Long-term objective
Comprehensive identification of genes involved in the adaptation of Saccharomyces cerevisiae to the winemaking environment.

Some limitations of transcriptomic approaches
• Genes relevant for many biological processes are not subject to transcriptional regulation in response to environmental conditions that influence these processes
(Birrell et al. 2002; PNAS).
• Not all genes showing a transcriptional change in response to a given culture condition are required for fitness under these conditions (Tai et al., 2007;
Microbiology SGM).

Some alternative genome-wide approaches (wine)
Proteomics
• Complementary information
• Usually no direct correlation with transcription data
Comparative genomics by hybridization
(aCGH or low coverage sequencing)
• Strains showing different fermentation phenotypes
• Wine vs. non-wine strains
Whole genome sequencing
• Horizontal transfer
• ¿New mobile elements?

HaploInsuficiency Profiling/HOmozygous Profiling
HIP/HOP
Construction of YKO S. cerevisiae collections
Heterozygous strains x6000
X
Homozygous strains x4500

HaploInsuficiency Profiling/HOmozygous Profiling
HIP/HOP

Some considerations about HIP/HOP analysis of wine fermentation
• Environmental conditions experiment dramatic changes
• Low number of generations in similar conditions
Alternative approach
Continuous culture

Simulation of wine fermentation in continuous culture
Continuo EC1118 CENIT 24%
25,00
20,00
15,00
10,00
5,00
CTR3
VCT3
600,00
500,00
400,00
300,00
200,00
100,00
0,00
0:00:00 4:48:00 9:36:00 14:24:00 19:12:00 24:00:00
Inoculation Time
28:48:00 33:36:00 38:24:00
0,00
43:12:00

Simulation of first step of wine fermentation in continuous culture
• 10 generation times for homozygous competition (SM)
• 20 generation times for heterozygous competition (SM)
• Controls for 10 and 20 generation times in YPD
 3 biological replicates for each of the above

HIP-HOP results for the first step of alcoholic fermentation
At least 150 heterozygous deleted strains showed deficient growth in synthetic must after 20 generations
(>2-fold reduced fitness as compared to fitness in YPD)
At least 126 homozygous deleted strains showed deficient growth in synthetic must after 10 generations
(>2-fold reduced fitness as compared to fitness in YPD)

Individual phenotypic characterization of selected strains.

Relevant functions from HIP analysis
• Vacuolar functions, including autophagy
• Different functions in the “DNA-to-protein” pathway o mRNA processing and stability o Protein synthesis o Secretion (ER functions)
Relevant functions from HOP analysis
• Adenine and lysine biosynthesis
• Inositol biosynthesis
• Biosynthesis of phospholipids

Apparent limitations of the HIP/HOP approach
• Limited to loss-of-function phenotypes
• Difficulty to estimate wine-related phenotypes in a BY4743 background
Complementary approaches
• Directed evolution of laboratory strains
• QTL mapping by high throughput methods

Directed evolution of laboratory strains
• Haploid laboratory strain (BY4741)
• Continuous culture in conditions emulating the first step of alcoholic fermentation
• Working volume 40-50 ml
• 150-250 generations (three biological replicates)
• Verification of the “evolved” phenotype
• Whole genome sequence analysis of the evolved strains

Phenotype of evolved strains. Batch culture
Strain Growth rate
BY 4741 0,1683
AV 8
BV 19
E18
AV 16
0,2254
0,2569
0,2760
0,2779
1,4
1,2
1
0,8
0,6
0,4 w t-1 w t-2 w t-3
AV16.1
AV 16.2
AV 16.3
AV 18.1
AV 18.2
AV 18.3
AV 21.1
AV 21.2
AV 21.3
AV 22.1
AV 22.2
AV 23.3
0,2
0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38
Adaptation to first steps does not involve improved overall fermentation performance, rather the opposite

Phenotype of evolved strains. Continuous culture
D=0,20 h -1
D=0,25 h -1
BY4741
Av16
BY4741
Av16
OD 600
0.69
1.28
0.28
0.74

Summary of mutations already identified
• SNPs in non-coding regions
• Nonsense mutations
• Missense mutations
• 50% mutations
Strain
E18
BV19
AV8
AV16*
6
1
SNPs 50%
4
2
4
1
2
0
Mutations requiring further confirmation
• Deletions/insertions
• Changes in copy-number
• Chromosomal rearrangements

RSP5: E3 Ubiquitin ligase

Ubiquitin-proteasome pathway mutants
Gene
RSP5
Strain Mutation
E18 Asn>Lys
CDC4 E18
BRE5
BV19 Glu>Asp
AV16 Asn>Thr
E18
Ser>Leu
(50%)
Glu>STOP
UBC6 BV19 Small deletion*
BUL1 AV8 Asp>His
E3 Ubiquitin ligase
E3 Ubiquitin ligase
E3 Ubiquitin ligase
Part of a complex with ubiquitin ligase activity on a CDK inhibitor
Ubiquitin protease cofactor
Ubiquitin-conjugating enzyme
Ubiquitin-binding component of the Rsp5p E3-ubiquitin ligase complex

www.icvv.es/winehiphop

Coincidences with previous studies
28 overlapping HOP genes
19 overlapping HIP genes
3 overlapping HIP genes

Previous reports of HIP/HOP analysis of wine fermentation
Delneri et al. 2008
• Commercial grape must ( 100 g/L sugar ) (among several other media)
• Chemostat
• Not supplemented with uridine. Aerobic .
• Only HIP analysis
• No unstressed contrast
• Concluded all nutritional requirements were provided by must

Previous reports of HIP/HOP analysis of wine fermentation
Piggott et al. 2011
• Synthetic must (200 g/L sugar)
• Single biological replicate each (HIP and HOP analyses)
• Time-course
• YPD amplification of samples
• No unstressed contrast
• Autophagy and ubiquitin-proteasome functions required
• Proficient deleted strains also identified (ribosomal and peroxisomal functions)
• FUR4

Some genes to watch
From the HIP analysis
SAM1 and SAM2; URE2; DUR1,2; MAL12, OCA6; CDC19; genes involved in Gap1p sorting; genes involved in chromatin remodeling and histone modification
From the HOP analysis
NPR2, NPR3 and RTC1; CAR1 and CAN1; GPD1 y GPD2; UBR1;
STB5; BCK1; BUL2; ADH3; AQR1; genes coding for ribosomal proteins; genes involved in protein folding in the ER

HIP
SAM1
SAM2
URE2
DUR1,2
MAL12
OCA6
GTR1
GTR2
SWD3
SGF29
RPH1
NHP6B
ITC1
GTR2
CAC2
RLF2
S-adenosylmethionine synthetase, catalyzes transfer of the adenosyl group of ATP to the sulfur atom of methionine; one of two differentially regulated isozymes (Sam1p and Sam2p)
S-adenosylmethionine synthetase, catalyzes transfer of the adenosyl group of ATP to the sulfur atom of methionine; one of two differentially regulated isozymes (Sam1p and Sam2p)
Nitrogen catabolite repression transcriptional regulator that acts by inhibition of GLN3 transcription in good nitrogen source; has glutathione peroxidase activity and can mutate to acquire GST activity; altered form creates [URE3] prion
Urea amidolyase, contains both urea carboxylase and allophanate hydrolase activities, degrades urea to CO2 and NH3; expression sensitive to nitrogen catabolite repression and induced by allophanate, an intermediate in allantoin degradation
Maltase (alpha-D-glucosidase), inducible protein involved in maltose catabolism; encoded in the MAL1 complex locus; hydrolyzes the disaccharides maltose, turanose, maltotriose, and sucrose
Cytoplasmic protein required for replication of Brome mosaic virus in S. cerevisiae, which is a model system for studying positivestrand RNA virus replication; null mutation confers sensitivity to tunicamycin and DTT
Cytoplasmic GTP binding protein and negative regulator of the Ran/Tc4 GTPase cycle; component of GSE complex, which is required for sorting of Gap1p; involved in phosphate transport and telomeric silencing; similar to human RagA and RagB
Putative GTP binding protein that negatively regulates Ran/Tc4 GTPase cycle; activates transcription; subunit of EGO and GSE complexes; required for sorting of Gap1p; localizes to cytoplasm and to chromatin; homolog of human RagC and RagD
Essential subunit of the COMPASS (Set1C) complex, which methylates histone H3 on lysine 4 and is required in transcriptional silencing near telomeres; WD40 beta propeller superfamily member and ortholog of mammalian WDR5
Component of the HAT/Core module of the SAGA, SLIK, and ADA complexes; HAT/Core module also contains Gcn5p, Ngg1p, and Ada2p; binds methylated histone H3K4; involved in transcriptional regulation through SAGA recruitment to target promoters and H3 acetylation
JmjC domain-containing histone demethylase; specifically demethylates H3K36 tri- and dimethyl modification states; associates with actively transcribed (RNA polymerase II) regions in vivo and specifically targets H3K36 in its trimethylation state as its substrate; transcriptional repressor of PHR1; Rph1p phosphorylation during DNA damage is under control of the MEC1-RAD53 pathway
High-mobility group (HMG) protein that binds to and remodels nucleosomes; involved in recruiting FACT and other chromatin remodelling complexes to the chromosomes; functionally redundant with Nhp6Ap; homologous to mammalian HMGB1 and
HMGB2
Subunit of the ATP-dependent Isw2p-Itc1p chromatin remodeling complex, required for repression of a-specific genes, repression of early meiotic genes during mitotic growth, and repression of INO1; similar to mammalian Acf1p, the regulatory subunit of the mammalian ATP-utilizing chromatin assembly and modifying factor (ACF) complex
Putative GTP binding protein that negatively regulates Ran/Tc4 GTPase cycle; activates transcription; subunit of EGO and GSE complexes; required for sorting of Gap1p; localizes to cytoplasm and to chromatin; homolog of human RagC and RagD
Subunit of chromatin assembly factor I (CAF-1), with Rlf2p and Msi1p; chromatin assembly by CAF-1 is important for multiple processes including silencing at telomeres, mating type loci, and rDNA; maintenance of kinetochore structure; deactivation of the
DNA damage checkpoint after DNA repair; and chromatin dynamics during transcription
Largest subunit (p90) of the Chromatin Assembly Complex (CAF-1); chromatin assembly by CAF-1 is important for multiple processes including silencing at telomeres, mating type loci, and rDNA; maintenance of kinetochore structure; deactivation of the
DNA damage checkpoint after DNA repair; and chromatin dynamics during transcription

HOP
GPD1
GPD2
NPR2
NPR3
RTC1
CAR1
CAN1
UBR1
STB5
STB5
BUL2
ADH3
AQR1
NAD-dependent glycerol-3-phosphate dehydrogenase, key enzyme of glycerol synthesis, essential for growth under osmotic stress; expression regulated by high-osmolarity glycerol response pathway; homolog of Gpd2p
NAD-dependent glycerol 3-phosphate dehydrogenase, homolog of Gpd1p, expression is controlled by an oxygen-independent signaling pathway required to regulate metabolism under anoxic conditions; located in cytosol and mitochondria
Subunit of SEA (Seh1-associated), Npr2/3, and Iml1p complexes; Npr2/3 complex mediates downregulation of TORC1 activity upon amino acid limitation; SEA complex is a coatomer-related complex that associates dynamically with the vacuole; Iml1p complex (Iml1p-Npr2p-Npr3p) is required for non-nitrogen-starvation (NNS)-induced autophagy; Iml1p interacts primarily with phosphorylated Npr2p; homolog of human NPRL2; target of Grr1p; required for growth on urea and proline
Subunit of SEA (Seh1-associated), Npr2/3, and Iml1p complexes; Npr2/3 complex mediates downregulation of TORC1 activity upon amino acid limitation; SEA complex is a coatomer-related complex that associates dynamically with the vacuole; Iml1p complex (Iml1p-Npr2p-Npr3p) is required for non-nitrogen-starvation (NNS)-induced autophagy; required for Npr2p phosphorylation and Iml1p-Npr2p interaction; null mutant shows delayed meiotic DNA replication and double-strand break repair
Subunit of the SEA (Seh1-associated) complex, a coatomer-related complex that associates dynamically with the vacuole; null mutation suppresses cdc13-1 temperature sensitivity; has N-terminal WD-40 repeats and a C-terminal RING motif
Arginase, responsible for arginine degradation, expression responds to both induction by arginine and nitrogen catabolite repression; disruption enhances freeze tolerance
Plasma membrane arginine permease, requires phosphatidyl ethanolamine (PE) for localization, exclusively associated with lipid rafts; mutation confers canavanine resistance
E3 ubiquitin ligase (N-recognin), forms heterodimer with Rad6p to ubiquitinate substrates in the N-end rule pathway; regulates peptide transport via Cup9p ubiquitination; mutation in human UBR1 causes Johansson-Blizzard Syndrome (JBS)
Transcription factor, involved in regulating multidrug resistance and oxidative stress response; forms a heterodimer with Pdr1p; contains a Zn(II)2Cys6 zinc finger domain that interacts with a pleiotropic drug resistance element in vitro
Transcription factor, involved in regulating multidrug resistance and oxidative stress response; forms a heterodimer with Pdr1p; contains a Zn(II)2Cys6 zinc finger domain that interacts with a pleiotropic drug resistance element in vitro
Component of the Rsp5p E3-ubiquitin ligase complex, involved in intracellular amino acid permease sorting, functions in heat shock element mediated gene expression, essential for growth in stress conditions, functional homolog of BUL1
Mitochondrial alcohol dehydrogenase isozyme III; involved in the shuttling of mitochondrial NADH to the cytosol under anaerobic conditions and ethanol production
Plasma membrane multidrug transporter of the major facilitator superfamily, confers resistance to short-chain monocarboxylic acids and quinidine; involved in the excretion of excess amino acids