Expression systems in yeast MCQs With Answer

Introduction

This quiz set on Expression systems in yeast is designed for M.Pharm students specializing in Advanced Pharmaceutical Biotechnology. It emphasizes practical and mechanistic aspects of yeast platforms such as Saccharomyces cerevisiae, Pichia (Komagataella) pastoris, Yarrowia lipolytica and other non-conventional yeasts used for recombinant protein production. The questions probe promoter selection, secretion signals, glycosylation patterns, vector strategies, host engineering (including glycoengineering and protease knockouts), fermentation parameters and regulatory considerations relevant to biopharmaceutical manufacture. Each MCQ includes plausible distractors and a precise correct answer to help deepen conceptual understanding and prepare for both exams and lab design decisions.

Q1. Which yeast promoter is tightly regulated and strongly induced by methanol, commonly used in Komagataella (Pichia) pastoris expression systems?

• GAP promoter
• AOX1 promoter
• GAL1 promoter
• TEF1 promoter

Correct Answer: AOX1 promoter

Q2. The Saccharomyces cerevisiae alpha-factor prepro leader is most frequently used in yeast expression systems for which purpose?

• Driving high transcriptional levels of the gene
• Enhancing plasmid replication
• Directing efficient secretion of heterologous proteins
• Improving chromosomal integration frequency

Correct Answer: Directing efficient secretion of heterologous proteins

Q3. One key disadvantage of using wild-type S. cerevisiae for therapeutic glycoproteins is:

• Insufficient secretion capacity
• Hypermannosylated N-glycans that differ from human patterns
• Inability to perform disulfide bond formation
• Complete lack of N-glycosylation machinery

Correct Answer: Hypermannosylated N-glycans that differ from human patterns

Q4. Which strategy is commonly applied to reduce proteolytic degradation of secreted recombinant proteins in yeast?

• Overexpressing vacuolar proteases
• Deleting major extracellular protease genes or using protease-deficient host strains
• Increasing culture temperature to inactivate proteases
• Replacing signal peptide with transmembrane anchor

Correct Answer: Deleting major extracellular protease genes or using protease-deficient host strains

Q5. In Pichia pastoris, what is the effect of integrating multiple tandem copies of an expression cassette into the genome?

• Guaranteed linear increase in specific productivity with each copy
• Often higher expression but may cause genetic instability or metabolic burden at high copy number
• Complete suppression of expression due to promoter occlusion
• No change because only single-copy expression is possible

Correct Answer: Often higher expression but may cause genetic instability or metabolic burden at high copy number

Q6. Which promoter is commonly used for strong constitutive expression in yeast when induction is not desired?

• AOX1 promoter
• GAP promoter
• GAL1 promoter
• CUP1 promoter

Correct Answer: GAP promoter

Q7. Glycoengineering of yeast to produce human-like N-glycans typically requires which of the following modifications?

• Overexpression of yeast mannosyltransferases only
• Knockout of yeast hypermannosylation pathways and introduction of heterologous mammalian glycosyltransferases
• Elimination of the unfolded protein response
• Replacement of the secretory signal peptide with a bacterial leader

Correct Answer: Knockout of yeast hypermannosylation pathways and introduction of heterologous mammalian glycosyltransferases

Q8. Which yeast species is favored for secretion of lipases and enzymes and is notable for lipid metabolism engineering?

• Komagataella phaffii (Pichia pastoris)
• Yarrowia lipolytica
• Saccharomyces cerevisiae
• Kluyveromyces lactis

Correct Answer: Yarrowia lipolytica

Q9. The primary advantage of using integrative vectors over episomal vectors in industrial yeast production is:

• Episomal vectors lead to higher genetic stability
• Integration provides stable genomic insertion and reduced plasmid loss without selection pressure
• Integrative vectors are always multi-copy by default
• Integrative vectors eliminate the need for promoters

Correct Answer: Integration provides stable genomic insertion and reduced plasmid loss without selection pressure

Q10. Which endoplasmic reticulum chaperone is typically co-expressed to enhance folding and secretion of disulfide-bonded recombinant proteins in yeast?

• BiP (Kar2)
• RNA polymerase II
• Glycosyltransferase OST
• Histone H3

Correct Answer: BiP (Kar2)

Q11. In methanol-induced Pichia systems, what is a principal fermentation safety and process concern at scale?

• Methanol is non-flammable and inert
• Methanol is volatile and flammable, requiring careful handling and explosion-proof equipment
• Methanol causes immediate cell lysis at any concentration
• Methanol cannot be metabolized by Pichia, so it accumulates

Correct Answer: Methanol is volatile and flammable, requiring careful handling and explosion-proof equipment

Q12. Which of the following best describes the typical N-glycan structure difference between Pichia pastoris and human cells?

• Pichia synthesizes complex-type N-glycans identical to humans
• Pichia adds short, high-mannose-type glycans but shorter than S. cerevisiae hypermannosylation
• Pichia lacks N-glycosylation entirely
• Pichia attaches only O-linked glycans and never N-linked

Correct Answer: Pichia adds short, high-mannose-type glycans but shorter than S. cerevisiae hypermannosylation

Q13. Which selection system is commonly used in Saccharomyces and Kluyveromyces to select transformants without antibiotic resistance markers?

• Auxotrophic markers such as HIS3, LEU2, or URA3
• Zeocin resistance only
• Tetracycline resistance
• Amphotericin B selection

Correct Answer: Auxotrophic markers such as HIS3, LEU2, or URA3

Q14. The unfolded protein response (UPR) activation in yeast during high-level recombinant protein production is mainly associated with:

• Excessive cytosolic DNA replication stress
• Accumulation of misfolded proteins in the ER leading to induction of ER-resident chaperones
• Increased mitochondrial ATP production with no ER involvement
• Direct proteasomal secretion of proteins outside the cell

Correct Answer: Accumulation of misfolded proteins in the ER leading to induction of ER-resident chaperones

Q15. Which genetic engineering tool has recently accelerated precise genome editing and pathway engineering in yeast expression hosts?

• Random chemical mutagenesis only
• CRISPR/Cas9-based genome editing
• UV irradiation exclusively
• Classical transposon mutagenesis without selection

Correct Answer: CRISPR/Cas9-based genome editing

Q16. For secreted therapeutic proteins, why is controlling N-terminal processing (signal peptide cleavage and propeptide removal) important?

• N-terminal processing has no effect on activity or immunogenicity
• Correct cleavage ensures proper folding, biological activity and reduces heterogeneous N-termini that can affect efficacy and immunogenicity
• Leaving signal peptides increases secretion efficiency
• Propeptide retention always improves protein stability

Correct Answer: Correct cleavage ensures proper folding, biological activity and reduces heterogeneous N-termini that can affect efficacy and immunogenicity

Q17. Which fermentation parameter most directly influences yeast secretion capacity and proper protein folding due to its effect on oxidative protein formation?

• Agitation and dissolved oxygen levels
• Trace metal absence only
• Light intensity in the bioreactor
• Magnetic stirring frequency only

Correct Answer: Agitation and dissolved oxygen levels

Q18. A common method to assess the glycosylation profile of a recombinant yeast-produced protein in process development is:

• Flow cytometry of whole yeast cells
• Mass spectrometry or HPLC analysis of released glycans
• Measuring optical density at 600 nm only
• DNA sequencing of the production strain

Correct Answer: Mass spectrometry or HPLC analysis of released glycans

Q19. Which yeast host is naturally capable of lactose utilization and has been used for expression of secreted proteins with relatively human-like glycosylation after engineering?

• Kluyveromyces lactis
• Saccharomyces cerevisiae
• Pichia pastoris
• Schizosaccharomyces pombe

Correct Answer: Kluyveromyces lactis

Q20. To improve secretion of a difficult-to-express heterologous protein in yeast, which combined approach is most appropriate?

• Use a stronger promoter only and ignore folding or secretion steps
• Optimize signal peptide, co-express ER chaperones/PDI, modulate cultivation conditions and consider host protease knockouts
• Remove all glycosylation sites without further optimization
• Use antibiotics in the culture to boost secretion

Correct Answer: Optimize signal peptide, co-express ER chaperones/PDI, modulate cultivation conditions and consider host protease knockouts

Download