Using the CRISPR/Cas9 system to eliminate native plasmids of <i>Zymomonas mobilis</i> ZM4

Cao, Qinghua; Shao, Huanhuan; Qiu, Hui; Li, Tao; Zhang, Yizheng; Tan, Xuemei

doi:10.1080/09168451.2016.1189312

Cited by 29 publications

(18 citation statements)

References 28 publications

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“…Finally, sophisticated and efficient tools for genome editing need to be developed instantaneously. For example, although CRISPR/Cas system has been investigated in Z. mobilis recently (Cao et al 2017a;Dong et al 2016), more efficient strategies are needed for efficient recreation of SNPs for genetics studies to identify the association of SNPs with improved phenotypes in tolerant mutants.…”

Section: Discussionmentioning

confidence: 99%

Progress and perspective on lignocellulosic hydrolysate inhibitor tolerance improvement in Zymomonas mobilis

Yang

Tang

et al. 2018

Bioresour. Bioprocess.

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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.

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Section: Discussionmentioning

confidence: 99%

Progress and perspective on lignocellulosic hydrolysate inhibitor tolerance improvement in Zymomonas mobilis

Yang

Tang

et al. 2018

Bioresour. Bioprocess.

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“…CRISPR/Cas systems can also target plasmid backbone genes such as replicase genes (Cao et al. 2017 ). CRISPR/Cas systems targeted and removed multiple AMR plasmids simultaneously (Yosef et al.…”

Section: Crispr/cas-based Plasmid Curing Systemsmentioning

confidence: 99%

Strategies to combat antimicrobial resistance: anti-plasmid and plasmid curing

Buckner

Ciusa

Piddock

2018

FEMS Microbiology Reviews

168

103

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Antimicrobial resistance (AMR) is a global problem hindering treatment of bacterial infections, rendering many aspects of modern medicine less effective. AMR genes (ARGs) are frequently located on plasmids, which are self-replicating elements of DNA. They are often transmissible between bacteria, and some have spread globally. Novel strategies to combat AMR are needed, and plasmid curing and anti-plasmid approaches could reduce ARG prevalence, and sensitise bacteria to antibiotics. We discuss the use of curing agents as laboratory tools including chemicals (e.g. detergents and intercalating agents), drugs used in medicine including ascorbic acid, psychotropic drugs (e.g. chlorpromazine), antibiotics (e.g. aminocoumarins, quinolones and rifampicin) and plant-derived compounds. Novel strategies are examined; these include conjugation inhibitors (e.g. TraE inhibitors, linoleic, oleic, 2-hexadecynoic and tanzawaic acids), systems designed around plasmid incompatibility, phages and CRISPR/Cas-based approaches. Currently, there is a general lack of in vivo curing options. This review highlights this important shortfall, which if filled could provide a promising mechanism to reduce ARG prevalence in humans and animals. Plasmid curing mechanisms which are not suitable for in vivo use could still prove important for reducing the global burden of AMR, as high levels of ARGs exist in the environment.

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“…Sequencing analysis revealed that all four plasmids encode replicases that are required for their replication. Thus, if these replicase genes were inactivated using the CRISPR-Cas12a-based toolkit, the plasmid would lose the ability for replication and will then be consequently cured [9,33].…”

Section: Application Of Crispr-cas12a System For Native Plasmid Curinmentioning

confidence: 99%

“…Plasmids play pivotal role in the advancement of molecular biology, and various plasmid vectors for genetic and metabolic engineering purposes have been developed using an origin of replication region from native plasmids of Z. mobilis [42]. Eight Z. mobilis strains have been completely sequenced and contain 2-8 native plasmids with different sizes [9,33]. Plasmid stability and compatibility issues will occur when an engineered plasmid with the same origin of replication is introduced into the host strain, which will limit the application of this powerful tool for strain improvement [43].…”

Section: Application Of Crispr-cas12a System For Native Plasmid Curinmentioning

confidence: 99%

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Establishment and application of a CRISPR–Cas12a assisted genome-editing system in Zymomonas mobilis

et al. 2019

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Background: Efficient and convenient genome-editing toolkits can expedite genomic research and strain improvement for desirable phenotypes. Zymomonas mobilis is a highly efficient ethanol-producing bacterium with a small genome size and desirable industrial characteristics, which makes it a promising chassis for biorefinery and synthetic biology studies. While classical techniques for genetic manipulation are available for Z. mobilis, efficient genetic engineering toolkits enabling rapidly systematic and high-throughput genome editing in Z. mobilis are still lacking. Results: Using Cas12a (Cpf1) from Francisella novicida, a recombinant strain with inducible cas12a expression for genome editing was constructed in Z. mobilis ZM4, which can be used to mediate RNA-guided DNA cleavage at targeted genomic loci. gRNAs were then designed targeting the replicons of native plasmids of ZM4 with about 100% curing efficiency for three native plasmids. In addition, CRISPR-Cas12a recombineering was used to promote gene deletion and insertion in one step efficiently and precisely with efficiency up to 90%. Combined with single-stranded DNA (ssDNA), CRISPR-Cas12a system was also applied to introduce minor nucleotide modification precisely into the genome with high fidelity. Furthermore, the CRISPR-Cas12a system was employed to introduce a heterologous lactate dehydrogenase into Z. mobilis with a recombinant lactate-producing strain constructed. Conclusions: This study applied CRISPR-Cas12a in Z. mobilis and established a genome editing tool for efficient and convenient genome engineering in Z. mobilis including plasmid curing, gene deletion and insertion, as well as nucleotide substitution, which can also be employed for metabolic engineering to help divert the carbon flux from ethanol production to other products such as lactate demonstrated in this work. The CRISPR-Cas12a system established in this study thus provides a versatile and powerful genome-editing tool in Z. mobilis for functional genomic research, strain improvement, as well as synthetic microbial chassis development for economic biochemical production.

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Using the CRISPR/Cas9 system to eliminate native plasmids of Zymomonas mobilis ZM4

Cited by 29 publications

References 28 publications

Progress and perspective on lignocellulosic hydrolysate inhibitor tolerance improvement in Zymomonas mobilis

Progress and perspective on lignocellulosic hydrolysate inhibitor tolerance improvement in Zymomonas mobilis

Strategies to combat antimicrobial resistance: anti-plasmid and plasmid curing

Establishment and application of a CRISPR–Cas12a assisted genome-editing system in Zymomonas mobilis

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