CRISPR-Cas12a-Mediated Gene Deletion and Regulation in <i>Clostridium ljungdahlii</i> and Its Application in Carbon Flux Redirection in Synthesis Gas Fermentation

Zhao, Ran; Liu, Yanqiang; Zhang, Huan; Chai, Chaochao; Wang, Jin; Jiang, Weihong; Gu, Yang

doi:10.1021/acssynbio.9b00033

Cited by 61 publications

(80 citation statements)

References 33 publications

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“…To disrupt ljungdin production, the ljuB gene was repressed via the Cas12a‐mediated CRISPRi system developed for C. ljungdahlii . [ 32 ] Triplicate batch fermentations with the resulting mutant and the wild type C. ljungdahlii were then performed, and the growth curve as well as ethanol and acetate production were measured. The results showed that ljuB ‐repressed mutant apparently grew much faster than the wild type, as the OD 600 values were about 1.6‐, 1.3‐, and 1.1‐folds higher than those of wild type at 24, 48, and 72 h, respectively (Figure 5A).…”

Section: Resultsmentioning

confidence: 99%

The SCIFF‐Derived Ranthipeptides Participate in Quorum Sensing in Solventogenic Clostridia

Chen

Yang

et al. 2020

Biotechnology Journal

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Ranthipeptides, defined as radical non-thioether-containing peptides, are a newly emerging class of natural products belonging to the ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily. Ranthipeptides are shown to be widespread in the bacterial kingdom, whereas heretofore their biological functions remain completely elusive. In this work, putative ranthipeptides are investigated from two solventogenic clostridia, Clostridium beijerinckii and Clostridium ljungdahlii, which are derived from the so-called six Cys in forty-five residues (SCIFF) family of precursor peptides. A series of analysis show that these two ranthipeptides participate in quorum sensing and controlling cellular metabolism. These results highlight the diverse biological functions of the ever-increasing family of RiPP natural products and showcase the potential to engineer industrially interesting organisms by manipulating their RiPP biosynthetic pathways.

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Section: Resultsmentioning

confidence: 99%

The SCIFF‐Derived Ranthipeptides Participate in Quorum Sensing in Solventogenic Clostridia

Chen

Yang

et al. 2020

Biotechnology Journal

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“…Nevertheless, plasmid-based gene expression methods developed for Clostridium species have been shown to be functional in other acetogens. In addition, many metabolic engineering efforts have recently been made using homologous recombination (HR) [ 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ], ClosTron [ 28 , 36 , 37 , 38 , 39 ] and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) along with its CRISPR-associated (Cas) protein (CRISPR-Cas) system [ 32 , 40 , 41 , 42 , 43 , 44 , 45 ] to improve the production of value-added biochemicals from C1 gases.…”

Section: Development Of Genetic Manipulation Tools In Acetogensmentioning

confidence: 99%

“…43%). Cas12a recognizes AT-rich PAM sequences, “TTN”, which are more abundant than “NGG” for Cas9 in the genome [ 42 ]. One advantage of Cas12a is that its crRNA processing activity enhances the simplicity of multiplex genome editing [ 83 , 84 ].…”

Section: Development Of Genetic Manipulation Tools In Acetogensmentioning

confidence: 99%

“…A similar approach has been shown in C. ljungdahlii , where they redirected acetyl-CoA flux from acetate to increase the production of 3-hydroxybutyric acid (3HB) [ 145 ]. Likewise, an ethanol synthesis gene ( adhE1 ) was repressed with dCas12a instead of dCas9, and the redistributed carbon flux improved the titer of butyric acid [ 42 ].…”

Section: Synthetic Biology Approach On Acetogensmentioning

confidence: 99%

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Synthetic Biology on Acetogenic Bacteria for Highly Efficient Conversion of C1 Gases to Biochemicals

Jin

Bae

Song

et al. 2020

IJMS

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Synthesis gas, which is mainly produced from fossil fuels or biomass gasification, consists of C1 gases such as carbon monoxide, carbon dioxide, and methane as well as hydrogen. Acetogenic bacteria (acetogens) have emerged as an alternative solution to recycle C1 gases by converting them into value-added biochemicals using the Wood-Ljungdahl pathway. Despite the advantage of utilizing acetogens as biocatalysts, it is difficult to develop industrial-scale bioprocesses because of their slow growth rates and low productivities. To solve these problems, conventional approaches to metabolic engineering have been applied; however, there are several limitations owing to the lack of required genetic bioparts for regulating their metabolic pathways. Recently, synthetic biology based on genetic parts, modules, and circuit design has been actively exploited to overcome the limitations in acetogen engineering. This review covers synthetic biology applications to design and build industrial platform acetogens.

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“…Single CRISPR/Cpf1 system plasmid can make multiple mutants in a single application (Zetsche et al, 2017;Hong et al, 2018;Zhang et al, 2018a). CRISPR/Cpf1 system has been applied in C. ljungdahlii, C. difficile, and C. beijerinckii (Hong et al, 2018;Zhang et al, 2018a;Zhao et al, 2019).…”

Section: Crispr/cas Based Clostridia Genome Engineeringmentioning

confidence: 99%

Synthetic Biology Tools for Genome and Transcriptome Engineering of Solventogenic Clostridium

Kwon¹,

Paari²,

Malaviya³

et al. 2020

Front. Bioeng. Biotechnol.

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Strains of Clostridium genus are used for production of various value-added products including fuels and chemicals. Development of any commercially viable production process requires a combination of both strain and fermentation process development strategies. The strain development in Clostridium sp. could be achieved by random mutagenesis, and targeted gene alteration methods. However, strain improvement in Clostridium sp. by targeted gene alteration method was challenging due to the lack of efficient tools for genome and transcriptome engineering in this organism. Recently, various synthetic biology tools have been developed to facilitate the strain engineering of solventogenic Clostridium. In this review, we consolidated the recent advancements in toolbox development for genome and transcriptome engineering in solventogenic Clostridium. Here we reviewed the genome-engineering tools employing mobile group II intron, pyrE alleles exchange, and CRISPR/Cas9 with their application for strain development of Clostridium sp. Next, transcriptome engineering tools such as untranslated region (UTR) engineering and synthetic sRNA techniques were also discussed in context of Clostridium strain engineering. Application of any of these discussed techniques will facilitate the metabolic engineering of clostridia for development of improved strains with respect to requisite functional attributes. This might lead to the development of an economically viable butanol production process with improved titer, yield and productivity.

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CRISPR-Cas12a-Mediated Gene Deletion and Regulation in Clostridium ljungdahlii and Its Application in Carbon Flux Redirection in Synthesis Gas Fermentation

Cited by 61 publications

References 33 publications

The SCIFF‐Derived Ranthipeptides Participate in Quorum Sensing in Solventogenic Clostridia

The SCIFF‐Derived Ranthipeptides Participate in Quorum Sensing in Solventogenic Clostridia

Synthetic Biology on Acetogenic Bacteria for Highly Efficient Conversion of C1 Gases to Biochemicals

Synthetic Biology Tools for Genome and Transcriptome Engineering of Solventogenic Clostridium

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