A vector set for systematic metabolic engineering in Saccharomyces cerevisiae.

Area of Science:

• Molecular Biology
• Synthetic Biology
• Yeast Genetics

Background:

• Metabolic engineering in Saccharomyces cerevisiae often requires stable integration of multiple genes.
• Existing methods for gene expression and integration in yeast have limitations in scalability and flexibility.
• Development of versatile tools is crucial for advancing complex metabolic engineering strategies.

Purpose of the Study:

• To construct and characterize a set of shuttle vectors for efficient gene expression in Saccharomyces cerevisiae.
• To enable differential gene expression using various selectable markers and plasmid types (CEN/ARS and 2 µ).
• To facilitate genomic integration of expression cassettes via homologous recombination and enable marker recycling.

Main Methods:

• Construction of shuttle vectors with selectable markers (URA3, TRP1, MET15, LEU2-d8, HIS3, CAN1) and promoters (TEF1, PGK1, HXT7-391) driving gene expression.
• Utilized CreA recombinase for site-specific marker deletion and recycling via loxP sites.
• Generated integration-ready fragments using polymerase chain reaction (PCR) for homologous recombination into the yeast genome.
• Characterized expression levels and copy numbers of representative vectors.

Main Results:

• Developed shuttle vectors supporting differential gene expression with multiple selectable markers and promoter options.
• Demonstrated efficient genomic integration of expression cassettes by replacing endogenous retrotransposons via homologous recombination.
• Showcased comparable expression of integrated reporters to those at standard loci, expanding available integration sites.
• Validated marker recycling using CreA recombinase for efficient reuse of genetic elements.

Conclusions:

• The developed shuttle vectors and genomic integration strategy provide a robust platform for combinatorial gene expression in Saccharomyces cerevisiae.
• This expands the repertoire of stable integration sites, offering a virtually inexhaustible pool for complex metabolic engineering.
• The tools facilitate efficient gene stacking and marker recycling, crucial for advancing yeast-based synthetic biology and metabolic engineering applications.