Genome Engineering of <i>Eubacterium limosum</i> Using Expanded Genetic Tools and the CRISPR-Cas9 System

Shin, Jisoo; Kang, Sung-Jin; Song, Yoseb; Jin, Sangrak; Lee, Jin Soo; Lee, Jung-Kul; Kim, Dong Rip; Kim, Sun Chang; Cho, Suhyung; Cho, Byung-Kwan

doi:10.1021/acssynbio.9b00150

Cited by 47 publications

(79 citation statements)

References 46 publications

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“…To prepare the electrocompetent strains, a previously modified protocol was used (Shin et al, 2019). The cells were cultured in 100 mL of DSM 135 medium supplemented with 5 g/L glucose.…”

Section: Transformationmentioning

confidence: 99%

Adaptive Laboratory Evolution of Eubacterium limosum ATCC 8486 on Carbon Monoxide

et al. 2020

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Acetogens are naturally capable of metabolizing carbon monoxide (CO), a component of synthesis gas (syngas), for autotrophic growth in order to produce biomass and metabolites such as acetyl-CoA via the Wood-Ljungdahl pathway. However, the autotrophic growth of acetogens is often inhibited by the presence of high CO concentrations because of CO toxicity, thus limiting their biosynthetic potential for industrial applications. Herein, we implemented adaptive laboratory evolution (ALE) for growth improvement of Eubacterium limosum ATCC 8486 under high CO conditions. The strain evolved under syngas conditions with 44% CO over 150 generations, resulting in a significant increased optical density (600 nm) and growth rate by 2.14 and 1.44 folds, respectively. In addition, the evolved populations were capable of proliferating under CO concentrations as high as 80%. These results suggest that cell growth is enhanced as beneficial mutations are selected and accumulated, and the metabolism is altered to facilitate the enhanced phenotype. To identify the causal mutations related to growth improvement under high CO concentrations, we performed whole genome resequencing of each population at 50-generation intervals. Interestingly, we found key mutations in CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex coding genes, acsA and cooC. To characterize the mutational effects on growth under CO, we isolated single clones and confirmed that the growth rate and CO tolerance level of the single clone were comparable to those of the evolved populations and wild type strain under CO conditions. Furthermore, the evolved strain produced 1.34 folds target metabolite acetoin when compared to the parental strain while introducing the biosynthetic pathway coding genes to the strains. Consequently, this study demonstrates that the mutations in the CODH/ACS complex affect autotrophic growth enhancement in the presence of CO as well as the CO tolerance of E. limosum ATCC 8486.

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“…To prepare the electrocompetent strains, a previously modified protocol was used (Shin et al, 2019). The cells were cultured in 100 mL of DSM 135 medium supplemented with 5 g/L glucose.…”

Section: Transformationmentioning

confidence: 99%

Adaptive Laboratory Evolution of Eubacterium limosum ATCC 8486 on Carbon Monoxide

et al. 2020

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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%

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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“…We designed a modularized system to enable fast generation of the base-editing plasmid series. The employed plasmid backbone, replicon for clostridia, antibiotic resistances markers, and the dCas9 protein have been separately demonstrated to be functional in various species in the order Clostridiales, including Acetobacterium woodii (dCas9 has not yet been validated) (31,32), Eubacterium limosum (13), and Clostridium autoethanogenum (14). Accordingly, the system could be easily generalized in acetogenic bacteria, which mainly belong to the order Clostridiales.…”

Section: An Expanded Synthetic Biology Toolkit For Acetogenic Bacteriamentioning

confidence: 99%

“…This would improve the production of certain platform chemicals that require acetate as an intermediate (33,34). For instance, our acetateproducing strain can be considered to further improve the two-stage bioprocess for singlecell protein production from C1 gases, with acetate as the carbon-fixing intermediate 13 product before being fed to aerobic yeasts, especially for industrial gases that contain CO (4). Importantly, single-nucleotide variations generated by base editing are clean mutations in the genome, which may also occur in natural evolution.…”

Section: Linking Base Editing With Microbial C1 Utilizationmentioning

confidence: 99%

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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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Genome Engineering of Eubacterium limosum Using Expanded Genetic Tools and the CRISPR-Cas9 System

Cited by 47 publications

References 46 publications

Adaptive Laboratory Evolution of Eubacterium limosum ATCC 8486 on Carbon Monoxide

Adaptive Laboratory Evolution of Eubacterium limosum ATCC 8486 on Carbon Monoxide

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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