TargeTron Technology Applicable in Solventogenic Clostridia: Revisiting 12 Years’ Advances

Wen, Zhiqiang; Lu, Minrui; Ledesma-Amaro, Rodrigo; Qi, Li; Jin, Mingjie; Yang, Sheng

doi:10.1002/biot.201900284

Cited by 15 publications

(15 citation statements)

References 128 publications

(242 reference statements)

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“…For example, it usually takes a long period (about 8 days) to complete one round of gene editing. Besides, the low efficiency of the second single crossover prevents the desired mutant to be obtained, even after a large number of colony PCR screening (Wen et al, 2020). To ensure the availability of desired mutant strains, counter-selectable markers and nuclease systems are introduced.…”

Section: Classic Allelic-exchange-based Genome Editing Toolsmentioning

confidence: 99%

“…Base editing can create a missense mutation or null mutation in a gene via base substitution without introducing a DSB (Komor et al, 2016;Nishida et al, 2016), which is especially suitable for strains with inefficient HR (like C. glutamicum), and has attracted increasing attention (Wen et al, 2020). developed a cytosine base editor (CBE) applicable in C. glutamicum based on activation-induced cytidine deaminase (AID) and the CRISPR/Cas9 system (Figure 1E), which can efficiently achieve C-T conversion with efficiencies up to 100%, 87.2%, and 23.3% for single-, double-, and triple-locus editing, respectively.…”

Section: Revolutionary Crispr/cas-based Genome Editing Toolsmentioning

confidence: 99%

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Advances and Perspectives for Genome Editing Tools of Corynebacterium glutamicum

et al. 2021

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Corynebacterium glutamicum has been considered a promising synthetic biological platform for biomanufacturing and bioremediation. However, there are still some challenges in genetic manipulation of C. glutamicum. Recently, more and more genetic parts or elements (replicons, promoters, reporter genes, and selectable markers) have been mined, characterized, and applied. In addition, continuous improvement of classic molecular genetic manipulation techniques, such as allelic exchange via single/double-crossover, nuclease-mediated site-specific recombination, RecT-mediated single-chain recombination, actinophages integrase-mediated integration, and transposition mutation, has accelerated the molecular study of C. glutamicum. More importantly, emerging gene editing tools based on the CRISPR/Cas system is revolutionarily rewriting the pattern of genetic manipulation technology development for C. glutamicum, which made gene reprogramming, such as insertion, deletion, replacement, and point mutation, much more efficient and simpler. This review summarized the recent progress in molecular genetic manipulation technology development of C. glutamicum and discussed the bottlenecks and perspectives for future research of C. glutamicum as a distinctive microbial chassis.

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Section: Classic Allelic-exchange-based Genome Editing Toolsmentioning

confidence: 99%

Section: Revolutionary Crispr/cas-based Genome Editing Toolsmentioning

confidence: 99%

Advances and Perspectives for Genome Editing Tools of Corynebacterium glutamicum

et al. 2021

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“…The TargeTron gene-editing technique, which depends on the mobility of group-II introns, was also found to be suitable for genetic manipulations in Clostridium. In addition, mesophilic-TargeTron and thermo-TargeTron technology was used for metabolic engineering and identification of functional genes (Wen et al, 2019).…”

Section: Utilization Of Extremophiles and Enzymes In Liquid Biofuelsmentioning

confidence: 99%

Recent Development of Extremophilic Bacteria and Their Application in Biorefinery

Zhu

Adebisi

Ahmad

et al. 2020

Front. Bioeng. Biotechnol.

116

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The biorefining technology for biofuels and chemicals from lignocellulosic biomass has made great progress in the world. However, mobilization of laboratory research toward industrial setup needs to meet a series of criteria, including the selection of appropriate pretreatment technology, breakthrough in enzyme screening, pathway optimization, and production technology, etc. Extremophiles play an important role in biorefinery by providing novel metabolic pathways and catalytically stable/robust enzymes that are able to act as biocatalysts under harsh industrial conditions on their own. This review summarizes the potential application of thermophilic, psychrophilic alkaliphilic, acidophilic, and halophilic bacteria and extremozymes in the pretreatment, saccharification, fermentation, and lignin valorization process. Besides, the latest studies on the engineering bacteria of extremophiles using metabolic engineering and synthetic biology technologies for high-efficiency biofuel production are also introduced. Furthermore, this review explores the comprehensive application potential of extremophiles and extremozymes in biorefinery, which is partly due to their specificity and efficiency, and points out the necessity of accelerating the commercialization of extremozymes.

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“…Recently, Clostridium cellulovorans has been engineered to produce 4.96 g/L butanol by consolidated bioprocessing (CBP) using pull‐push modular metabolic engineering, which implied the great potential of engineered pure cultures (Wen, Ledesma‐Amaro, Lu, Jin, & Yang, 2020). However, this requires complicated genetic modification, which is rather difficult in Clostridium because of the lack of efficient and relevant genetic tools (Wen, Li, et al, 2020; Wen, Lu, et al, 2020). Compared with pure cultures, mixed‐cultures of cellulolytic and solventogenic Clostridia provide a more convenient and feasible approach to achieve butanol production by CBP (Jiang et al, 2018; Wen, Li, et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

Combined evolutionary engineering and genetic manipulation improve low pH tolerance and butanol production in a synthetic microbial Clostridium community

Wen

Ledesma-Amaro

et al. 2020

Biotech & Bioengineering

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Synthetic microbial communities have become a focus of biotechnological research since they can overcome several of the limitations of single‐specie cultures. A paradigmatic example is Clostridium cellulovorans DSM 743B, which can decompose lignocellulose but cannot produce butanol. Clostridium beijerinckii NCIMB 8052 however, is unable to use lignocellulose but can produce high amounts of butanol from simple sugars. In our previous studies, both organisms were cocultured to produce butanol by consolidated bioprocessing. However, such consolidated bioprocessing implementation strongly depends on pH regulation. Since low pH (pH 4.5–5.5) is required for butanol fermentation, C. cellulovorans cannot grow well and saccharify sufficient lignocellulose to feed both strains at a pH below 6.4. To overcome this bottleneck, this study engineered C. cellulovorans by adaptive laboratory evolution, inactivating cell wall lyases genes (Clocel_0798 and Clocel_2169), and overexpressing agmatine deiminase genes (augA, encoded by Cbei_1922) from C. beijerinckii NCIMB 8052. The generated strain WZQ36: 743B*6.0*3△lyt0798△lyt2169‐(pXY1‐Pthl‐augA) can tolerate a pH of 5.5. Finally, the alcohol aldehyde dehydrogenase gene adhE1 from Clostridium acetobutylicum ATCC 824 was introduced into the strain to enable butanol production at low pH, in coordination with solvent fermentation of C. beijerinckii in consortium. The engineered consortium produced 3.94 g/L butanol without pH control within 83 hr, which is more than 5‐fold of the level achieved by wild consortia under the same conditions. This exploration represents a proof of concept on how to combine metabolic and evolutionary engineering to coordinate coculture of a synthetic microbial community.

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TargeTron Technology Applicable in Solventogenic Clostridia: Revisiting 12 Years’ Advances

Cited by 15 publications

References 128 publications

Advances and Perspectives for Genome Editing Tools of Corynebacterium glutamicum

Advances and Perspectives for Genome Editing Tools of Corynebacterium glutamicum

Recent Development of Extremophilic Bacteria and Their Application in Biorefinery

Combined evolutionary engineering and genetic manipulation improve low pH tolerance and butanol production in a synthetic microbial Clostridium community

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