Figure 4.

Markerless deletion of the vdh gene with CRISPR-Cas12a-assisted DNA cleavage and HDR-mediated recombination in Amycolatopsis sp. CCTCC NO: M2011265. (A) Schematic diagram illustrating the workflow of vdh deletion using the CRISPR-Cas12a system in CCTCC NO: M2011265. The editing template repaired the DSBs cut by Cas12a via HDR. P_km and P_hsp60: the promoters for expression of crRNA and Cas12a, respectively. (B) The transformants with pULΔvdhLR are on the left and those with the pULcrRNAkm-Cas12a control plasmid (no vdh guide sequence) are on the right. The data are obtained from three independent transformation experiments, and only one replicate is shown. (C) Verification of vdh deletion mutants by colony PCR with paired primers P1 and P2. The PCR products for the wild type were 3925 bp and those for the mutants were 2464 bp. M represents the nucleotide size marker; WT represents the wild type; and 1–14 represent 14 colonies from the screening of the Bennet plate. (D) Confirmation of mutants by Sanger sequencing. All three amplicons are sequenced and found to be correct; only the results from one colony are shown. (E) Growth curves of the wild-type strain and Δvdh mutant in shake flask fermentation. Error bars (mean ± SD) were derived from triplicate samples. (F) Vanillin and vanillic acid titers from shake flask fermentation of the wild-type strain and Δvdh mutant.