Yeasts Flashcards
Products of fermentation
Ethanol
Carbon Dioxide
In presence of glucose, yeast prefers to grow by fermentation
Respiration: more energy efficient but slow
Fermentation to compete in microbial community:
- use up glucose quickly making ethanol
- poison competitors with ethanol
- metabolise ethanol at leisure
Check slides for diagram
Long history of alcoholic beverages, early form of biotechnology
From ~8000 years ago: brewing in Sumeria, China, Babylonia, Egypt
Pharaohs buried with bottles of beer to enjoy in afterlife.
Brewing recipes on walls of Egyptian tombs.
Yeast spores live incredibly long: Ancient yeast revived from jars, and from beer in shipwreck
History of yeast domestication: insights from genome sequencing
• Present-day industrial yeasts: from few ancestors
• Beer yeasts, genetic and phenotypic hallmarks of domestication
• Domestication predates microbe discovery
• Independent domestication of wine and sake yeasts.
Chocolate and coffee
Yeast is also essential for fermentation of cacao and coffee beans.
Yeast strains that hitchhiked with human migration are critical for the flavour of chocolate and coffee.
Ethanol as a biofuel
Biotech –ethanol from yeast fermentation
USA (maize)
Brazil, Columbia (sugar cane)
Fermentation – potentially carbon neutral Bioenergy
The mystery of fermentation
Antonie van Leeuwenhoek used simple microscopes to observe yeast in fermenting beer in 1680
Yeast is not motile, unlike many bacteria
Theodor Schwann showed that yeast was essential for fermentation (1837)
Justus von Liebig (1803-1873), along with other chemists, believed that fermentation was simple chemical reaction. They thought yeast was a non-living catalyst
Louis Pasteur showed that heating of unfermented brewing mixture killed the yeast and prevented fermentation (1857). He also showed that fermentation was anaerobic
Buchner: showed that yeast cells produce enzymes that can promote fermentation even without cells (1897).
Reconciliation of two scientific cam
Fermentation is chemical reaction but requires
enzymes produced by living cells
Birth of biochemistry
Brewing
By mid 1800s, new style of beer became fashionable: Lager (fermented and conditioned at low temperatures, German for ‘storage’)
Jacob Jacobsen obtained Lager yeast and started
Carlsberg (1845).
Carlsberg applied science to brewing (e.g. thermometers), beginning of biotechnology.
Emil Christian Hansen (1842-1909) learned to culture yeast from single cells.
Identified Lager yeast, Saccharomyces carlsbergensis (S. cerevisiae X S. bayanus)
Father of yeast genetics
Øjvind Winge (1886-1964) – Father of Yeast Genetics
1933: Director of Carlsberg Labs
Winge found Hansen’s 46 year old cultures still viable
Discovered that yeast has sex, and realized it would make a fantastic system for genetics research
Yeast as a model organism in research
• Well-characterized, small genome, rapid growth and breeding, easy to manipulate genetically
• Yeast genes homologous to human genes,
closer related to humans than to E. coli
• Yeast as host for experiments with human proteins
S. cerevisiae life cycle
Look at slides for diagram
Mating haploid cells grow towards each other to
become ‘shmoos’, named after cartoon character in Al Capp’s strip Li’l Abner
• The mating-type is controlled by genes at single locus, MAT.
- Haploids have either the MATa allele or MATα allele.
- The MAT genes control mating-type via expression of pheromones and receptors.
• Wild strains can change mating-type using a genetic switch.
• Diploids are heterozygous at MAT locus: MATa/MATα.
Classical yeast genetics
• Auxotrophic mutants
• Finding mutants defective in biological functions
– genetic complementation
• Crossing of strains
- Mendelian segregation
- Dissecting tetrads and linkage analysi
Isolating auxotrophic mutations in yeast
• An auxotrophic mutant requires a specific nutrient (e.g. amino acid) that a wild-type cell can produce itself
• Wild-type yeast strains are prototrophic
• Natural mutations are rare
How can we make mutants?
• Mutagenize liquid culture, dilute, plate on solid medium
• Cells are plated onto rich medium (not defined) contains yeast extract, everybody can grow.
• Replica-plate to minimal medium (defined)
Replica plating
Rewatch lecture
How can we find out what nutrient they need?
Some cells cannot grow on minimal medium —> auxotrophic mutations
Add defined nutrients to minimal medium, one at a time, to see what nutrient different mutants need to grow
What if you cross a lysine auxotroph to another
lysine auxotroph?
If the two lysine auxotrophs are mutated in different lys genes then the diploid can grow as they will complement each other: Complementation test
Tetrad analysis
Cross two different strains -> recombination, meiosis
Separate out four haploid spores: tetrad dissection
Germinate on rich medium
Replica-plate to minimal medium
Mendelian segregation 2:2
Two mutations (e.g. cross ura7 with lys2)
If mutations are far apart (or on different
chromosomes) – will segregate randomly with respect to each other
You get new combinations of mutations in spores
Mutations close together (e.g. lys2 crossed to tyr1)
Crossovers will only rarely occur between the 2 mutations
Spore genotypes are mostly same as parent genotypes – no new combinations
TYR1 and LYS2 are close together so crossovers
between them are rare
Segregation of 2 mutations is not random, new combinations rare
–> genetic linkage
Frequency of recombination between mutations
reflects separation on chromosome
By analyzing hundreds of tetrads for different combinations of. mutations, you can build up a genetic map (linkage map) for all auxotrophic mutations
Such studies resulted in a high-resolution genetic map of all 16 budding yeast chromosomes, long before the genome was fully sequenced
Rewatch lecture
Molecular yeast genetics:
• Plasmids – common in wild yeast
• Can be fused with an E. coli plasmid - add genetic markers to allow selection in either yeast or E. coli (e.g. AMP, LEU2)
‘shuttle vector’ (2 different host species)
How can we clone a specific gene?
e.g., find the gene defective in auxotrophic lys2 mutant
Principle:
Cut up genomic DNA from wild-type yeast and
put pieces into plasmids
Test each plasmid for ability to complement auxotrophic lys2 mutant to find plasmid with wild-type LYS2 gene
Genomic library construction
Restriction endonucleases are enzymes that cut DNA at specific sequence (protect bacteria from invading nucleic acids). They make sticky ends.
Sticky ends can be fit back together with DNA ligase.
Check slides
Genomic library screening
Transform lys2 mutant strain with genomic library
Plasmid with wild-type LYS2 gene can complement the lys2 mutant, allowing growth in absence of lysin
Testing of candidate genes: 1 step gene replacement
Having found a candidate clone, we want to make sure it really is the gene of interest (LYS2)
Cut candidate gene with restriction enzyme
Ligate in a known yeast marker gene – e.g. KanMX6, which gives resistance to the drug G418
Now cut out whole construct (insert), and transform into yeast, selecting with the drug G418.
Yeast will attempt to repair the bare ends of the transformed DNA by recombining with its homologous sequence.
Wild-type LYS2 gene gets replaced by lys2::KanMX4 construct, giving the cell resistance to drug G418 -> Gene deletion (knock-out)
Test phenotype of deletion mutant: cannot grow without lysine?
Same phenotype as original lys2 mutant.
But how can we be certain that it is same gene
Testing of candidate genes: complementation test
Complementation test to independently confirm that we really cloned the right gene
Double mutant does not grow without lysine —> same gene mutated
Biotechnology
Yeast is used to produce commercially important
proteins; it can be genetically engineered, is safe, and fermentation technology is highly advanced
Example: Artemisinin -> potent but expensive
malaria drug extracted from Artemisia plant
Yeast engineered to produce Artemisinin:
Inserted 12 synthetic genes into yeast (Artemisinin biosynthesis pathway) to scale up for industrial production: cheaper drug for wide distribution
-> Synthetic biology: engineer/design organisms
with new features
Yeasts as human pathogens
Opportunistic fungal infections
Candida albicans
Opportunistic fungal infections
are important causes of mortality in immuno-compromised patients (e.g., AIDS, cancer chemotherapy, organ or bone marrow transplantation).
Candida albicans
Among gut flora in 80% of us, no harmful
effects. Candidiasis (‘thrush’) is common vaginal or oral infection.
To infect host tissue, the usual uni-cellular yeast-like form switches into an invasive, multi-cellular filamentous form
Schizosaccharomyces pombe: Fission Yeast
• Genome : 14 Mb, ~5,000 genes
• Simple model system
• Easy to handle / genetics
• Distantly related to budding yeast, complementary model organis
The revolution of DNA sequencing
S. cerevisiae was the first eukaryote to be completely sequenced (1996).
Huge international collaboration, involving over 600 people spread about in 92 labs around the world, taking many years and costing millions of £.
55% of sequencing done by hand in 92 small labs
45% done by high-throughput sequencing centres
Next-generation sequencers:
One person can sequence 100 yeast strains in a
week, at low cost!
Genetic variation within species
Genomic and phenotypic diversity of S. pombe
Collection of 161 natural isolates from 20 countries
Human-associated samples: cultivated fruits,
various fermentation
Genetic diversity
- Mean of 3 mutations per 1000 bp (lowest in protein-coding regions) Why?
- All but 1 strain are haploid, all 57 strains are prototrophic
- Gene content:
17 proteins not present in laboratory strain,
most are similar to proteins in other fungi
-> ancient ancestry, lost in reference strain
Exception: protein most similar to OsmC family from
plant pathogenic enterobacterium Brenneria salicis
-> horizontal gene transfer
-> ecological association with plants
Conclusions
Comprehensive survey of genetic and phenotypic diversity
Ancestors of strains dispersed within human history and probably distributed across globe by humans, recent European origin of S. pombe in Americas
GWAS are effective with this strain collection, large number of potential mutants contributing to specific traits
-> Less blunt tool than gene deletions to explore complex relationship between genotypes and complex phenotypes (disease
Fission yeast as model for cellular ageing
Chronological Lifespan of non-dividing cells
Check slides
Intercrosses among parents differing in lifespan to create recombinant pool of genetically and phenotypically diverse segregants
Sequence samples at different times during ageing of pooled cells to identify genetic variants that become enriched as a function of age (longevity alleles)
Summary
- Molecular genetics: construction of genomic library to clone gene of interest, gene deletions
- Synthetic biology
- Yeasts as human pathogens, e.g. Candida albicans
- Additional model yeast: S. pombe (fission yeast)
- Rapid technological progress is transforming research: next-generation sequencing
- Genome sequences, population structure, genetic diversity, genome evolution, complex relationship between genotypes and phenotype
