (pdh) locus is screened with primers FC_PDH and RC_PDH (lane 4–6), with the expected size of 1716 bp for WT and 1495 bp for promoter replacement with G. stearothermophilus ldh promoter (LS003, TM003). The pyruvate formate lyase (pfl) locus is screened with primers FC_PFL and RC_PFL (lane 7–11), with the expected size of 3348 bp for WT, 1122 bp for pfl deletion (Δpfl) (LS004, TM004) and 2996 bp for pfl disruption (pfl ⁻) in the same context as that of TM242, involving a 360-bp central region disruption of pfl replaced by a NotI site (LS005, TM005). Solvent profiles (ethanol, acetate, lactate, formate and glucose) of the constructed strains, in three biological replicates, are characterised by HPLC after 24 h of fermentation using 40 ml of ASYE with 1% yeast extract and 2% glucose in a 50-ml Falcon tube at 60 °C. AA indicates the addition of 0.1 mM acetic acid