scholarly journals Multiplexed CRISPR-Cas9-Based Genome Editing of Rhodosporidium toruloides

mSphere ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Peter B. Otoupal ◽  
Masakazu Ito ◽  
Adam P. Arkin ◽  
Jon K. Magnuson ◽  
John M. Gladden ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Disruption ◽  
Genome Engineering ◽  
Carbon Sources ◽  
Rhodosporidium Toruloides ◽  
Optimized Design ◽  
Content Type ◽  
Wide Range ◽  
Fuels And Chemicals ◽  
Microbial Biofuel

ABSTRACT Microbial production of biofuels and bioproducts offers a sustainable and economic alternative to petroleum-based fuels and chemicals. The basidiomycete yeast Rhodosporidium toruloides is a promising platform organism for generating bioproducts due to its ability to consume a broad spectrum of carbon sources (including those derived from lignocellulosic biomass) and to naturally accumulate high levels of lipids and carotenoids, two biosynthetic pathways that can be leveraged to produce a wide range of bioproducts. While R. toruloides has great potential, it has a more limited set of tools for genetic engineering relative to more advanced yeast platform organisms such as Yarrowia lipolytica and Saccharomyces cerevisiae. Significant advancements in the past few years have bolstered R. toruloides’ engineering capacity. Here we expand this capacity by demonstrating the first use of CRISPR-Cas9-based gene disruption in R. toruloides. Transforming a Cas9 expression cassette harboring nourseothricin resistance and selecting transformants on this antibiotic resulted in strains of R. toruloides exhibiting successful targeted disruption of the native URA3 gene. While editing efficiencies were initially low (0.002%), optimization of the cassette increased efficiencies 364-fold (to 0.6%). Applying these optimized design conditions enabled disruption of another native gene involved in carotenoid biosynthesis, CAR2, with much greater success; editing efficiencies of CAR2 deletion reached roughly 50%. Finally, we demonstrated efficient multiplexed genome editing by disrupting both CAR2 and URA3 in a single transformation. Together, our results provide a framework for applying CRISPR-Cas9 to R. toruloides that will facilitate rapid and high-throughput genome engineering in this industrially relevant organism. IMPORTANCE Microbial biofuel and bioproduct platforms provide access to clean and renewable carbon sources that are more sustainable and environmentally friendly than petroleum-based carbon sources. Furthermore, they can serve as useful conduits for the synthesis of advanced molecules that are difficult to produce through strictly chemical means. R. toruloides has emerged as a promising potential host for converting renewable lignocellulosic material into valuable fuels and chemicals. However, engineering efforts to improve the yeast’s production capabilities have been impeded by a lack of advanced tools for genome engineering. While this is rapidly changing, one key tool remains unexplored in R. toruloides: CRISPR-Cas9. The results outlined here demonstrate for the first time how effective multiplexed CRISPR-Cas9 gene disruption provides a framework for other researchers to utilize this revolutionary genome-editing tool effectively in R. toruloides.

scholarly journals Multiplexed CRISPR-Cas9 based genome editing of Rhodosporidium toruloides

2019 ◽  
Author(s):  
Peter B. Otoupal ◽  
Masakazu Ito ◽  
Adam P. Arkin ◽  
Jon K. Magnuson ◽  
John M. Gladden ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Disruption ◽  
Genome Engineering ◽  
Carbon Sources ◽  
Carotenoid Biosynthesis ◽  
Rhodosporidium Toruloides ◽  
Optimized Design ◽  
Wide Range ◽  
Fuels And Chemicals ◽  
Microbial Biofuel

ABSTRACTMicrobial production of biofuels and bioproducts offers a sustainable and economic alternative to petroleum-based fuels and chemicals. The basidiomycete yeast Rhodosporidium toruloides is a promising platform organism for generating bioproducts due to its ability to consume a broad spectrum of carbon sources (including those derived from lignocellulosic biomass) and to naturally accumulate high levels of lipids and carotenoids, two biosynthetic pathways that can be leveraged to produce a wide range of bioproducts. While R. toruloides has great potential, it has a more limited set of tools for genetic engineering relative to more advanced yeast platform organisms such as Yarrowia lipolytica and Saccharomyces cerevisiae. Significant advancements in the past few years have bolstered R. toruloides’ engineering capacity. Here we expand this capacity by demonstrating the first use of CRISPR-Cas9 based gene disruption in R. toruloides. Stably integrating a Cas9 expression cassette into the genome brought about successful targeted disruption of the native URA3 gene. While editing efficiencies were initially low (0.002%), optimization of the cassette increased efficiencies 364-fold (to 0.6%). Applying these optimized design conditions enabled disruption of another native gene involved in carotenoid biosynthesis, CAR2, with much greater success; editing efficiencies of CAR2 deletion reached roughly 50%. Finally, we demonstrated efficient multiplexed genome editing by disrupting both CAR2 and URA3 in a single transformation. Together, our results provide a framework for applying CRISPR-Cas9 to R. toruloides that will facilitate rapid and high throughput genome engineering in this industrially relevant organism.IMPORTANCEMicrobial biofuel and bioproduct platforms provide access to clean and renewable carbon sources that are more sustainable and environmentally friendly than petroleum-based carbon sources. Furthermore, they can serve as useful conduits for the synthesis of advanced molecules that are difficult to produce through strictly chemical means. R. toruloides has emerged as a promising potential host for converting renewable lignocellulosic material into valuable fuels and chemicals. However, engineering efforts to improve the yeast’s production capabilities have been impeded by a lack of advanced tools for genome engineering. While this is rapidly changing, one key tool remains unexplored in R. toruloides; CRISPR-Cas9. The results outlined here demonstrate for the first time how effective multiplexed CRISPR-Cas9 gene disruption provides a framework for other researchers to utilize this revolutionary genome-editing tool effectively in R. toruloides.

scholarly journals TALEN and CRISPR/Cas Genome Editing Systems: Tools of Discovery

Acta Naturae ◽  
2014 ◽  
Vol 6 (3) ◽  
pp. 19-40 ◽  
Author(s):  
A. A. Nemudryi ◽  
K. R. Valetdinova ◽  
S. P. Medvedev ◽  
S. M. Zakian
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Genome Engineering ◽  
Disease Modeling ◽  
Regulation Of Gene Expression ◽  
Hereditary Disease ◽  
Transcription Activator ◽  
Wide Range ◽  
High Standards ◽  
Phenotype Formation

Precise studies of plant, animal and human genomes enable remarkable opportunities of obtained data application in biotechnology and medicine. However, knowing nucleotide sequences isnt enough for understanding of particular genomic elements functional relationship and their role in phenotype formation and disease pathogenesis. In post-genomic era methods allowing genomic DNA sequences manipulation, visualization and regulation of gene expression are rapidly evolving. Though, there are few methods, that meet high standards of efficiency, safety and accessibility for a wide range of researchers. In 2011 and 2013 novel methods of genome editing appeared - this are TALEN (Transcription Activator-Like Effector Nucleases) and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems. Although TALEN and CRISPR/Cas9 appeared recently, these systems have proved to be effective and reliable tools for genome engineering. Here we generally review application of these systems for genome editing in conventional model objects of current biology, functional genome screening, cell-based human hereditary disease modeling, epigenome studies and visualization of cellular processes. Additionally, we review general strategies for designing TALEN and CRISPR/Cas9 and analyzing their activity. We also discuss some obstacles researcher can face using these genome editing tools.

scholarly journals Draft Genome Sequence of a Medium- and Long-Chain n-Alkane-Degrading Bacterium, Tsukamurella tyrosinosolvens Strain PS2, with Two Genetic Systems for Alkane Degradation

2019 ◽  
Vol 8 (17) ◽  
Author(s):  
Valeriya A. Romanova ◽  
Eugenia A. Boulygina ◽  
Maria N. Siniagina ◽  
Maria I. Markelova ◽  
Tatiana V. Grigoryeva ◽  
...  
Keyword(s):  
Organic Synthesis ◽  
Genome Sequence ◽  
Draft Genome ◽  
Carbon Sources ◽  
Draft Genome Sequence ◽  
Alkane Degradation ◽  
Content Type ◽  
Degrading Bacterium ◽  
Wide Range ◽  
Long Chain

Here, we report the genome sequence of Tsukamurella tyrosinosolvens strain PS2, which was isolated from hydrocarbon sludge of an organic synthesis factory. This strain was able to utilize a wide range of n-alkanes, from C16 to C35, as sole carbon sources.

A methodology for the optimized design of power transformer insulation system

2018 ◽  
Vol 37 (3) ◽  
pp. 1002-1010 ◽  
Author(s):  
Hugo Rodriguez-Ignacio ◽  
Xose M. Lopez-Fernandez ◽  
Casimiro Álvarez-Mariño
Keyword(s):  
Power Transformer ◽  
Safety Margin ◽  
Analytical Calculation ◽  
Optimized Design ◽  
Insulation System ◽  
Content Type ◽  
Candidate Solution ◽  
Wide Range ◽  
Two Stages ◽  
Transformer Insulation

Purpose The purpose of this paper is to present a methodology based on an optimizer linked with electric finite element method (FEM) for automating the optimized design of power transformer insulation system structures. Design/methodology/approach The proposed methodology combines two stages to obtain the optimized design of transformer insulation system structures. First, an analytical calculation is carried out with the optimizer to search a candidate solution. Then, the candidate solution is numerically checked in detail to validate its consistency. Otherwise, these two steps are iteratively repeated until the optimizer finds a candidate solution according to the objective function. Findings The solutions found applying the proposed methodology reduce the inter-electrode distances compared to those insulation designs referenced in the literature for the same value of safety margin. Originality/value The proposed methodology explores a wide range of insulation system structures in an automated way which is not possible to do with the classical trial-and-error approach based on personal expertise.

scholarly journals L-Rhamnose Metabolism inClostridium beijerinckiiStrain DSM 6423

2018 ◽  
Vol 85 (5) ◽  
Author(s):  
Mamou Diallo ◽  
Andre D. Simons ◽  
Hetty van der Wal ◽  
Florent Collas ◽  
Bwee Houweling-Tan ◽  
...  
Keyword(s):  
First Generation ◽  
Major Product ◽  
Green Macroalgae ◽  
Content Type ◽  
Sugar Fermentation ◽  
Bacterial Microcompartment ◽  
General Metabolism ◽  
Wide Range ◽  
Biomass Resources ◽  
Fuels And Chemicals

ABSTRACTMacroalgae (or seaweeds) are considered potential biomass feedstocks for the production of renewable fuels and chemicals. Their sugar composition is different from that of lignocellulosic biomasses, and in green species, includingUlva lactuca, the major sugars arel-rhamnose andd-glucose.C. beijerinckiiDSM 6423 utilized these sugars in aU. lactucahydrolysate to produce acetic acid, butyric acid, isopropanol, butanol, and ethanol (IBE), and 1,2-propanediol.d-Glucose was almost completely consumed in diluted hydrolysates, whilel-rhamnose ord-xylose was only partially utilized. In this study, the metabolism ofl-rhamnose byC. beijerinckiiDSM 6423 was investigated to improve its utilization from natural resources. Fermentations ond-glucose,l-rhamnose, and a mixture ofd-glucose andl-rhamnose were performed. Onl-rhamnose, the cultures showed low growth and sugar consumption and produced 1,2-propanediol, propionic acid, andn-propanol in addition to acetic and butyric acids, whereas ond-glucose, IBE was the major product. On ad-glucose–l-rhamnose mixture, both sugars were converted simultaneously andl-rhamnose consumption was higher, leading to high levels of 1,2-propanediol (78.4 mM), in addition to 59.4 mM butanol and 31.9 mM isopropanol. Genome and transcriptomics analysis ofd-glucose- andl-rhamnose-grown cells revealed the presence and transcription of genes involved inl-rhamnose utilization and in bacterial microcompartment (BMC) formation. These data provide useful insights into the metabolic pathways involved inl-rhamnose utilization and the effects on the general metabolism (glycolysis, early sporulation, and stress response) induced by growth onl-rhamnose.IMPORTANCEA prerequisite for a successful biobased economy is the efficient conversion of biomass resources into useful products, such as biofuels and bulk and specialty chemicals. In contrast to other industrial microorganisms, natural solvent-producing clostridia utilize a wide range of sugars, including C5, C6, and deoxy-sugars, for production of long-chain alcohols (butanol and 2,3-butanediol), isopropanol, acetone,n-propanol, and organic acids. Butanol production by clostridia from first-generation sugars is already a commercial process, but for the expansion and diversification of the acetone, butanol, and ethanol (ABE)/IBE process to other substrates, more knowledge is needed on the regulation and physiology of fermentation of sugar mixtures. Green macroalgae, produced in aquaculture systems, harvested from the sea or from tides, can be processed into hydrolysates containing mixtures ofd-glucose andl-rhamnose, which can be fermented. The knowledge generated in this study will contribute to the development of more efficient processes for macroalga fermentation and of mixed-sugar fermentation in general.

scholarly journals EngineeringKluyveromyces marxianusas a Robust Synthetic Biology Platform Host

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Paul Cernak ◽  
Raissa Estrela ◽  
Snigdha Poddar ◽  
Jeffrey M. Skerker ◽  
Ya-Fang Cheng ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Synthetic Biology ◽  
Genome Editing ◽  
Kluyveromyces Marxianus ◽  
Lipid Production ◽  
Carbon Sources ◽  
Industrial Applications ◽  
Human Society ◽  
Content Type ◽  
Renewable Chemicals

ABSTRACTThroughout history, the yeastSaccharomyces cerevisiaehas played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However,S. cerevisiaehas proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineered the thermotolerant yeastKluyveromyces marxianusto create a new synthetic biology platform. Using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, we show that wild isolates ofK. marxianuscan be made heterothallic for sexual crossing. By breeding two of these mating-type engineeredK. marxianusstrains, we combined three complex traits—thermotolerance, lipid production, and facile transformation with exogenous DNA—into a single host. The ability to crossK. marxianusstrains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering ofK. marxianusisolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establishK. marxianusas a synthetic biology platform comparable toS. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.IMPORTANCEThe yeastKluyveromyces marxianusgrows at high temperatures and on a wide range of carbon sources, making it a promising host for industrial biotechnology to produce renewable chemicals from plant biomass feedstocks. However, major genetic engineering limitations have kept this yeast from replacing the commonly used yeastSaccharomyces cerevisiaein industrial applications. Here, we describe genetic tools for genome editing and breedingK. marxianusstrains, which we use to create a new thermotolerant strain with promising fatty acid production. These results open the door to usingK. marxianusas a versatile synthetic biology platform organism for industrial applications.

scholarly journals New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi

mSphere ◽  
2018 ◽  
Vol 3 (2) ◽  
Author(s):  
Valmik K. Vyas ◽  
G. Guy Bushkin ◽  
Douglas A. Bernstein ◽  
Matthew A. Getz ◽  
Magdalena Sewastianik ◽  
...  
Keyword(s):  
Fungal Pathogens ◽  
Genome Engineering ◽  
High Efficiency ◽  
Selection Marker ◽  
Important Species ◽  
Content Type ◽  
Guide Rna ◽  
Wide Range ◽  
Repair Mechanisms ◽  
The Creation

ABSTRACT We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in Candida albicans , Saccharomyces cerevisiae , Candida glabrata , and Naumovozyma castellii . These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each of these species and reveal different dependencies for repair of the Cas9 double-stranded break. Both C. albicans and S. cerevisiae rely heavily on homology-directed repair, whereas C. glabrata and N. castellii use both homology-directed and nonhomologous end-joining pathways. The high efficiency of these vectors permits the creation of unmarked deletions in each of these species and the recycling of the dominant selection marker for serial mutagenesis in prototrophs. A further refinement, represented by the "Unified" Solo vectors, incorporates Cas9, guide RNA, and repair template into a single vector, thus enabling the creation of vector libraries for pooled screens. To facilitate the design of such libraries, we have identified guide sequences for each of these species with updated guide selection algorithms. IMPORTANCE CRISPR-mediated genome engineering technologies have revolutionized genetic studies in a wide range of organisms. Here we describe new vectors and guide sequences for CRISPR mutagenesis in the important human fungal pathogens C. albicans and C. glabrata , as well as in the related yeasts S. cerevisiae and N. castellii . The design of these vectors enables efficient serial mutagenesis in each of these species by leaving few, if any, exogenous sequences in the genome. In addition, we describe strategies for the creation of unmarked deletions in each of these species and vector designs that permit the creation of vector libraries for pooled screens. These tools and strategies promise to advance genetic engineering of these medically and industrially important species.

Chryseobacterium frigidisoli sp. nov., a psychrotolerant species of the family Flavobacteriaceae isolated from sandy permafrost from a glacier forefield

2013 ◽  
Vol 63 (Pt_7) ◽  
pp. 2666-2671 ◽  
Author(s):  
Felizitas Bajerski ◽  
Lars Ganzert ◽  
Kai Mangelsdorf ◽  
Lisa Padur ◽  
André Lipski ◽  
...  
Keyword(s):  
Type Species ◽  
Carbon Sources ◽  
Dry Weight ◽  
Rrna Gene ◽  
Aerobic Bacterium ◽  
Content Type ◽  
Optimum Growth Temperature ◽  
Glacier Forefield ◽  
Link Type ◽  
Wide Range

During diversity studies of the glacier forefields of the Larsemann Hills, East Antarctica, a novel psychrotolerant, non-motile Gram-negative, shiny yellow, rod-shaped, aerobic bacterium, designated strain PB4T was isolated from a soil sample. Strain PB4T produces indole from tryptophan and hydrolyses casein. It grows between 0 and 25 °C with an optimum growth temperature of 20 °C. A wide range of substrates are used as sole carbon sources and acid is produced from numerous carbohydrates. The major menaquinone is MK-6. Identified polar lipids are ethanolamines and ornithine lipids. Major fatty acids (>10 %) are iso-C15 : 0 (13.0 %) and iso-2OH-C15 : 0 (51.2 %). G+C content is 33.7 mol%. The polyamine pattern is composed of sym-homospermidine (25.1 µmol g−1 dry weight), minor amounts of cadaverine (0.2 µmol g−1 dry weight) and spermidine (0.4 µmol g−1 dry weight) and traces of putrescine and spermine (<0.1 µmol g−1 dry weight). Strain PB4T had highest 16S rRNA gene similarities with the type strains of Chryseobacterium humi (97.0 %) and Chryseobacterium marinum (96.5 %). Considering phenotypic and genotypic characterization, strain PB4T represents a novel species in the genus Chryseobacterium (family Flavobacteriaceae ), for which the name Chryseobacterium frigidisoli sp. nov. is proposed. The type strain is PB4T ( = DSM 26000T = LMG 27025T).

scholarly journals mSphere of Influence: Ushering in the CRISPR Revolution to Toxoplasma Biology

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Alfredo J. Guerra
Keyword(s):  
Toxoplasma Gondii ◽  
Structural Biology ◽  
Gene Disruption ◽  
Gene Editing ◽  
Genome Engineering ◽  
Content Type ◽  
Efficient Gene ◽  
Implementing Strategies ◽  
Molecular Parasitology

ABSTRACT Alfredo J. Guerra works in the field of molecular parasitology and structural biology. In this mSphere of Influence article, he reflects on how “Efficient Gene Disruption in Diverse Strains of Toxoplasma gondii Using CRISPR/CAS9” by Bang Shen et al. (mBio 5:e01114-14, 2014, https://doi.org/10.1128/mBio.01114-14) and “Efficient Genome Engineering of Toxoplasma gondii using CRISPR/CAS9” by Saima M. Sidik et al. (PLoS One 9:e100450, 2014, https://doi.org/10.1371/journal.pone.0100450) made an impact on him by successfully implementing strategies to genetically manipulate T. gondii using CRISPR/CAS9 gene editing technology.

scholarly journals Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

2016 ◽  
Vol 82 (17) ◽  
pp. 5421-5427 ◽  
Author(s):  
Josef Altenbuchner
Keyword(s):  
Bacillus Subtilis ◽  
Genome Editing ◽  
Genome Engineering ◽  
Shuttle Vector ◽  
Temperature Sensitive ◽  
Target Sequence ◽  
Target Specificity ◽  
Content Type ◽  
A Genome ◽  
Single Plasmid

ABSTRACTThe clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems are adaptive immune systems of bacteria. A type II CRISPR-Cas9 system fromStreptococcus pyogeneshas recently been developed into a genome engineering tool for prokaryotes and eukaryotes. Here, we present a single-plasmid system which allows efficient genome editing ofBacillus subtilis. The plasmid pJOE8999 is a shuttle vector that has a pUC minimal origin of replication forEscherichia coli, the temperature-sensitive replication origin of plasmid pE194tsforB. subtilis, and a kanamycin resistance gene working in both organisms. For genome editing, it carries thecas9gene under the control of theB. subtilismannose-inducible promoter PmanPand a single guide RNA (sgRNA)-encoding sequence transcribed via a strong promoter. This sgRNA guides the Cas9 nuclease to its target. The 20-nucleotide spacer sequence at the 5′ end of the sgRNA sequence, responsible for target specificity, is located between BsaI sites. Thus, the target specificity is altered by changing the spacer sequences via oligonucleotides fitted between the BsaI sites. Cas9 in complex with the sgRNA induces double-strand breaks (DSBs) at its target site. Repair of the DSBs and the required modification of the genome are achieved by adding homology templates, usually two PCR fragments obtained from both sides of the target sequence. Two adjacent SfiI sites enable the ordered integration of these homology templates into the vector. The function of the CRISPR-Cas9 vector was demonstrated by introducing two large deletions in theB. subtilischromosome and by repair of thetrpC2mutation ofB. subtilis168.IMPORTANCEIn prokaryotes, most methods used for scarless genome engineering are based on selection-counterselection systems. The disadvantages are often the lack of a suitable counterselection marker, the toxicity of the compounds needed for counterselection, and the requirement of certain mutations in the target strain. CRISPR-Cas systems were recently developed as important tools for genome editing. The single-plasmid system constructed for the genome editing ofB. subtilisovercomes the problems of counterselection methods. It allows deletions and introduction of point mutations. It is easy to handle and very efficient, and it may be adapted for use in other firmicutes.

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