Expanding the molecular toolkit for the homoacetogen Clostridium ljungdahlii

Molitor, Bastian; Kirchner, Kristina; Henrich, Alexander; Schmitz, Simone; Rosenbaum, Miriam A.

doi:10.1038/srep31518

Cited by 54 publications

(41 citation statements)

References 43 publications

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“…So far, the technique has not been used to systematically quantify gene expression but rather to label proteins and track their intracellular localization 46,47 . pasteurianum 50 and in Clostridium ljungdahlii 51 .…”

Section: Gfp-family Fluorescent Proteinsmentioning

confidence: 99%

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Part by Part: Synthetic Biology Parts Used in Solventogenic Clostridia

et al. 2017

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The solventogenic Clostridia are of interest to the chemical industry because of their natural ability to produce chemicals such as butanol, acetone and ethanol from diverse feedstocks. Their use as whole cell factories presents multiple metabolic engineering targets that could lead to improved sustainability and profitability of Clostridium industrial processes. However, engineering efforts have been held back by the scarcity of genetic and synthetic biology tools. Over the past decade, genetic tools to enable transformation and chromosomal modifications have been developed, but the lack of a broad palette of synthetic biology parts remains one of the last obstacles to the rapid engineered improvement of these species for bioproduction. We have systematically reviewed existing parts that have been used in the modification of solventogenic Clostridia, revealing a narrow range of empirically chosen and nonengineered parts that are in current use. The analysis uncovers elements, such as promoters, transcriptional terminators and ribosome binding sites where increased fundamental knowledge is needed for their reliable use in different applications. Together, the review provides the most comprehensive list of parts used and also presents areas where an improved toolbox is needed for full exploitation of these industrially important bacteria.

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Section: Gfp-family Fluorescent Proteinsmentioning

confidence: 99%

“…ljungdahlii and C. acetobutylicum 51 . Segregation and transformation frequencies are available; however, more work is needed to determine copy number and compatibility groups.…”

Section: Plasmid Origins Of Replicationmentioning

confidence: 99%

Part by Part: Synthetic Biology Parts Used in Solventogenic Clostridia

et al. 2017

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“…Acetogens have high potential as biocatalysts, but there have been many difficulties in their industrial application due to the limited availability of genetic manipulation tools. This is because most acetogens are Gram-positive bacteria and have a thick cell wall, which makes them recalcitrant to receiving foreign DNA molecules [ 10 , 23 , 24 ]. Nevertheless, plasmid-based gene expression methods developed for Clostridium species have been shown to be functional in other acetogens.…”

Section: Development Of Genetic Manipulation Tools In Acetogensmentioning

confidence: 99%

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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“…Therefore, it is essential that a sufficient number of cells receive the CRISPR-Cas system to ensure that enough cells survive a DNA cleavage by undergoing the inefficient homology-directed repair process with donor DNAs (15). This renders the CRISPR-Cas system even more difficult for acetogenic bacteria, which are typically recalcitrant to receiving foreign DNA (10,12,17). Consequently, the process of cleavage-and-repairing, which is typically considered the important advantage of conventional CRISPR-Cas systems, becomes a bottleneck to perform CRISPR-Casbased genome editing in acetogenic bacteria.…”

Section: Introductionmentioning

confidence: 99%

Reprogramming acetogenic bacteria with CRISPR-targeted base editingviadeamination

Xia

Casini

Schulz

et al. 2020

Preprint

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Acetogenic bacteria are rising in popularity as chassis microbes in biotechnology due to their capability of converting inorganic one-carbon (C1) gases to organic chemicals. To fully uncover the potential of acetogenic bacteria, synthetic-biology tools are imperative to either engineer designed functions or to interrogate the physiology. Here, we report a genome-editing tool at a one-nucleotide resolution, namely base editing, for acetogenic bacteria based on CRISPR-targeted deamination. This tool combines nuclease deactivated Cas9 with activation-induced cytidine deaminase to enable cytosine-tothymine substitution without DNA cleavage, homology-directed repair, and donor DNA, which are generally the bottlenecks for applying conventional CRISPR-Cas systems in bacteria. We designed and validated a modularized base-editing tool in the model acetogenic bacterium Clostridium ljungdahlii. The editing principles were investigated, and an in-silico analysis revealed the capability of base editing across the genome.Moreover, genes related to acetate and ethanol production were disrupted individually by installing premature STOP codons to reprogram carbon flux towards improved acetate production. This resulted in engineered C. ljungdahlii strains with the desired phenotypes and stable genotypes. Our base-editing tool promotes the application and research in acetogenic bacteria and provides a blueprint to upgrade CRISPR-Cas-based genome editing in bacteria in general. SignificanceAcetogenic bacteria metabolize one-carbon (C1) gases, such as industrial waste gases, to produce fuels and commodity chemicals. However, the lack of efficient genemanipulation approaches hampers faster progress in the application of acetogenic bacteria in biotechnology. We developed a CRISPR-targeted base-editing tool at a onenucleotide resolution for acetogenic bacteria. Our tool illustrates great potential in engineering other A-T-rich bacteria and links designed single-nucleotide variations with biotechnology. It provides unique advantages for engineering industrially relevant bacteria without creating genetically modified organisms (GMOs) under the legislation of many countries. This base-editing tool provides an example for adapting CRISPR-Cas systems in bacteria, especially those that are highly sensitive to heterologously expressed Cas proteins and have limited ability of receiving foreign DNA.

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Expanding the molecular toolkit for the homoacetogen Clostridium ljungdahlii

Cited by 54 publications

References 43 publications

Part by Part: Synthetic Biology Parts Used in Solventogenic Clostridia

Part by Part: Synthetic Biology Parts Used in Solventogenic Clostridia

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

Reprogramming acetogenic bacteria with CRISPR-targeted base editingviadeamination

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