Figure 5.

Markerless deletion of the phdB gene with CRISPR-Cas12a-assisted DNA cleavage and HDR-mediated recombination in the Δvdh mutant. (A) Schematic for pULΔphdBLR-mediated gene knockout from the Δvdh mutant. The donor DNA is located upstream and downstream of the target region; P_kasO*pand P_gapdh are the promoters for expression of crRNA and Cas12a, respectively. (B) The transformants with pULΔphdBLR are on the left and those with the pULcrRNAkasO*p-Cas12a plasmid (no phdB guide sequence) are on the right. The data are obtained from three independent transformation experiments, and only one replicate is shown. (C) Verification of phdB deletion mutants by colony PCR with paired primers P3 and P4. A fragment of 1674 bp was amplified from the edited colonies, and a fragment of 2436 bp was amplified from the wild type. M represents the nucleotide size marker, WT represents the wild type, and 1–12 represent randomly selected clone colonies from the Bennet plate. (D) Sanger sequencing results of the amplicon from (C). All seven amplicons are sequenced and found to be correct; only the results from one colony are shown. (E) Growth curves of the wild type strain, Δvdh mutant, and ΔvdhΔphdB mutant in shake flask fermentation. The error bars (mean ± SD) were derived from triplicate samples. (F) Vanillin and vanillic acid titers from shake flask fermentation of the wild type strain, Δvdh mutant, and ΔvdhΔphdB mutant. The error bars (mean ± SD) were derived from triplicate samples.