Konckout Yeast

Konckout Yeast

Yeast is a eukaryotic single-celled microorganism classified as a member of the kingdom Fungi. The first yeast originated hundreds of millions of years ago, and 1,500 species have been identified. It is estimated that they account for 1% of all described fungal species. Most yeasts reproduce asexually through mitosis, and many reproduce through asymmetric division (i.e. budding). Through fermentation, the yeast species Saccharomyces cerevisiae converts carbohydrates into carbon dioxide and alcohol. The history of beer, wine and bread is related to yeast fermentation. It is also the most important model organism in modern cell biology research, and one of the most in-depth eukaryotic microorganisms. Researchers have used it to gather information about eukaryotic cell biology and ultimately human biology.

Genetic engineering of unconventional yeast for renewable biofuel and biochemical production

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Due to its unique physiological and metabolic characteristics, this unconventional yeast is not only a good model for studying the basic properties of fungal differentiation, but also a promising microbial platform for biochemical production and various biotechnology applications, requiring a wide range of knockout cell lines . However, the genetic manipulation of Yarrowia lipolytica is limited due to the lack of an effective and stable genetic transformation system and a very high rate of non-homologous recombination, which is mainly attributed to the KU70 gene. Researchers report a simple and rapid protocol for effective genetic transformation and gene deletion in Yarrowia lipolytica Po1g. Firstly, a protocol for efficiently transforming foreign DNA into Yarrowia lipolytica Po1g was established. Second, in order to obtain an improved double-crossover homologous recombination rate for further deletion of the target gene, the KU70 gene was deleted by transforming a destruction cassette carrying a 1kb homology arm. Third, in order to prove the improved gene deletion efficiency after deleting the KU70 gene, the researchers used the same procedure on the KU70 knockout platform strain to delete 11 target genes encoding alcohol dehydrogenase and alcohol oxidase, respectively. It was observed that the rate of precise homologous recombination increased significantly from less than 0.5% for deletion of KU70 genes in Po1g to 33%-71% for single gene deletions of 11 target genes in Po1gKU70Δ. Construct a replicating plasmid carrying the hygromycin B resistance marker and Cre/LoxP system, and finally remove the selectable marker gene in the yeast knockout strain by expressing the Cre recombinase to promote multiple rounds of targeted genetic manipulation. The resulting single gene deletion mutant has potential applications in biofuel and biochemical production.

RNA-guided assembly of Cas9 combinatorial metabolic pathways in the yeast genome

Saccharomyces cerevisiae is an important industrial platform for the production of grain and cellulosic ethanol, isobutanol, butylene glycol, isoprenoids and other chemicals. The construction of successful production strains usually involves multiple gene knockouts and chromosomal integration of expression cassettes to redirect metabolic flux to convert sugar and other raw materials into desired products. RNA-guided Cas9 genome editing has been confirmed in many prokaryotic and eukaryotic hosts, including Saccharomyces cerevisiae, where they are also used as tools for metabolic engineering. To expand the use of RNA-guided Cas9 as a metabolic pathway construction tool, the researchers demonstrated the direct assembly and chromosomal integration of up to 17 overlapping DNA fragments encoding the β-carotene biosynthetic pathway. In addition, the researchers generated a combinatorial strain library for the β-carotene biosynthetic pathway, which was directly integrated into the yeast genome to create a diverse strain library. In this way, combinatorial libraries can be screened in stable chromosomal integration strains to quickly increase product titer. This combined method of assembly will greatly speed up the current S metabolic engineering. As an industrial platform, Saccharomyces cerevisiae has increased the number of strains that can perform enzyme screening, expression optimization and protein engineering evaluation at the same time to achieve the titer, rate and yield required for the commercialization of new industrial fermentation products.

Developing a CRISPR/Cas9 System for Genome Editing in the Basidiomycetous Yeast Rhodosporidium Toruloides

The basidiomycetous yeast Rhodosporidium toruloides (R. toruloides) has been explored as a promising host for the production of lipids and carotenoids. However, the rational manipulation of this yeast remains difficult due to lack of efficient genetic tools. The development of a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas9) system for genome editing in R. toruloides is described. First, R. toruloides strains are generated with sufficient production of the Cas9 protein of Staphylococcus aureus origin by integrating a cassette containing a codon-optimized Cas9 gene into the genome. In parallel, two U6 genes are identified, predicting two U6 promoters and confirming better transcription of single-guide RNA (sgRNA) with the U6b promoter. Next, sgRNA cassettes are designed targeting CRTI, CAR2, and CLYBL gene, respectively, transforming into those Cas9-expressed strains, and finding over 60% transformants with successful insertion and deletion (indel) mutations. Furthermore, when the sgRNA cassette includes donor DNA flanked by two homologous arms of the gene CRTI, gene knockout occurs via homologous recombination. Thus, the CRISPR/Cas9 system is now established as a powerful genome-editing tool in R. toruloides, which should facilitate functional genomic study and advanced cell factory development.

Reference

Yu A Q , Pratomo N , Ng T K , et al. Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production[J]. Journal of Visualized Experiments, 2016(115).

EauClaire, Steve, Zhang, et al. Combinatorial metabolic pathway assembly in the yeast genome with RNA-guided Cas9.[J]. Journal of Industrial Microbiology & Biotechnology, 2016.

Xiang, Jiao, Yue,et al. Developing a CRISPR/Cas9 system for genome editing in the basidiomycetous yeast Rhodosporidium toruloides.[J]. Biotechnology Journal, 2019.

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We developed CRISPR-U™ and CRISPR-B™(based on CRISPR/Cas9 technology) which is more efficient than general CRISPR/Cas9 in double-strand breaking, CRISPR-U™ and CRISPR-B™ can greatly improve the efficiency of homologous recombination, easily achieve knockout (KO), point mutation (PM) and knockin (KI) in vitro and in vivo.