CRISPR/Cas9-coupled recombineering for metabolic engineering of Corynebacterium glutamicum

Cho, Jae Sung; Choi, Kyeong Rok; Prabowo, Cindy Pricilia Surya; Shin, Jae Ho; Yang, Dongsoo; Jang, Jaedong; Lee, Sang Yup

doi:10.1016/j.ymben.2017.06.010

Cited by 181 publications

(132 citation statements)

References 39 publications

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“…Even with development of the powerful CRISPR/Cas system, multiplex engineering of bacterial genome is hard to realize due to the low homologous recombination rate in most bacteria (J. Liu et al, ). C. glutamicum is a representative, which is hard to engineer but scientifically and industrially important (Cho et al, ; Y. Jiang et al, ). The base editing method is expected to revolutionize bacterial genome engineering and facilitate fast strain development because programmability and efficient editing can be achieved without double‐stranded DNA break or a donor template (Komor et al, ; Nishida et al, ).…”

Section: Discussionmentioning

confidence: 99%

Expanding targeting scope, editing window, and base transition capability of base editing in Corynebacterium glutamicum

Wang

Liu

et al. 2019

Biotech & Bioengineering

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CRISPR/Cas9‐guided cytidine deaminase enables C:G to T:A base editing in bacterial genome without introduction of lethal double‐stranded DNA break, supplement of foreign DNA template, or dependence on inefficient homologous recombination. However, limited by genome‐targeting scope, editing window, and base transition capability, the application of base editing in metabolic engineering has not been explored. Herein, four Cas9 variants accepting different protospacer adjacent motif (PAM) sequences were used to increase the genome‐targeting scope of bacterial base editing. After a comprehensive evaluation, we demonstrated that PAM requirement of bacterial base editing can be relaxed from NGG to NG using the Cas9 variants, providing 3.9‐fold more target loci for gene inactivation in Corynebacterium glutamicum. Truncated or extended guide RNAs were employed to expand the canonical 5‐bp editing window to 7‐bp. Bacterial adenine base editing was also achieved with Cas9 fused to adenosine deaminase. With these updates, base editing can serve as an enabling tool for fast metabolic engineering. To demonstrate its potential, base editing was used to deregulate feedback inhibition of aspartokinase via amino acid substitution for lysine overproduction. Finally, a user‐friendly online tool named gBIG was provided for designing guide RNAs for base editing‐mediated inactivation of given genes in any given sequenced genome (http://www.ibiodesign.net/gBIG).

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Section: Discussionmentioning

confidence: 99%

Expanding targeting scope, editing window, and base transition capability of base editing in Corynebacterium glutamicum

Wang

Liu

et al. 2019

Biotech & Bioengineering

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“…Deletion of a l ‐glutamate export factor ( Ncgl1221 ), a GABA reuptake system component ( gabP ), and GABA transaminase ( gabT ) decreased the export of l ‐glutamate and excretion of GABA into the culture medium and disrupted a competing pathway for the conversion of GABA into succinyl semialdehyde. The resulting strain, which used glutamate decarboxylase encoded by gadB2 from L. brevis, produced 27.5 ±0.30 g L −1 of GABA (Table ) . To improve glutamate decarboxylase activity, the availability of pyridoxal 5′‐phosphate cofactor was increased by the heterologous expression of pyridoxal kinase ( plk ) from L. plantarum .…”

Section: Production Of Polyamide Monomersmentioning

confidence: 99%

Recent advances in metabolic engineering of Corynebacterium glutamicum as a potential platform microorganism for biorefinery

Baritugo

Kim

David

et al. 2018

Biofuels Bioprod Bioref

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The fermentative production of platform chemicals in biorefineries is a sustainable alternative to current petroleum‐refining processes. Industrial microorganisms, such as Escherichia coli, Saccharomyces cerevisiae, and Corynebacterium glutamicum, have been engineered as microbial cell factories that are able to utilize biomass for the production of value‐added platform chemicals and polymers. Compared to E. coli and S. cerevisiae, C. glutamicum displays weak carbon catabolite repression and can co‐utilize mixed sugars as carbon sources, without any significant growth retardation. Pathways for the utilization of alternative carbon sources, such as d‐xylose and l‐arabinose from lignocellulosic biomass, lactose and galactose from whey, glycerol from biodiesel, and methanol from natural gas refineries, have been evaluated for chemical production. However, the application of C. glutamicum in biorefineries is limited because it does not secrete hydrolases for the efficient utilization of cellulose, xylan, and starch from lignocellulosic and starch biomass. To solve the limitation, C. glutamicum has been engineered for the consolidated bioprocessing of biomass by the heterologous expression of amylolytic and cellulolytic enzymes. Recently, C. glutamicum has been extensively engineered for polyamide monomer production owing to its ability to produce l‐lysine and l‐glutamate. This review summarizes recent advances in the development of C. glutamicum strains that can utilize renewable biomass resources for the production of industrially important chemicals. It highlights recent progress in metabolic engineering for the production of polyamide monomers. © 2018 Society of Chemical Industry and John Wiley & Sons, Ltd

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“…By extension, 'paired nickases', i.e. using two adjacent gRNAs with Cas9n, can efficiently introduce both indel mutations and HR events with a single-stranded DNA oligo-nucleotide donor template in mammalian cells [28,10,80]. Complete disruption of the endonuclease activities (RuvC D10A along with HNH H840A ) results in a catalytically inactive Cas9, or dead-Cas9 (dCas9) [78,79].…”

Section: Expanding Cas9 Features Through Enzyme Engineeringmentioning

confidence: 99%

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

Ferreira

David

Nielsen

2018

Journal of Industrial Microbiology and Biotechnology

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Clustered regularly interspaced short palindromic repeats (CRISPR) is poised to become one of the key scientific discoveries of the twenty-first century. Originating from prokaryotic and archaeal immune systems to counter phage invasions, CRISPRbased applications have been tailored for manipulating a broad range of living organisms. From the different elucidated types of CRISPR mechanisms, the type II system adapted from Streptococcus pyogenes has been the most exploited as a tool for genome engineering and gene regulation. In this review, we describe the different applications of CRISPR/Cas9 technology in the industrial biotechnology field. Next, we detail the current status of the patent landscape, highlighting its exploitation through different companies, and conclude with future perspectives of this technology.

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CRISPR/Cas9-coupled recombineering for metabolic engineering of Corynebacterium glutamicum

Cited by 181 publications

References 39 publications

Expanding targeting scope, editing window, and base transition capability of base editing in Corynebacterium glutamicum

Expanding targeting scope, editing window, and base transition capability of base editing in Corynebacterium glutamicum

Recent advances in metabolic engineering of Corynebacterium glutamicum as a potential platform microorganism for biorefinery

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

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