*4.9. Growth Studies and Analysis of Ampicillin*−*Resistant Gene Mutants*

Cell growth was monitored by measuring the cell optical density (OD) values using a Bioscreen C high−throughput growth measurement instrument (Bioscreen C MBR, Helsinki, Finland). The single colony was inoculated in 1 mL of RMG5 at 30 ◦C overnight as the seed culture. Then, the seed culture of *Z. mobilis* was transferred to RMG5 until reaching the exponential phase. Subsequently, the cultures were inoculated to the well of the Bioscreen C plate containing 300 µL bacterium suspension with an initial OD600 nm value of 0.05. Three technical replicates were used for each condition.

#### *4.10. Cell Morphology Observation*

*Z. mobilis* strains were cultured in RMG5 or RMG5 with 150 µg/mL ampicillin overnight at 30 ◦C. Bacterial pellets were collected at the exponential phase, washed twice with 1× phosphate buffer saline (1× PBS), resuspended in the same buffer, and observed under a light microscope (Leica Dmi8, Buffalo Grove, IL, USA) at 400 magnifications. Each image was taken using the image−based autofocus system LAS X software of the Leica Dmi8 system, and the cell size was measured using ImageJ software [41].

#### **5. Conclusions**

In summary, genes associated with antibiotic resistance in *Z. mobilis* were systematically investigated, and six ampicillin−resistant genes in *Z. mobilis* were identified. Five of them were knocked out individually, and four ampicillin−resistant gene deletion mutant ZM4∆Ars was also constructed to verify their functions. The strains of ZM4∆0103 and ZM4∆Ars were sensitive to ampicillin at a lower concentration of 150 µg/mL with a long and filament shape. The putative membrane protein ZMO0103 is probably the essential β−lactamase, and other ampicillin−resistant genes may have a synergetic effect with it. This study not only identified the ampicillin−resistant genes in *Z. mobilis* ZM4, but also verified their functions on cell growth, morphology, and transformation efficiency. In addition, ampicillin−sensitive mutants can serve as the parental strains for metabolic engineering practices in *Z. mobilis* enabling the usage of antibiotics, such as ampicillin, that are routinely used for genetic engineering.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/antibiotics11111476/s1, Figure S1: Secondary structure prediction of ZMO0103, ZMO0893, ZMO1094, ZMO1650, ZMO1866, and ZMO1967 in NCBI database; Figure S2: Multiple sequence alignment of the AmpC superfamily and β−lactamase class A protein in Z. mobilis; Figure S3: Loss of editing plasmids in ampicillin−resistant (AR) gene knockout strains; Figure S4: Cell growth of single and multiple ampicillin−resistant gene knockout strains cultured under 0 (A), 150 (B), and 300 (C) µg/mL of ampicillin; Figure S5: Electroporation efficiency of ZM4∆0103, ZM4∆Ars, and ZM4 by pEZ15A; Figure S6: Electroporation efficiency of ZM4∆0103, ZM4∆Ars, and ZM4 by pEZ15A and pE39−MVA; Figure S7: Distance tree of ZMO0103 after multiple sequence alignment with other proteins collected in NCBI using BALSTP; Figure S8: The detailed result of multiple sequence alignment with other proteins collected in NCBI using BALSTP; Table S1: List of the putative efflux pump−type antibiotic−resistant genes by BLASTP with CARD and MEGARes databases; Table S2: List of the putative β−lactamase genes by BLASTP in the UniProt database; Table S3: Strains used in this study; Table S4: Plasmids used in this study; Table S5: Primers used in this study.

**Author Contributions:** Conceptualization, S.Y.; methodology, B.G., X.H. and Y.W.; validation, X.H.; formal analysis, B.G. and X.H.; investigation, B.G., X.H., Y.W., Q.H. and S.Y.; data curation, B.G. and X.H.; writing—original draft preparation, B.G. and X.H.; writing— review and editing, Q.H. and S.Y.; visualization, Y.W.; supervision and project administration, S.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** Supported by National Key Research and Development Program of China (2018YFC1801200), the National Natural Science Foundation of China (21978071 and U1932141), Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang Province (2018R01014), and the Innovation Base for Introducing Talents of Discipline of Hubei Province (2019BJH021).

**Institutional Review Board Statement:** Ethical review and approval were waived for this study as the study did not involve animal interventions.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** No new data were created or analyzed in this study. Data sharing does not apply to this article.

**Acknowledgments:** We acknowledge the support from the State Key Laboratory of Biocatalysis and Enzyme Engineering.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

