Yeast Expression Systems in Bioprocessing

Learning Outcomes

• Students will be able to describe yeast expression systems and their use in bioprocessing.
• Students will be able to identify and differentiate between various yeast expression systems.
• Students will be able to discuss the advantages and disadvantages of yeast expression.
• Students will be able to describe the process of protein expression in yeast, from transformation to purification.

Prescribed Reading

• New England Biolabs (2024): Protein Expression in Yeast. Available at: [website address]. Accessed: July 25, 2024.
• Merck (2024): Protein Expression Systems. Available at: [website address]. Accessed: July 25, 2024.
• Roghayyeh Baghban, et al. (2019): Yeast Expression Systems: Overview and Recent Advances. Molecular Biotechnology. 61: 365–384. DOI: [DOI]. Accessed: July 25, 2024.

Introduction

• Yeast protein expression is a common alternative to bacterial and higher eukaryotic expression systems.
• Yeast cells offer advantages like growth speed, easy genetic manipulation, and low-cost media, similar to bacterial systems, while also offering post-translational modifications and secretory expression abilities.
• Several yeast expression systems exist in organisms like Saccharomyces, Pichia, Kluyveromyces, Hansenula and Yarrowia.
• Saccharomyces cerevisiae is the workhorse of yeast expression systems, preferred for its ease of use, reduced time and costs.

Yeast Expression Systems

• Gene of interest is inserted into a bacterial plasmid.
• Plasmid is introduced into the yeast host.
• Yeast replicates and produces the desired protein.

Expression Vectors/Plasmids and Selection Markers

• Important vector system for this strategy.
• Expression vectors contain a strong yeast promoter/terminator and a selectable marker cassette.
• Many yeast expression vectors can optionally clone a gene downstream of that efficiently directs a heterologous protein to be secreted from the cell.
• Selection markers help in bacterial and yeast vector selection.
• Targeting sequences, multiple cloning sites, promoters, and selection markers are key vector components.
• Integration vectors and episomal Vectors (Ylp or YEp) integrate into or replicate independently of the genome.

Yeast Cloning Vectors

• Integrative and episomal vectors use for expressing recombinant proteins.
• Integrated vectors have high stability but low copy number.
• Episomal vectors demonstrate higher copy numbers and simple transformation protocols but may be unstable in the absence of selection pressure.
• Shuttle vectors are able to propagate in both prokaryotic and eukaryotic hosts.

Promoters for Protein Production in Various Yeast Strains

• Well-characterized inducible or constitutive promoters with strong transcriptional activity are preferred for overexpressing heterologous proteins (inducible are induced by a specific environmental condition).
• Great constitutive promoters are available (e.g. GAL1, GAL10, JUB1, SNR52, MET17, TDH3, TPI1, and PDC1).

Creating a Yeast Expression System

• Use competent E. coli cells to take up DNA sequence of interest.
• Integrate DNA into bacterial genome, or circularize to become a plasmid.
• Select transformed E. coli using a selection marker (antibiotic).
• Expand selected E. coli in an appropriate culture.
• Isolate DNA or plasmid.
• Transform into yeast.
• Screen transformants for DNA integration.
• Select and scale high-expressing yeast clones.
• Isolate and purify intracellular/secreted proteins.

Yeast Classification

• Methylotrophic yeasts (e.g., Pichia pastoris, Hansenula polymorpha) utilize methanol as a carbon and energy source
• Non-methylotrophic yeasts (e.g., Saccharomyces cerevisiae) utilize the normal sugars such as glucose and fructose.

Advantages of Saccharomyces cerevisiae

• Preferred host for bacteria, other yeasts, and filamentous fungi.
• Tolerates wide pH ranges and high concentrations of ethanol and sugar.
• Resistance to elevated osmotic pressure.
• Commonly studied for gene expression regulation, signal transduction, aging, apoptosis, metabolism, cell cycle control, programmed cell death, neurodegenerative diseases, and secretory pathways.

Important Advantages and Disadvantages of Yeast Expression Systems

• Advantages
  • Yeast exhibits rapid growth.
  • Relatively easy to manipulate genetically
  • Moderate rapid expression
  • Works well for secreted and intracellular proteins.
  • Less expensive
  • Easy to scale up fermentations
• Disadvantages
  • N-linked glycan structures differs from typical mammalian proteins
  • Use of yeast secretion signal peptide is necessary.
  • High biomass production is possible, and production is safe.