CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae

Mans, Robert; Rossum, Harmen M. van; Wijsman, Melanie; Backx, Antoon H.; Kuijpers, Niels G. A.; Broek, Marcel van den; Daran‐Lapujade, Pascale; Pronk, Jack T.; Maris, Antonius J. A. van; Daran, Jean‐Marc

doi:10.1093/femsyr/fov004

Cited by 376 publications

(463 citation statements)

References 76 publications

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“…The p414-KanMX-TEF1p-Cas9-CYCt plasmid was transformed into the DS69473 strain, and the expression of the CAS9 gene was analyzed using qPCR. Herein, we used the CRISPR/Cas9 protocol described by Mans et al (40) and pMEL16 to express the guide RNA targeting the CYC8 gene at the Y353 position (Fig. S4) and targeting a PAM site 6 bp in front of the Y353 position.…”

Section: Methodsmentioning

confidence: 99%

Improved Xylose Metabolism by a CYC8 Mutant of Saccharomyces cerevisiae

Nijland

Shin

Boender

et al. 2017

Appl Environ Microbiol

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Engineering Saccharomyces cerevisiae for the utilization of pentose sugars is an important goal for the production of second-generation bioethanol and biochemicals. However, S. cerevisiae lacks specific pentose transporters, and in the presence of glucose, pentoses enter the cell inefficiently via endogenous hexose transporters (HXTs). By means of in vivo engineering, we have developed a quadruple hexokinase deletion mutant of S. cerevisiae that evolved into a strain that efficiently utilizes D-xylose in the presence of high D-glucose concentrations. A genome sequence analysis revealed a mutation (Y353C) in the general corepressor CYC8, or SSN6, which was found to be responsible for the phenotype when introduced individually in the nonevolved strain. A transcriptome analysis revealed altered expression of 95 genes in total, including genes involved in (i) hexose transport, (ii) maltose metabolism, (iii) cell wall function (mannoprotein family), and (iv) unknown functions (seripauperin multigene family). Of the 18 known HXTs, genes for 9 were upregulated, especially the low or nonexpressed HXT10, HXT13, HXT15, and HXT16. Mutant cells showed increased uptake rates of D-xylose in the presence of D-glucose, as well as elevated maximum rates of metabolism (V max ) for both D-glucose and D-xylose transport. The data suggest that the increased expression of multiple hexose transporters renders D-xylose metabolism less sensitive to D-glucose inhibition due to an elevated transport rate of D-xylose into the cell. IMPORTANCEThe yeast Saccharomyces cerevisiae is used for second-generation bioethanol formation. However, growth on xylose is limited by pentose transport through the endogenous hexose transporters (HXTs), as uptake is outcompeted by the preferred substrate, glucose. Mutant strains were obtained with improved growth characteristics on xylose in the presence of glucose, and the mutations mapped to the regulator Cyc8. The inactivation of Cyc8 caused increased expression of HXTs, thereby providing more capacity for the transport of xylose, presenting a further step toward a more robust process of industrial fermentation of lignocellulosic biomass using yeast.KEYWORDS sugar transporter, xylose transport, evolutionary engineering, transcriptome, yeast A n increasing energy demand and concerns of obtaining this energy from fossil fuels have stimulated the development of liquid fuels from renewable feedstock. Bioethanol, mostly used as a fuel additive, produced from readily fermentable agricultural feedstocks, such as sugar cane and corn, is less desired because the production of these feedstocks requires large amounts of arable land and competes with the food supply (1). A more sustainable source of feedstock is lignocellulosic biomass from hardwood, softwood, and agricultural residues (2). However, a major drawback of lignocellulosic feedstocks is the inability of the most commonly used yeast in industry,

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Section: Methodsmentioning

confidence: 99%

Improved Xylose Metabolism by a CYC8 Mutant of Saccharomyces cerevisiae

Nijland

Shin

Boender

et al. 2017

Appl Environ Microbiol

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“…In practice, unintended genome rearrangements caused by HR, either between the engineered genetic elements themselves or between engineered genetic elements and the native yeast genome, were not observed. These results highlight the amazing efficiency and versatility of in vivo assembly and CRISPR/Cas9-facilitated genome editing in S. cerevisiae (6,24).…”

Section: Discussionmentioning

confidence: 78%

“…Applications ranging from functional analysis of heterologous proteins, testing of kinetic models (now hindered by the multiplicity of paralogs) (46) or screening drugs, to more technical aspects such as exploring the effect of genomic location of highly expressed native pathways, are now within reach. Continued improvements in CRISPR-mediated removal of scattered genes (24) should even further facilitate functional clustering and fast, modular swapping of key pathways/processes.…”

Section: Discussionmentioning

confidence: 99%

Pathway swapping: Toward modular engineering of essential cellular processes

Kuijpers

Solis-Escalante

Luttik

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Recent developments in synthetic biology enable one-step implementation of entire metabolic pathways in industrial microorganisms. A similarly radical remodelling of central metabolism could greatly accelerate fundamental and applied research, but is impeded by the mosaic organization of microbial genomes. To eliminate this limitation, we propose and explore the concept of "pathway swapping," using yeast glycolysis as the experimental model. Construction of a "single-locus glycolysis" Saccharomyces cerevisiae platform enabled quick and easy replacement of this yeast's entire complement of 26 glycolytic isoenzymes by any alternative, functional glycolytic pathway configuration. The potential of this approach was demonstrated by the construction and characterization of S. cerevisiae strains whose growth depended on two nonnative glycolytic pathways: a complete glycolysis from the related yeast Saccharomyces kudriavzevii and a mosaic glycolysis consisting of yeast and human enzymes. This work demonstrates the feasibility and potential of modular, combinatorial approaches to engineering and analysis of core cellular processes.pathway swapping | glycolysis | Saccharomyces cerevisiae | modular genomes R eplacement of petrochemistry by bio-based processes is a key element for sustainable development and requires microbes equipped with novel-to-nature capabilities. Recent developments in synthetic biology enable introduction of entire metabolic pathways and, thereby, new functionalities for product formation and substrate consumption, into microbial cells (1). However, industrial relevance of the resulting strains critically depends on optimal interaction of the newly introduced pathways with the core metabolism of the host cell. Central metabolic pathways such as glycolysis, tricarboxylic acid cycle, and pentose phosphate pathways, are essential for synthesis of precursors, for providing free energy (ATP), and for redox-cofactor balancing. Optimization of productivity, product yield, and robustness therefore requires modifications in the configuration and/or regulation of these core metabolic functions.Engineering of central metabolism is in some respects more challenging than the functional expression of heterologous product pathways. Millions of years of evolution of microorganisms have endowed their metabolic and regulatory networks with a level of complexity that cannot be efficiently reengineered by iterative, single-gene modifications. Enzymes of central metabolism are encoded by hundreds of genes that, especially in eukaryotes, are scattered across microbial genomes. Moreover, inactivation and subsequent replacement of genes involved in central metabolism is complicated by functional redundancy of isoenzymes (2, 3) as well as by the essential role of many of the corresponding biochemical reactions. Microbial platforms in which the configuration of key pathways can be remodelled in a swift, combinatorial manner would provide an invaluable asset for fundamental research and engineering of central metabolism.Wherea...

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“…The CRISPR/Cas9 system of Mans et al (2015) 1 should be cited. Any differences between that system and the one described in this manuscript, should be emphasized.…”

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confidence: 99%

A set of novel CRISPR-based integrative vectors for Saccharomyces cerevisiae

Daniels¹,

Mukherjee²,

Goldman³

et al. 2018

Wellcome Open Res

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Integrating a desired DNA sequence into yeast genomes is a widely-used genetic manipulation in the budding yeast Saccharomyces cerevisiae. The conventional integration method is to use an integrative plasmid such as pRS or YIplac series as the target DNA carrier. The nature of this method risks multiple integrations of the target DNA and the potential loss of integrated DNA during cell proliferation. In this study, we developed a novel yeast integration strategy based on the widely used CRISPR-Cas9 system and created a set of plasmids for this purpose. In this system, a plasmid bearing Cas9 and gRNA expression cassettes will induce a double-strand break (DSB) inside a biosynthesis gene such as Met15 or Lys2. Repair of the DSB will be mediated by another plasmid bearing upstream and downstream sequences of the DSB and an integration sequence in between. As a result of this repair the sequence is integrated into genome by replacing the biosynthesis gene, the disruption of which leads to a new auxotrophic genotype. The newly-generated auxotroph can serve as a traceable marker for the integration. In this study, we demonstrated that a DNA fragment up to 6.3 kb can be efficiently integrated into the Met15 or Lys2 locus using this system. This novel integration strategy can be applied to various yeasts, including natural yeast isolated from wild environments or different yeast species such as Candida albicans.

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CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae

Cited by 376 publications

References 76 publications

Improved Xylose Metabolism by a CYC8 Mutant of Saccharomyces cerevisiae

Improved Xylose Metabolism by a CYC8 Mutant of Saccharomyces cerevisiae

Pathway swapping: Toward modular engineering of essential cellular processes

A set of novel CRISPR-based integrative vectors for Saccharomyces cerevisiae

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