Genome Engineering of Saccharomyces cerevisiae

1. Service Overview

Saccharomyces cerevisiae is a model eukaryotic microorganism with the following notable characteristics:

(1) High fermentation efficiency: Under anaerobic conditions, it efficiently converts glucose into ethanol, making it acore strain for traditional brewing and modern biofuel production.

(2) Well-defined genetic background: Asone of the first eukaryotes to have its genome fully sequenced, its genefunctions are relatively well-studied.

(3) High tolerance: Exhibits strongtolerance to environmental stresses such as ethanol and osmotic pressure, making it suitable for industrial-scale cultivation.

(4) Ease of manipulation: Possesses amature genetic transformation system, facilitating gene editing operations.

2. Application Areas

(1) Biofuel production: A primarystrain for bioethanol production. Optimizing metabolic pathways through geneediting can significantly improve ethanol yield and production efficiency. Forexample, editing the ADH2, GPD1, and ALD4 genes resulted in a 1.41-fold increase in ethanol yield compared to the wild-type strain.

(2) Food and Brewing Industry: Used inthe fermentation production of beer, wine, bread, and other food products. Geneediting can improve flavor compound synthesis and enhance fermentation performance.

(3) Chemical Synthesis: Serves as a"cell factory" for producing various chemicals such as organic acids,amino acids, and proteins.

(4) BasicResearch Model: Widely used in basic life science research areas including cellcycle, signal transduction, and metabolic regulation.

3. S.cerevisiae Gene Editing Workflow

3.1 CRISPR/Cas9 System Design

(1) Target Selection: Design specific sgRNAs targeting the open reading frame (ORF) region of the target gene, typically selecting regions containing the PAM (protospacer adjacent motif) sequence to ensure editing efficiency and specificity.

(2) sgRNA Expression Cassette Construction: Amplify the sgRNA codingsequence via PCR and construct expression vectors for single or multiple sgRNAs.

3.2 Gene Editing Vector Construction

(1) Single Gene Editing Vector: Integrate a single sgRNA expression cassette and Cas9 expression elements into a suitable plasmid vector.

(2) Multi-Gene Editing Vector: Utilize a multiple cloning site (MCS)design, linking multiple sgRNA expression cassettes via restriction enzyme digestion and ligation to construct a vector capable of editing multiple genes simultaneously.

3.3 Yeast Transformation

Use chemical transformation methods(e.g., LiAc/PEG method) or electroporation to introduce the editing vector intoS. cerevisiae cells, achieving efficient uptake of foreign DNA.

3.4 Mutant Strain Screening and Identification

(1) Primary Screening: Select transformants on selective media (e.g., SDmedium).

(2) Molecular Identification: Verify gene mutations by PCR amplificationof the target gene region combined with Sanger sequencing, confirming thesuccess of gene knockout, knock-in, or site-specific mutation.

Fig.1 CRISPR-based genome modification strategies for S.cerevisiae

4. S.cerevisiae Gene Editing ServiceTypes

4.1 Single Gene Editing Services

(1) Gene Knockout: For a single target gene, design sgRNA and constructan editing vector to achieve efficient gene inactivation. Commonly used for studying gene function or blocking byproduct synthesis pathways.

(2) Gene Knock-in / Site-Directed Mutagenesis: Introduce foreign genesor perform site-directed mutations at specific genomic loci, used for gene function studies or protein engineering.

4.2 Multi-Gene Combinatorial Editing Services

(1) Dual-Gene Editing: Simultaneously edit two genes to study gene synergy and optimize metabolic networks.

(2) Triple-Gene and Multi-Gene Editing: Achieve simultaneous editing ofthree or more genes, regulating complex metabolic pathways through combinatorial effects to increase target product yield.

4.3 Metabolic Pathway Optimization Custom Services

Based on client requirements, designgene editing strategies for specific metabolic pathways. Optimize carbon flux distribution through multi-gene combinatorial editing to improve the yield andproduction efficiency of target products (e.g., ethanol). Provides end-to-endservices from target design to strain performance validation.

4.4 Custom Strain Construction Services

Tailored to the client's specific application scenario (e.g., industrial fermentation, chemical production),provide custom S. cerevisiae strain construction services, including gene editing scheme design, vector construction, strain screening, and performance optimization.

5. Technical Advantages

5.1 High Editing Efficiency

(1) Single gene knockout efficiency can reach 80%-100%.

(2) Dual-gene and triple-gene editing efficiencies are 85%-95% and70%-90% respectively, enabling efficient genomic engineering.

(3) Multi-gene editing can be completed in a single transformation step,avoiding the tedious process of multiple transformations and significantly improving experimental efficiency.

5.2 Strong Multi-Gene EditingCapability

Based on a multi-gRNA vector construction strategy using the CRISPR/Cas9 system, simultaneous editing of 1-3 genes can be achieved, facilitating the study of gene combinatorial effects andthe regulation of complex metabolic networks.

5.3 Significant Metabolic RegulationEffects

Through multi-gene combinatorial editing, carbon flux distribution can be effectively regulated, reducing byproduct (e.g., glycerol, acetate) formation and increasing the yield and production efficiency of target products (e.g., ethanol).

5.4 Good Technical Versatility

Applicable to various S. cerevisiae strains, including laboratory model strains and industrial production strains. Can be customized according to different needs, offering broad application prospects.

6. Project Timeline

(1) Typical S. cerevisiae strains (e.g., Y1H, BY4741/4742): 2-3months

(2) Wild S. cerevisiae strains: Custom projects, require specific evaluation.

7. Deliverables

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

(2) Two glycerol stocks of the engineered strain.

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

(4) Project final report.