EasyCloneYALI: CRISPR/Cas9‐Based Synthetic Toolbox for Engineering of the Yeast <i>Yarrowia lipolytica</i>

Holkenbrink, Carina; Dam, Marie Inger; Kildegaard, Kanchana Rueksomtawin; Beder, Johannes; Dahlin, Jonathan; Belda, David Doménech; Borodina, Irina

doi:10.1002/biot.201700543

Cited by 172 publications

(223 citation statements)

References 28 publications

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“…Our previous work has shown that it is difficult to express heterologous genes in C. tropicalis , so codon optimization of the entire gene would be the best way forward (L. Zhang et al, ). Using either the monocistronic or polycistronic transcript version, the efficiency of double gene deletion in C. tropicalis was lower than that in S. cerevisiae (Y. P. Zhang et al, ) and industrial yeast (Lian et al, ), but higher than that in Yarrowia lipolytica (Holkenbrink et al, ). Part of the reason for this lack of efficiency may be that the “HH‐N20 target sequence‐sgRNA scaffold‐HDV” construction is not the most suitable for multiple RNA processing in C. tropicalis .…”

Section: Discussionmentioning

confidence: 99%

A CRISPR–Cas9 system for multiple genome editing and pathway assembly in Candida tropicalis

Zhang

Liu

et al. 2019

Biotech & Bioengineering

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Genetic manipulation is among the most important tools for synthetic biology; however, modifying multiple genes is extremely time‐consuming and can sometimes be impossible when dealing with gene families. Here, we present a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR‐associated protein 9 (Cas9) system for use in the diploid yeast Candida tropicalis that is vastly superior to traditional techniques. This system enables the rapid and reliable introduction of multiple genetic deletions or mutations, as well as a stable expression using an integrated CRISPR–Cas9 cassette or a transient CRISPR–Cas9 cassette, together with a short donor DNA. We further show that the system can be used to promote the in vivo assembly of multiple DNA fragments and their stable integration into a target locus (or loci) in C. tropicalis. Based on this system, we present a platform for the biosynthesis of β‐carotene and its derivatives. These results enable the practical application of C. tropicalis and the application of the system to other organisms.

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Section: Discussionmentioning

confidence: 99%

A CRISPR–Cas9 system for multiple genome editing and pathway assembly in Candida tropicalis

Zhang

Liu

et al. 2019

Biotech & Bioengineering

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“…Very recently, Holkenbrink et al . () developed a toolkit based on an alternative USER cloning approach, which allows the integration of one to two TUs at intergenic regions called EasyCloneYALI. The system largely utilizes a ku70 mutant background and a CRISPR/Cas9 strategy, and it carries out improved homologous recombination at specific non‐coding loci.…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, with a one-pot reaction, we are able to assemble three TUs, a marker and various integration site sequences, which correspond to the assembly of twelve fragments in a destination vector. Very recently, Holkenbrink et al (2018) developed a toolkit based on an alternative USER cloning approach, which allows the integration of one to two TUs at intergenic regions called EasyCloneYALI. The system largely utilizes a ku70 mutant background and a CRISPR/Cas9 strategy, and it carries out improved homologous recombination at specific non-coding loci.…”

Section: Discussionmentioning

confidence: 99%

A modular Golden Gate toolkit for Yarrowia lipolytica synthetic biology

Larroude

Park

Soudier

et al. 2019

Microbial Biotechnology

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Summary The oleaginous yeast Yarrowia lipolytica is an established host for the bio‐based production of valuable compounds and an organism for which many genetic tools have been developed. However, to properly engineer Y. lipolytica and take full advantage of its potential, we need efficient, versatile, standardized and modular cloning tools. Here, we present a new modular Golden Gate toolkit for the one‐step assembly of three transcription units that includes a selective marker and sequences for genome integration. Perfectly suited to a combinatorial approach, it contains nine different validated promoters, including inducible promoters, which allows expression to be fine‐tuned. Moreover, this toolbox incorporates six different markers (three auxotrophic markers, two antibiotic‐resistance markers and one metabolic marker), which allows the fast sequential construction and transformation of multiple elements. In total, the toolbox contains 64 bricks, and it has been validated and characterized using three different fluorescent reporter proteins. Additionally, it was successfully used to assemble and integrate a three‐gene pathway allowing xylose utilization by Y. lipolytica. This toolbox provides a powerful new tool for rapidly engineering Y. lipolytica strains and is available to the community through Addgene.

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“…This methodology combined with the multigene overexpression system for heterologous genes (Ledesma-Amaro et al, 2018) will allow to avoid the use of selectable markers for metabolic engineering applications in A. gossypii. Similar toolkits have been described in other biotechnological microorganisms such as Yarrowia lypolytica, thus enabling the rapid engineering of metabolic pathways in that oleaginous yeast (Wong et al, 2017;Holkenbrink et al, 2018;Larroude et al, 2018).…”

Section: Introduction Of Point Mutations Using the Crispr/cas9 Systemmentioning

confidence: 98%

One‐vector CRISPR/Cas9 genome engineering of the industrial fungus Ashbya gossypii

Jiménez

Muñoz‐Fernández

Ledesma‐Amaro

et al. 2019

Microbial Biotechnology

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Summary The filamentous fungus Ashbya gossypii is currently used for the industrial production of vitamin B2. Furthermore, the ability of A. gossypii to grow using low‐cost substrates together with the inexpensive downstream processing makes this fungus an attractive biotechnological chassis. Indeed, the production in A. gossypii of other high‐added value compounds such as folic acid, nucleosides and biolipids has been described. Hence, the development of new methods to expand the molecular toolkit for A. gossypii genomic manipulation constitutes an important issue for the biotechnology of this fungus. In this work, we present a one‐vector CRISPR/Cas9 system for genomic engineering of A. gossypii. We demonstrate the efficiency of the system as a marker‐less approach for nucleotide deletions and substitutions both with visible and invisible phenotypes. Particularly, the system has been validated for three types of genomic editions: gene inactivation, the genomic erasure of loxP scars and the introduction of point mutations. We anticipate that the use of the CRISPR/Cas9 system for A. gossypii will largely contribute to facilitate the genomic manipulations of this industrial fungus in a marker‐less manner.

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EasyCloneYALI: CRISPR/Cas9‐Based Synthetic Toolbox for Engineering of the Yeast Yarrowia lipolytica

Cited by 172 publications

References 28 publications

A CRISPR–Cas9 system for multiple genome editing and pathway assembly in Candida tropicalis

A CRISPR–Cas9 system for multiple genome editing and pathway assembly in Candida tropicalis

A modular Golden Gate toolkit for Yarrowia lipolytica synthetic biology

One‐vector CRISPR/Cas9 genome engineering of the industrial fungus Ashbya gossypii

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