CRISPR/Cas system for yeast genome engineering: advances and applications

Šťovíček, Vratislav; Holkenbrink, Carina; Borodina, Irina

doi:10.1093/femsyr/fox030

Cited by 151 publications

(136 citation statements)

References 78 publications

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“…Of course one cannot also fail to mention the variants of CRISPR/Cas9 gene editing technology that allow almost unlimited tinkering (e.g. in yeast [309,310]). …”

Section: Control Factor Expression Engineeringmentioning

confidence: 99%

Control of metabolite efflux in microbial cell factories: current advances and future prospects

Kell¹

2018

Preprint

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The yield and ease of purification of biotechnological products are typically greatly enhanced if they can be secreted into the external medium. Although not universally appreciated, however, small molecule biotechnological products do not ‘float across’ the phospholipid bilayer portion of biological membranes, and thus they need transporters to assist their passage into the extramembrane and extracellular spaces. Some of these transporters may be reversible, equilibrative transporters that might more normally be used for uptake, while others may have an efflux directionality imposed on them via suitable energy coupling mechanisms. Despite the energetic costs of small molecule synthesis, natural evolution does in fact provide a number of mechanisms by which the secretion of such products can actually enhance fitness. Where available these provide useful starting points, and assaying for such activities is crucial. In particular, in a systems biology approach, we first need to identify such/suitable activities before we can seek to increase them. They may then be improved through promoter engineering, via manipulation of control elements, or by directed evolution of the transporter proteins themselves. Modern methods of synthetic biology provide enormous opportunities for all kinds of efflux transporter engineering; they are just beginning to be realised.

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“…Of course one cannot also fail to mention the variants of CRISPR/Cas9 gene editing technology that allow almost unlimited tinkering (e.g. in yeast [309,310]). …”

Section: Control Factor Expression Engineeringmentioning

confidence: 99%

Control of metabolite efflux in microbial cell factories: current advances and future prospects

Kell¹

2018

Preprint

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“…Another major limitation is the relatively low copy numbers of gRNA expressing plasmid in industrial yeast strains. So far, several strategies have been adopted for the expression of Cas9 and gRNA and most of the studies have found it necessary to express gRNA on a 2 μ based multicopy plasmid for efficient genome engineering in S. cerevisiae (DiCarlo et al, ; Stovicek et al, ; Stovicek, Holkenbrink, & Borodina, ). Interestingly, although the 2 μ‐based plasmids generally have relatively high copy numbers (20–30 copies/cell) with auxotrophic markers (i.e., HIS3 , TRP1 , LEU2 , and URA3 ), they can be as low as 3–5 copies/cell when antibiotic markers (i.e., KanMX and HygB ) are included (Karim, Curran, & Alper, ; Lian, Jin, & Zhao, ).…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains

Lian

Bao

et al. 2018

Biotech & Bioengineering

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The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. However, its application in manipulating industrial yeast strains is less successful, probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to construct xylose-fermenting, lactate-producing industrial yeast strains, in which ALD6, PHO13, LEU2, and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories.

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“…Of particular relevance is the development of optimized CRISPR-Cas9 methods to engineer diploid and polyploid industrial yeast strains (Stovicek, Borja, Forster, & Borodina, 2015;Zhang et al, 2014). The history and various applications of genome editing have been discussed in-depth in the following reviews by Fraczek, Naseeb, and Delneri (2018) and Stovicek, Holkenbrink, and Borodina (2017).…”

Section: Crispr For Rapid Multiplex Engineering Of Cell Factoriesmentioning

confidence: 99%

The renaissance of yeasts as microbial factories in the modern age of biomanufacturing

Payen

Thompson

2019

Yeast

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Yeasts are essential for many processes, including the production of some of our most beloved foods and beverages. Less is known to the public about the far‐reaching impacts of yeasts on other products such as biofuels, pharmaceuticals, and industrial products. By leveraging and combining newly discovered yeast genetic diversity with now affordable and efficient genetic engineering and synthetic biology tools, academic and industrial yeast labs have designed yeast cell factories for a wide range of novel applications such as the production of medicines, components of human breast milk, heme for meat substitutes, bioplastics, and other biomaterials. This review covers the newest technologies developed for yeast research including synthetic biology and their use in the engineering of yeast cell factories for emerging applications.

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CRISPR/Cas system for yeast genome engineering: advances and applications

Cited by 151 publications

References 78 publications

Control of metabolite efflux in microbial cell factories: current advances and future prospects

Control of metabolite efflux in microbial cell factories: current advances and future prospects

Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains

The renaissance of yeasts as microbial factories in the modern age of biomanufacturing

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