Cell Factories - Yeast Systems

Question 1

Advantages of yeast as a bioreactor

Answer 1

• Well characterised = genome completely sequenced & precision genetic eng is possible by gene targeting
• Lots of use in food (GRAS certified)
• Eukaryotic = carries out some PT processing steps & secretes polypeptides (using standard eukaryotic secretion pathway)
• Maintain ^ level of recombinant activity - one of major DNA repair pathways utilised homology dependent repair = key factor in yeast matin system

Question 2

Yeast Lifecycle

Answer 2

Exist as haploid and diploid cells
- Haploid undergo sexual fusion = diploid which can undergo meiosis which = haploid ascospores ((A tetrad of the 4 meiotic products in each ascus: individual spores can be microdissected and inoculated to initiate haploid colonies and to carry out segregation analysis)

Question 3

Sexual fusion between haploid cells depends on…

Answer 3

Having TWO mating types designated “a” and “α” (alpha).

• Cells of the “a” type produce a mating pheromone that attracts and stimulates cells of the alpha strain, and vice-versa.
• Cells of the “a” strain bear surface receptors that bind to the alpha pheromone and vice-versa

Binding of the pheromone promotes cell fusion to produce diploid strains

Question 4

What allows yeast to change sex?

Answer 4

Due to MAT (= biallelic, and on chromosome 3 of 16)
- Cells either MAT-A or MAT-Aplha
2 additional silenced MAT loci:
-HML = MAT-alpha locus
-HMR = MAT-A locus
(Hidden Mat Left or Right)

Question 5

How does mating-type switching occur?

Answer 5

Initiated by an endonuclease called HO, which makes a DNA double-strand break at the MAT locus.
- This is then repaired by homology-dependent DNA repair (homologous recombination) with one of the silent, but homologous HM loci.
MAT-A locus = repaired by HML = MAT-Alpha
MAT-Alpha locus = repaired by HMR = MAT- A

T/ most yeast colonies are usually made up of diploid cells.

Question 6

Selectable markers in yeast as a bioreactor

Answer 6

Nutrition associated genes = good candidates for selectable markers. Lab strains can’t always be used industrially b/ they are maintained using nutritional genes
URA3 = encodes an enzyme in the uracil biosynthetic pathway.
It converts Orotic acid to uracil & can also convert the analogue (FOA) to 5-fluorouracil, which is toxic.

BUT ura3- mutants can grow = t/ use this property to select ura3- mutants of industrial strains, that can be transformed with vectors using the wt URA3 gene as a selection marker.

Question 7

Vectors used for yeast in bioreactors

Answer 7

YIPs - Integrating
YEPs - Episomal
YRPs - Replicating
YCPs - Centromeric

Question 8

YIPs

Answer 8

Stable but low-copy
Stable and high-copy number

Make use of the very high efficiency of homologous recombination that occurs in yeast.
Plasmids containing a gene with homology with an endogenous yeast gene will integrate at that locus within the yeast genome, and be maintained as part of the yeast chromosome.

Question 9

YEPs

Answer 9

Replicate autonomously: contain
-origin of replication &
- a stability determinant
derived from an endogenous yeast plasmid, the “2-micron” plasmid.

Higher copy expression = 2 micron plasmid encodes FLP recombinase
- undertakes recombination between 2 direct repeat target sites which are exploited in disintegration vectors to remove bacterial vector sequences from YEPs

Question 10

YRPs

Answer 10

Less stable and low -copy number

Like YEPs - replicate autonomously b/ they contain genomic ‘ARS’ sequences

Question 11

YCPs

Answer 11

Low copy
Contain centromeric sequences = ensure partition into daughter cells and behave like mini chromosomes - segregating at meiosis in a Mendelian manner

Question 12

Transgene expression in yeast as a bioreactor

Answer 12

3 types of expression:

• high-level gene expression, but with little regulatability
• Low- level gene expression but tightly controlled gene expression
• Hybrid promoters = high levels of expression coupled with tight regulation.

Question 13

High expression in Yeast as a bioreactor

Answer 13

Strong promoters exist in yeast, typically driving expression of genes involved in major biochemical pathways, such as the glycolytic pathway (essential in fermentation!) - include promoters for PGK, PYK, ADH1, GPD

In continuous fermentation = cellular levels of proteins can be 1-5% of the total cellular protein.
When the PGK gene was cloned on multi-copy plasmid, the PGK protein comprised up to 50% of the total cellular protein.

Question 14

Regulation in yeast when being used as a bioreactor - GAL 1

Answer 14

GAL1 gene expression regulated by GAL4 and GAL80
- By using galactose as a replacement for glucose by in the medium, you can get a 1,000x up-regulation of the GAL1 gene
- Good promoter for laboratory use, but not on an
industrial scale: galactose is an expensive sugar, and causes excessive foaming in fermenters

Question 15

Regulation in yeast when being used as a bioreactor - PHO5

Answer 15

PHO5 (acid phosphatase) gene expression is induced by phosphate starvation.

Also inconvenient on a large scale b/ easier to add compounds to growth medium than it is to remove them.
However, if a tsPho80 mutant strain is used, the PHO80 repressor is inactivated at higher temp - so transgenes under the control of the tsPHO5 promoter will be active at higher temp - even in the presence of phosphate.
This promoter will become temperature regulated under low-phosphate conditions.

Question 16

Hybrid promoter when using yeast as a bioreactor

Answer 16

Uses ^ expression of PGK = tight regulation of yeast mating type switch.

Fusing PGK promoter to MAT-Alpha cis acting DNA sequence (MAT alpha operator) -> PGK-driven gene can be controlled by MAT locus

Temp-sensitive mat-12-gene = expression of transgene stopped at 23C but induced at 34C

Question 17

Secretion of recombinant proteins when using yeast as a bioreactor

Answer 17

Yeasts have eukaryotic secretion systems -> recognises sequences from other eukaryotes = protein export

• Helps in purification b/ initial medium is not contaminated w/ large amounts of cellular protein
• Correct folding is enhanced by sending recomb proteins through endomembrane systems w/ chaperones
  This makes it more likely that bioactive proteins will be isolate

Question 18

Hep B Vaccine: history

Answer 18

Vaccination is the principal means of protection against Hep. B.

Historically, vaccines have been developed using attenuated strains of virus, but such strains are not necessarily harmless: where vaccines have been developed from heat-killed strains, there is a fine line between producing:

• effective vaccine (too much heat treatment can denature the viral protein resulting in loss of immunogenicity) and
• killing the virus (too little heat treatment may not kill the virus!).

Growth of virus is often problematic (and often dangerous: in the 1978 a lab worker died following the accidental escape of smallpox virus in a Birmingham laboratory – the head of the lab committed suicide)

Question 19

Hep B Vaccine: What do rDNA approaches allow…

Answer 19

Us to identify immunogenic epitopes, and express these in isolation from the rest of the virus. This can result in the production of large quantities of safe and highly effective vaccines.

Question 20

Hep B Vaccine: Hep B virus structure

Answer 20

human pathogenic virus with the smallest known viral DNA genome (3.2kbp).
- 4 genes, encoding proteins “C”, “X”, “P”” and the main viral surface antigen “HBSAg”

Antigen is actually encoded by a single long open reading frame that has three in-frame ATG codons, that act as independent translation initiation codons when the mRNA is translated = 3 polypeptides produced are called S1, S2 and S. The most abundant is the smallest – the “S” protein.

The virus has some similarities to Cauliflower mosaic virus,

• a reverse transcriptase step is involved in its life cycle.
• A supragenomic initial transcript is copied back into DNA and imported into the nucleus for the rest of the replication cycle.
• This Reverse transcription stem can result in reactivation of “dormant” virus long after an infection has been apparently cleared up.

The HBSAg sequence is, additionally, variable (again, likely a result of the high mutation rate consequent on the Rtase-mediated replication step). There are thus several serotypic strain variants.

Question 21

Recombinax HB

Answer 21

Very effective recombinant vaccine against Hep B

By expressing the HBSAg DNA sequence in yeast, we can obtain the production and export of empty shells that can subsequently – and safely – be used as a highly effective vaccine

Question 22

Gardasil

Answer 22

Yeast derived vaccine against HPV = genital wart and cervical cancer
A more versatile vaccine is the quadrivalent vaccine
- protects not only against strains 16 and 18
- also against HPV-6 and HPV11.
These 4 strains account for ca. 90% of all cases of genital warts.

Contains traces of yeast proteins, but these are very low (about 7 micrograms/ml)

Question 23

Recombinant vaccines and auto-immune diseases?

Answer 23

Dr. Andrew Wakefield published claims that the measles/mumps/rubella (MMR) vaccine could cause autism = large no. of children withdrawn from MMR vaccination programme.

Some activist groups now claim that yeast proteins present in recombinant yeast-produced vaccines cause allergic reactions, and in some cases trigger autoimmune diseases.

No evidence supports these claims,

• although yeast proteins can be present at quite high concentrations (up to 5mg/ml). Anti-HepB vaccines have been used since 1981, and adverse reactions to the vaccine are low (about 1 in 600,000).
• None of these adverse reactions appear to be a consequence of the yeast protein in the vaccine (no anti-yeast antibodies were found in the patients).