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Antibiotics can inhibit or kill microorganisms, while microorganisms have evolved antibiotic resistance strategies to survive antibiotics. Zymomonas mobilis is an ideal industrial microbial chassis and can tolerate multiple antibiotics. However, the mechanisms of antibiotic resistance and genes associated with antibiotic resistance have not been fully analyzed and characterized. In this study, we investigated genes associated with antibiotic resistance using bioinformatic approaches and examined genes associated with ampicillin resistance using CRISPR/Cas12a−based genome−editing technology. Six ampicillin−resistant genes (ZMO0103, ZMO0893, ZMO1094, ZMO1650, ZMO1866, and ZMO1967) were identified, and five mutant strains ZM4∆0103, ZM4∆0893, ZM4∆1094, ZM4∆1650, and ZM4∆1866 were constructed. Additionally, a four−gene mutant ZM4∆ARs was constructed by knocking out ZMO0103, ZMO0893, ZMO1094, and ZMO1650 continuously. Cell growth, morphology, and transformation efficiency of mutant strains were examined. Our results show that the cell growth of ZM4∆0103 and ZM4∆ARs was significantly inhibited with 150 μg/mL ampicillin, and cells changed to a long filament shape from a short rod shape. Moreover, the transformation efficiencies of ZM4∆0103 and ZM4∆ARs were decreased. Our results indicate that ZMO0103 is the key to ampicillin resistance in Z. mobilis, and other ampicillin−resistant genes may have a synergetic effect with it. In summary, this study identified and characterized genes related to ampicillin resistance in Z. mobilis and laid a foundation for further study of other antibiotic resistance mechanisms. [ABSTRACT FROM AUTHOR]

Zymomonas mobilis is a native ethanologen with the unique anaerobic Entner Doudoroff pathway. In this study, a heterologous poly-3-hydroxybutyrate (PHB) pathway was engineered into Z. mobilis. Genetic strategies were applied to improve PHB production including overexpression of the phaCAB operon by using a strong promoter and/or increasing its copy numbers, cofactor balance by overexpressing the endogenous genes for NADPH balance, and competition of acetaldehyde for acetyl-CoA generation through the ethanol utilization pathway (EUP). PHB contents in recombinant strains were enhanced gradually. The highest PHB content of 37.68 ± 2.56% (dry cell weight, DCW) was achieved in the ZMPtN2-EUP strain, which contains copies of the phaCAB operon in both the chromosome and plasmid, an extra copy of the native zwf gene for cofactor balance, and genes for the EUP. The PHB production in ZMPtN2-EUP was further improved by optimizing the C/N ratio, and the PHB content was increased to 50.30 ± 3.39% (DCW) at a C/N ratio of 20/2 (20 g L−1 glucose and 2 g L−1 yeast extract). ZMPtN2-EUP was then empowered with the self-flocculating phenotype using a native CRISPR-Cas genome editing system to generate ZMPt-FloN2-EUP for biomass recovery through gravity sedimentation. The PHB content of 40.57 ± 0.60% (DCW) in ZMPt-FloN2-EUP with self-flocculation capability was slightly higher than that of ZMPtN2-EUP, which further increased to 74.03 ± 1.81% (DCW) at a C/N ratio of 20/2. Moreover, ZMPtN2-EUP was able to be used as a microbial biocatalyst for continuous co-production of high-titer ethanol and PHB without affecting ethanol percentage productivity. Our study thus demonstrated that the strategy to combine genetic and process engineering is effective to develop non-model microorganisms for heterologous production of bio-based chemicals, and the capability of Z. mobilis for continuous co-production of high-titer ethanol and PHB exhibited that Z. mobilis is a promising chassis for economic biochemical production. [ABSTRACT FROM AUTHOR]

Abstract Biorefinery of biomass-based biofuels and biochemicals by microorganisms is a competitive alternative of traditional petroleum refineries. Zymomonas mobilis is a natural ethanologen with many desirable characteristics, which makes it an ideal industrial microbial biocatalyst for commercial production of desirable bioproducts through metabolic engineering. In this review, we summarize the metabolic engineering progress achieved in Z. mobilis to expand its substrate and product ranges as well as to enhance its robustness against stressful conditions such as inhibitory compounds within the lignocellulosic hydrolysates and slurries. We also discuss a few metabolic engineering strategies that can be applied in Z. mobilis to further develop it as a robust workhorse for economic lignocellulosic bioproducts. In addition, we briefly review the progress of metabolic engineering in Z. mobilis related to the classical synthetic biology cycle of "Design-Build-Test-Learn", as well as the progress and potential to develop Z. mobilis as a model chassis for biorefinery practices in the synthetic biology era. Highlights • Z. mobilis has attractive features for lignocellulosic biofuels and biochemicals. • Progress achieved for expanded substrate utilization and robustness improvement. • Knowledge learned from Z. mobilis can be employed in other microorganisms. • Z. mobilis can be developed as a synthetic chassis. [ABSTRACT FROM AUTHOR]

Pretreatment is the key step to overcome the recalcitrance of lignocellulosic biomass making sugars available for subsequent enzymatic hydrolysis and microbial fermentation. During the process of pretreatment and enzymatic hydrolysis as well as fermentation, various toxic compounds may be generated with strong inhibition on cell growth and the metabolic capacity of fermenting strains. Zymomonas mobilis is a natural ethanologenic bacterium with many desirable industrial characteristics, but it can also be severely affected by lignocellulosic hydrolysate inhibitors. In this review, analytical methods to identify and quantify potential inhibitory compounds generated during lignocellulose pretreatment and enzymatic hydrolysis were discussed. The effect of hydrolysate inhibitors on Z. mobilis was also summarized as well as corresponding approaches especially the high-throughput ones for the evaluation. Then the strategies to enhance inhibitor tolerance of Z. mobilis were presented, which include both forward and reverse genetics approaches such as classical and novel mutagenesis approaches, adaptive laboratory evolution, as well as genetic and metabolic engineering. Moreover, this review provided perspectives and guidelines for future developments of robust strains for efficient bioethanol or biochemical production from lignocellulosic materials. [ABSTRACT FROM AUTHOR]