Genome Engineering of Yarrowia lipolytica

1. Strain Characteristics and Biological Background

Yarrowia lipolytica is a non-conventional oleaginous yeast, first isolated in 1942, and is classified as a Generally Recognized As Safe (GRAS) microorganism. It possesses a broad substrate spectrum, enabling it to utilize various low-cost substances such as citric acid, isocitric acid, and alkanes as feedstocks. In industrial applications, it can be used to produce a variety of high-value products, including organic acids (e.g., citric acid, succinic acid, α-ketoglutaric acid), terpenoids (e.g., geraniol, limonene), lipids, biofuels, and natural pigments like red beet pigment. Concurrently, due to its strong ability to degrade oily substances, it can also be employed for the bioremediation of waste oil pollution. In the food and pharmaceutical industries, Yarrowia lipolytica is an ideal host for producing related products owing to its non-pathogenic nature and excellent metabolic capabilities.

2. CRISPR Editing Workflow

(1) Design and Construction: Design specific sgRNAs based on the target gene and editing requirements, and construct a vector containing the sgRNA expression cassette and the Cas9 expression cassette.

(2) Transformation: Introduce the constructed plasmid vector into Yarrowia lipolytica strains using the lithium acetate method. After transformation, strains are cultured and selected on medium containing the appropriate selection marker (e.g., SC medium).

(3) Editing Verification: Selected transformants are further cultured in liquid medium for a period to facilitate gene editing. Subsequently, random colonies are picked, genomic DNA is extracted, and the efficacy of the gene editing (such as knockout, integration, or mutation) is verified using techniques like PCR.

Fig.1 Yarrowia lipolytica Genome Engineering Strategy

3. Service Types

(1) Gene Knockout Service: Design and implement gene knockout strategies for specific genes in Yarrowia lipolytica to efficiently disrupt target genes. This aids in functional gene studies or the construction of strains with defects in specific metabolic pathways, such as URA3 or TRP1 knockouts.

(2) Gene Integration Service: Precisely integrate exogenous genes or specific DNA fragments into the Yarrowia lipolytica genome for stable expression, supporting the construction of engineered strains for producing specific compounds, e.g., integrating a GFP expression cassette for visualization studies.

(3) Multiplex Gene Editing Service: Utilize tRNA-sgRNA array strategies to simultaneously edit multiple genes, accelerating metabolic engineering of strains.

4. Technical Advantages

(1) High Editing Efficiency: The introduction of CRISPR/Cas9 technology enhances gene editing efficiency to over 90%.

(2) High Precision: The CRISPR/Cas9 system uses gRNA to precisely guide Cas9 to the target site, enabling precise editing. Furthermore, by designing different gRNAs and donor DNA, various types of precise genetic manipulations can be achieved, such as single-base mutations, fragment insertions, or deletions.

(3) Diverse Strategies: Flexible choice between plasmid-based or genome-integrated Cas9 expression strategies. Editing outcomes can be optimized by modulating DNA repair pathways (e.g., knocking out KU70 or overexpressing ScRad52) to meet different experimental and production needs.

5. Project Timeline

(1) Standard Yarrowia lipolytica Strains: 2-3 months

(2) Wild Yarrowia lipolytica Strains: Custom projects requiring specific evaluation

6. Delivery Standards

(1) Target site PCR and DNA sequencing identification data.

(2) Two glycerol stock vials of the engineered strain.

(3) mRNA transcription level data (for overexpression strains).

(4) Project final report.

7. References

[1] Liu J, Zhu Y, Hou J. Optimizing the CRISPR/Cas9 system for gene editing in Yarrowia lipolytica. Engineering Microbiology, 2025, 5:100193.