*2.2. Ampicillin Resistance*−*Related Gene Knockout in Z. mobilis ZM4*

Subsequently, six ampicillin−resistant (AR) candidate genes based on the above bioinformatics study were selected for knockout using the CRISPR−Cas12a genome−editing system. Except for *ZMO1967,* which may be an essential gene and cannot be knocked out, five other genes of *ZMO0103*, *ZMO0893*, *ZMO1094*, *ZMO1650*, and *ZMO1866* were successfully knocked out with the CRISPR−Cas12a system. As demonstrated in Figure 1, the expected sizes of the amplicon of *ZMO0103*, *ZMO0893*, *ZMO1094*, *ZMO1650*, and *ZMO1866* in the wild type (WT) strain were obtained, which were ~4021, 3314, 3122, 3746, and 3768 bp, respectively. As a contrast, the corresponding amplified fragments in the knockout strains were ~2247, 2232, 2361, 2221, and 2080 bp, respectively (Figure 1). The inactivation of the AR genes was further confirmed by Sanger sequencing of the PCR products. Then, the knockout strains harboring the editing plasmids were cultured in RMG5 for several passages to obtain the final strains with the editing plasmid cured. Except for *ZMO1866*, *ZMO0103*, *ZMO0893*, *ZMO1094,* and *ZMO1650,* the knockout strains lost the editing plasmid successfully, which can only grow in RMG5 after 24 h cultivation, but not in the medium with chloramphenicol (Supplementary Materials Figure S2).

**Figure 1.** Confirmation of ampicillin−resistant (AR) knockout strains in *Z. mobilis* ZM4 by PCR. The mutants of ZM4∆0103 (**A**), ZM4∆0893 (**B**), ZM4∆1650 (**C**), ZM4∆1094 (**D**), ZM4∆1866 (**E**), and ZM4∆ARs (**F**) were confirmed by colony PCR using their corresponding primers. The sizes of PCR products (bp) of WT and knockout strains were 4021, 2247 (ZM4∆0103); 3314, 2232 (ZM4∆0893); 3122, 2361 (ZM4∆1650); 3746, 2221 (ZM4∆1094); 3768, 2080 (ZM4∆1866).

To obtain the strain with all the ampicillin−resistant (AR) candidate genes knocked out, we further conducted six rounds of genome editing continuously with the CRISPR−Cas12a system. Consistent with the above single−gene deletion experiments, we only obtained a mutant strain, ZM4∆Ars, with four ampicillin−resistant (AR) genes of *ZMO0103*, *ZMO0893*, *ZMO1094,* and *ZMO1650* knocked out continuously, while ZMO1967 and ZMO1866 were not able to be deleted. The ZM4∆ARs was further identified by colony PCR using the primers for each gene. The results of the correct PCR products of four genes indicate that these four ampicillin−resistant (AR) genes were knocked out successfully in ZM4∆ARs (Figure 1F).
