*4.3. Construction of Editing Plasmids of Ampicillin*−*Resistant (AR) Genes in Z. mobilis ZM4*

The spacers were designed to bear the entire 23 bp sequences containing a 50−NTTN−3 0 PAM for six genes of *ZMO0103*, *ZMO0893*, *ZMO1094*, *ZMO1358*, *ZMO1866,* and *ZMO1967*. The oligonucleotides of spacers were ordered from TsingKe Biotechnology Co., Ltd. (Beijing, China). CRISPR−Cas12a−editing plasmids were constructed following the previous description [25]. The Cas12a−targeting gRNA sequence was annealed using two single−stranded oligonucleotides by first heating the reaction mixture to 95 ◦C for 5 min and subsequently cooling down gradually to room temperature. Then, the annealed spacer was ligated into *Bsa* I−linearized pEZ−sgr by T4 ligase at 22 ◦C for 3 h. The resulting plasmids were named as pEZ−sgr−0103, pEZ−sgr−0893, pEZ−sgr−1094, pEZ−sgr−1385, pEZ−sgr−1866, and pEZ−sgr−1967.

Gibson assembly method was utilized for donor construction. Donor sequences including extra ~800 bp upstream and downstream flank sequences of the candidate gene were amplified using Primer STAR polymerase (Takara, Japan) from the genomic DNA of *Z. mobilis* ZM4 and then cloned into pEZ−sgr vector by T5 exonuclease (NEB, WA, USA). The resulting plasmids were named as pEZ−sgr−0103−D, pEZ−sgr−0893−D, pEZ−sgr−1094−D, pEZ−sgr−1385−D, pEZ−sgr−1866−D, and pEZ−sgr−1967−D. All plasmids used in this work are provided in the Supplementary Materials Table S4. The sequences of primers used in this study are shown in the Supplementary Materials Table S5.

#### *4.4. Curing of Editing Plasmids*

Transformants harboring editing plasmids were cultured in RMG5 broth without the supplement of antibiotics. After 6 consecutive passages in the nonresistant RMG5 liquid medium, 100 µL cultures were diluted and plated on nonresistant RMG5 plates. Then, single colonies were picked to conduct colony PCR using primers pEZ15A−F/R for amplifying the editing plasmid. Editing plasmids were lost if no PCR product was amplified compared with the control. Single colonies with correct PCR results were then inoculated on RMG5 with or without chloramphenicol for further verification. The knockout strains losing the editing plasmids can only grow in RMG5, but not in the medium with the supplementation of chloramphenicol.

#### *4.5. Electroporation of Editing Plasmids to Z. mobilis ZM4*

Transformation of *Z. mobilis* ZM4 with the editing plasmid was achieved by preparing electrocompetent cells using a modified protocol as previously described. *Z. mobilis* ZM4 was inoculated with 40 mL RMG5 in a 100 mL flask and was grown to an OD600 nm = 0.4~0.6. Cells were harvested by centrifugation at 4000× *g* for 10 min at room temperature. The supernatant was discarded, and the cell pellet was washed with Milli−Q® ultrapure water and 10% (*v*/*v*) glycerol before being resuspended in a final volume of 400 µL of 10% (*v*/*v*) glycerol. Cells were stored as 50 µL aliquots on ice for immediate use in electroporation experiments. Then, 500 ng plasmid DNAs was used for electroporation

(Bio−Rad, CA, USA). After pulsing at 16 kV/cm, 25 µF and 200 Ω, 1 mL of RMG5 was added to the electroporated solutions and then incubated at 30 ◦C for 4~6 h. Finally, the cells were plated and selected on RM plates supplemented with chloramphenicol until colonies were visible (≤2 d).

#### *4.6. Construction of ZM4*∆*ARs by Continuous Gene Editing*

We constructed ZM4∆ARs with four genes of *ZMO0103*, *ZMO0893*, *ZMO1094,* and *ZMO1650* knocked out following these steps:

Step 1. A CRISPR−Cas12a−editing plasmid pEZ−sgr−0103−D was transformed into ZM4−Cas12a. Colony PCR was then performed with the primers 0103−out−F and 0103−out−R.

Step 2. The positive single clone with the correct PCR size was cultured in RMG5, and the editing plasmid was then cured following Section 4.4.

Step 3. After curing the editing plasmid, the electrocompetent cells of knockout strain ZM4∆0103 were then prepared for the next−round knockout.

Knockout of *ZMO0893*, *ZMO1094*, *ZMO1650*, *ZMO1866*, and *ZMO1967* followed the above steps 1 to 3. After each gene was edited and the corresponding editing plasmid was lost, the competent cells of the corresponding mutant were prepared for transferring to the next editing plasmid.
