Divergence) of) alternative) sugar) preferences) through) modulation) of) the)
expression)and)activity)of)the)Gal3)sensor)in)yeast)
)
Josep& Fita-Torró
1
,& Krishna& B.& S.& Swamy
2
,& Amparo& Pascual-Ahuir
3*
,& and& Markus&
Proft
1*&
&
1
& Department& of& Molecular& and& Cellular& Pathology& and& Therapy,& Instituto& de&
Biomedicina&de&Valencia&IBV-CSIC,&46010&Valencia,&Spain&
2
&Division&of&Biological&and&Life&Sciences,&School&of&Arts&and&Sciences,&Ahmedabad&
University,&Ahmedabad,&380009,&India&
3
& Department& of& Biotechnology,& Instituto& de& Biología& Molecular& y& Celular& de&
Plantas,&Universitat&Politècnica&de&València,&46022&Valencia,&Spain&
&
*
&Correspondence:&[email protected]&(AP-A);&[email protected]&(MP)&
&
&
&
Abstract)
Optimized&nutrient&utilization&is&crucial&for&the&progression&of&microorganisms&in&
competing&communities.&Here&we&investigate&how&different&budding&yeast&species&
and&ecological&isolates&have&established&divergent&preferences&for&two&alternative&
sugar& substrates:& Glucose,& which& is& fermented& preferentially& by& yeast,& and&
galactose,&which&is&alternatively&used&upon&induction&of&the&relevant&GAL&metabolic&
genes.&We&quantified&the&dose-dependent&induction&of&the&GAL1&gene&encoding& the&
central& galactokinase& enzyme,& and& found& that& a& very& large& diversification& exists&
between& different& yeast& ecotypes& and& species.& The& sensitivity& of& GAL1& induction&
correlates& with& the& growth& performance& of& the& respective& yeasts& with& the&
alternative& sugar.& We& further& define& some& of& the& mechanisms,& which& have&
established& different& glucose/galactose& consumption& strategies& in& representative&
yeast&strains&by&modulating&the&activity&of&the&Gal3&inducer.&(1)&Optimal&galactose&
consumers,&such&as&Saccharomyces/bayanus,&contain&a&hyperactive&GAL3&promoter,&
sustaining& highly&sensitive& GAL1&expression,&which&is&not & further& improved& upon&
repetitive& galactose& encounters.& (2)& Desensitized& galactose& consumers,& such& as& S./
cerevisiae&Y12,&contain&a&less&sensitive&Gal3&sensor,&causing&a&shift&of&the&galactose&
response&towards&higher&suga r&concentrations&even&in&galactose&experienced&cells.&
(3)& Galactose& insensitive& sugar& consumers,& such& as& S./ cerevisiae& DBVPG6044,&
contain& an& interrupted& GAL3& gene,& causing& extremely& reluctant& galactose&
consumption,& which& however& still& is& improved& upon& repeated& galactose&
availability.&In&summary,&different&yeast&strains&and&natural&isolates&have&evolved&
galactose& utilization& strategies,& which& cover& the& whole& range& of& possible&
sensitivities& by& modulating& the& expression& and/or& activity& of& the& inducible&
galactose&sensor&Gal3.&
&
Keywords:) Budding& yeast,& sugar& utilization,& GAL& sw it ch,& diauxic& growth,& GAL&
evolution,&GAL1,&GAL3,&Saccharomyces/bayanus/
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preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted January 16, 2023. ; https://doi.org/10.1101/2023.01.12.523712doi: bioRxiv preprint
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted January 16, 2023. ; https://doi.org/10.1101/2023.01.12.523712doi: bioRxiv preprint
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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Introduction&
Microorganisms& have& evolved& sophisticated& metabolic& regulation& to& adapt& to&
environments& with& continuously& changing& availability& of& nutrients.& An& important&
example& is& the& usage& of& different& sugar& sources& by& unicellular& fungi& such& as&
budding& yeast& (1).& Saccharomyces/ cerevisiae& and& evolutionarily& related& species&
have&specialized&towards&the&preferential&fermentation&of&glucose&over&alternative&
sugar& moieties& (2).& This& metabolic& behavior& is& driven& by& a& regulatory& network&
called&glucose&repression,&which&inhibits&the&expression&of&genes&necessary&for&the&
uptake& and&breakdown&of&alternative&carbohydrates&in&the&presence&of&glucose&(2).&
One& alternative& sugar,& which& is& frequently& found& in& natural& environments& is& the&
monosaccharide& galactose& (3).& Budding& yeasts& can& assimilate& galactose& by& the&
induced& expression& of& three& clustered& structural& genes,& GAL1,& GAL7& and& GAL10,&
whose&enzymatic&products&are&responsible&for&converting&galactose&into&glucose-1-
phosphate&in&order&to&generate&energy&for&the&cell&via&glycolysis&(4).&The&galactose-
utilization& (GAL)& pathway& in& Saccharomyces/ cerevisiae& has& b een& established& as& a&
model& system& to& understand& the& function& and& evolution& of& eukaryotic& metabolic&
regulation&and&to&unravel&the&principles&of&microbial&decision-making&(5–7).&&
GAL&gene&expression&in&S./ cerevisiae&is&strictly&repressed&in&the&presence&of&glucose&
(8,&9),&and&massively&induced&when&glucose&levels&decline&and&galactose&is&a vailab le&
(10).&The&Zn-cluster&transcription&factor&Gal4&is&the&master&activator&of&GAL&genes&
binding&to&a&highly&enriched&recognition&site&with&the&consensus&sequence&5´-CGG-
N
11
-CCG-3´& (11–13).& However,& in& the& absence& of& galactose,& Gal4& is& kept& in& an&
inactive& state& by& direct& binding& of& the& Gal80& repressor& protein& (14,& 15).& This&
inhibition& is& counteracted& by& the& additional& binding& of& t he& Gal3& sensor& upon& its&
activation& by& galactose& (16,& 17).& Thus,& Gal3& operates& the& GAL& on/off& switch& in& a&
galactose& concentration-dependent& manner& (18).& Since& the& expression& of& GAL3&
itself&is&inducible&by&ga l a ct ose&and&Gal4&,&the&GAL&switch&acquires&additional& modes&
of& plasticity,& which& might& fine-tune& the& sensitivity& and& efficiency& of& the& GAL&
response&during&the&adaptation&to&different&galactose&availability&(19,&20).&Indeed,&
at& certain& galactose& limiting& conditions& and& dependent& on& the& carbon& source&
history&and&the&genetic&background,&the&transition&from&the&uninduced&(off)&to&the&
induced&(on)&state&is&not&uniform&within&a&yeast&population,&which&leads&to&the&co-
existence&of& responders&and& non-responders&or& bimodal&response&(19,&21,&22).& It&
has&been&very&recently&show n&that&the&levels&of&the&Gal3&sensor,&either&artificially&
modulated& or& dictated& by& different& natural& alleles,& dominantly& mediates& the&
modality&of&t he&GAL&switch&(23).&The&constitutive&overexpression&of&Gal3&leads&to&a&
galactose& independent& activation& of& the& GAL& response,& highlighting& the& dominant&
function&of&the&sensor&in&the&control&of&GAL&gene&induction&(24).&
Another&important&feature&of&the&S./cerevisiae/GAL&response,&which&accounts&for&its&
environmental& adaptability,& is& its& enhanced& activation& after& previous& galactose&
encounter& or& transcriptional& memory& (25,& 26).& In& naïve& yeast& cells,& GA L& gene&
induction&is&slow&and&inefficient&especially&at&low&galactose&concentrations.&After&a&
previous& galactose& encounter,& transcriptional& activation& of& GAL& is& faster& and&
sensitive& to& low& inducer& concentrat ions,& and& this& memory& state& is& maintained&
during& several& generations& (27–29).& Enhanced& GAL& induction& during& memory&
depends& on& the& accumulation& of& the& Gal1& galactokinase& or& the& Gal3& galactose&
sensor&(28,&30,&31).&Both&proteins&recognize&galactose&and&associate&with&the&Gal80&
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repressor,& thus& switching& Gal4& to& the& active& form& and& unleashing& GAL& gene&
expression& (18,& 32).& Faster& re-activation& of& the& GAL& response& might& be&
advantageous& in& environments& with& frequent& galactose& availability.& Induction& of&
Gal1/Gal3& in& the& previous& galactose& encounter& is& a& way& to& promote& faster&
adaptation&to& the& alternative& sugar,& and& accordingly,& the& Gal1& or& Gal3& expression&
levels&have&been&found&to&correlate&with&the&efficiency&of&GAL&memory.&&
In& this& work,& we& investigate& to& what& degree& different& galactose& recognition& and&
consumption& behaviors& have& b een& evolved& in& natural& S./ cerevisiae& isolates& from&
different&ecological&niches&and&evolutionarily&related&species&such&as&S./bayanus,&S./
mikatae&and&S./paradoxus.&We&found&that&across&12&different&species&a&very&broad&
spectrum& of& galactose& preferences& exists,& from& almost& complete& galactose&
reluctance& to& highly& sensit ive& GAL& respon ses. & These& b ehaviors& correlate& with&
divergent& dose-response& profiles& of& GAL1& induction& and& have& consequences& for&
glucose/galactose& growth& performance,& diauxic& shift,& galactose& memory& and&
susceptibility& t o& toxic& sugar& analogues.& We& further& characterize& representative&
galactose& consumption& strategies,& which& have& evolved& by& tuning& Gal3&
expression/activity:& the& ultra-sensitive& galactose& response& of& S./ bayanus,& the&
specialization&towards&higher&galactose&concentrations&of&S./cerevisiae&sake&strain&
Y12,&and&galactose&insensitivity&in&West&African&S./cerevisiae&strains.&&
&
Materials)and)methods)
Yeast&strains&and&growth&conditions&
The&yeast&strains&used&in&this&work&are&described&in&Table&1.&Yeast&cultures&were&
grown&at&28
o
C&in&Yeast&Extract&Peptone&Dextrose&(YPD)&or&Galactose&(YPGal)&media&
containing& 2%& glucose& or& galactose& or& indicated& mixtures& of& both& sugars,& or& in&
Synthetic& Dextrose& (SD),& Raffinose& (SRaf)& or& Galactose& (SGal)& media& containing&
0.67%& yeast& nitrogen& base& with& ammonium& sulfate& and& without& amino& acids,&
50mM& succinic& acid& (pH& 5.5)& and& 2%& of& the& respective& sugar.& According& to& the&
auxotrophies&of&each&st rain,&methionine&(10&mg/l),&histidine&(10&mg/l),&leucine&(10&
mg/l)&or&uracil&(25&mg/l)&were&supp lemented.&Yeast&cells&were&transformed&by&the&
lithium&acetate/PEG&method&described& by&(33).&The&glucose&analogue&glucosamine&
hydrochloride& (GlcN,& Sigma& Aldrich)& was& added& to& the& growth& medium& at& the&
indicated&final&concentrations&from&a&10%&stock&solution&in&H
2
O.&&
Plasmid&constructions&
The& single-copy& destabilized& luciferase& reporter& plasmid& pAG413-GAL1
BY4741
-
lucCP
+
& (34)& was& used& to& quantify& the& GAL1& dose& response& of& different& yeast&
isolates.& Multi-copy& reporter& plasmids& expressing& destabilized& luciferase& under&
control& of& different& GAL3& promoter& alleles& were& constructed& by& insertion& of& PCR&
amplified& fragments& containing& the& 900bp& upstream& of& the& ATG& into& luciferase&
vector& pAG423-lucCP
+
& (34).& For& the& quantification& of& GAL1& activation& by& flow&
cytometry,& a& single-copy& GFP& reporter& was& constructed& b y& insertion& of& the&
GAL1
BY4741
& upstream& regulatory& region& in& plasmid& pRS416-GFP& (35).& For& the&
quantitative& analysis& of& the& Gal3& sensors& of& BY4741& and& Y12,& we& cl oned& the&
respective& GAL3& ORF& regions& in& the& Gateway& single& copy& expression& vect or&
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pAG416-GAL1-ccdB& (36).& Complementation& of& yeast& cells& with& the& wild& type&
BY4741&copy&of&GAL3&was&done&using&the&single&copy&expression&plasmid&pAG416-
GPD-GAL3
BY4741
& (28).& Primers& used& for& plasmid& constructions& are& summarized& in&
Table&2.&All& GAL3& promoter& and& ORF&variants&used& in& this& study& were&sequenced&
for& verification& purposes& and& for& the& identification& of& specific& nucleotide& changes&
occurring&in&the&natural&yeast&variants.&
Live-cell&luciferase&assays&
Yeast&strains&containing&the&indicated&luciferase&fusion&genes&were&grown&at&28
o
C&
in&synthetic&raffinose&(SRaff)&medium&lacking&histidine&at&pH=3&to&low&exponential&
phase&(OD600&=&1-2).&Culture&aliquots&were&then&incubated&with&0.5mM& luciferin&
(free& acid,& Synchem,& Germany)& on& a& roller& at& 28
o
C& for& 1h.& The& cells& were& then&
transferred& in& 135µl& aliquots& to& white& 96& well& plates& (Nunc)& containing& the&
indicated& concentrations& of& galactose.& The& light& emission& was& continuously&
recorded&on& a&GloMax& microplate&luminometer&(Promega)& in&at& least&3&biological&
replicas.&Data&were&processed&with&Microsoft&Excel&software.&For&rep resen tation&of&
the&relative&light&units,&we&normalized&the&raw&data&for&the&number&of&cells&in&each&
individual& assay.& The& maximal& synthesis& rate& was& calculated& as& described&
previously&(34).&
Quantitative&growth&assays&
For&the&quantitative&estimation&of&growth&parameters,&fresh&overnight&precultures&
of&the&indicated&yeast&isolates&in&Y PD&or&YPGal&media&were&diluted&in&triplicate&in&
the&assay&medium&in&multiwell&pl ates&to&a&starting&OD600&=&0.1.&Growth&was&then&
constantly&monitored&at&28
o
C&on&a&Tecan&Spark&multiplate&reader&for&the&indicated&
times.&The&growth&curves&were&processed&in&Microsoft&Excel&and&absolute&growth&
ratios&and&lag&phases&calculated.&
Transcriptional&memory&experiments&
For&memory&experiments&at&GAL1,&cells&containing&the&GAL1p-lucCP
+
&reporter&gene&
were& grown& over& night& in& synthetic& Raffinose& (2%)& medium& lacking& histidine& to&
exponential&growth&phase.&A&first&round&of&induction&was&then&performed&for&2&h&&
with&1%& galactose,& while& naïve&cells&remained& in& SRaf& medium.&Both& cell& cutures&
were&then&pelleted,& washed&once&with&H2O,&and& then&incubated&in&SD&(2%&glucose)&
medium&for&1h.&Finally,&cell s&were&washed&again&and&resuspended&in&SRaf&medium&
containing&0.5&mM&luciferin&for&90&min&before&starting&the&next&induction&with&the&
indicated& galactose& concentrations.& Light& emission& was& then& monitored&
continuously&as&described&above&for&the&standard&luciferase&assays.&
Flow&cytometry&
Cells& harboring& a& single-copy& GAL1p-eGFP& reporter& were& pre-grown& in& synthetic&
raffinose& medium& (SRaf)& without& uracil& to& exponential& growth& phase.& Cells& were&
diluted& to& OD
600
& =& 0.1& and& induced& with& the& indicated& galactose& concentrations.&
Before& induct ion& and& at& the& indicated& times& of& galactose& supplementation,& cell&
aliquots&were&passed&through&a&MACS&Quant&10&flow&cytometer&(Miltenyi&Biotec).&
GFP& was& excited& with& a& 488nm& laser& and& emission& was& detected& applying& a&
525/550nm& band& pass& filter& set.& All& GAL1-GFP& induction& experiments& were&
performed& on& 3& independent& biological& samples.& 20000& cel l s& were& analyzed& for&
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each&time&point,&and&aggregated&cells&(<5%)&were&discarded&from&further&analysis.&
For& the& detection& of& the& induced& single& cell& fraction,& the& uninduced& fluorescence&
intensity& was& defined& for& the& cell& population& at& time& point& 0,& and& during& the&
induction& process,& all& cells& with& higher& fluorescence& intensities& were& counted& as&
“positive”.& The& mean& fluorescence& intensity& was& additionally& determined& in& the&
single&cell&fraction&at&each&time&point&of&galactose&induction.&&
Results)
Dose-dependent)GAL1)induction)profiles)reveal)a)broad) spectru m)of)different)
sugar) preferences) across) natural) budding) yeast) strains.& Laboratory&
Saccharomyces/cerevisiae&strains&typically&show&a&st rictly&galactose&concentration-
dependent&activation&of&GAL&genes&upon&the&switch&from&glucose&to&galactose&(28).&
High& galactose& inducer& concentrations& are& needed& to& reach& optimal& induction&
kinetics.& We& therefore& reasoned& that& divergent& galactose& preferences& could& be&
quantitatively&determined&by&comparing&the&dose&dependent&induction&profiles&of&
GAL1&across&a&variety&of&yeast&strains&from&different&ecological&origins.&In&order&to&
obtain& truly& quantitative& gene& expression& profiles& in& real& time,& we& employed& the&
extremely&unstable&lucCP
+
&luciferase&derivative&in&the&laboratory&reference&strain&
BY4741& (34),& as& well& as& in& 8& different& S./ cerevisiae& natural& isolates& and& in& the&
evolutionarily&close&S./paradoxus,&S./mikatae&and&S./bayanus&strains&(Figure&1A).&We&
recorded&the&dose-responsive&activation&profiles&for&GAL1&applying&an&exhaustive&
range&of&galactose&inducer&concentrations&(Figure&1B).&All&strains&tested&showed&a&
measurable&activation&of&luciferase&activity&over&the&initial&2.5&hours&of&induction,&
except&the&West&African&isolates&DBVPG6044&and&NCYC110.&We&noticed&important&
differences&when&we&compared&the&absolute&induction&levels&along&the&increasing&
galactose& concentrations& for& each& yeast& strain& (Figure& 1C).& S./ mikatae& and&
especially& S./ bayanus& respond& to& low& galactose& concentrations& much& more&
efficiently& and& reach& optimal& induction& velocities& at& much& lower& galact ose&
concentrations& as& compared& to& the& BY4741& laboratory& strain.& Both& yeast& species&
also& display& a& prolonged& activation& of& the& GAL1/ gene,& which& in& the& case& of& S.&
bayanus& leads& to& almost& 7& fold& enhanced& induction& ratios.& This& suggests& that& S./
mikatae& and& S./ bayanus& have& evolved& towards& a& more& sensitive& galactose&
recognition.& The& natural& Y12& S./ cerevisiae& isolate& has& yet& a& distinctive& pattern& of&
galactose& induction.& In& this& case,& GAL1& induction& is& very& efficient& at& high& inducer&
concentrations,&while&it&is&slow&and&inefficient&upon&low& gala ctose&stimulation.&This&
suggests& that& S./ cerevisiae& Y12& has& adapted& to& discriminate& more& different &
galactose& concentrations& and& recognizes& the& alternative& sugar& preferentially& at&
higher&doses.&Most&other&natural&S./cerevisiae&isolates&tested&here&showed&a&GAL1&
dose& response,& which& resembled& the& BY4741& reference.& However,& S./ paradoxus&
responds&to&increasing&galactose&concentrations&much&more&inefficiently&and&in&the&
DBVPG6044& and& NCYC110& isolates,& a& rapid& galactose& response& was& completely&
absent.& These& data& demonstrate& that& natural& yeast& isolates& have& evolved& very&
different& galactose& response& strategies,& whose& consequences& for& growth& in& the&
presence&of&the&alternative&sugar&will&be&investigated&next.&&
Sensitivity) of) GAL) induction) correlates) with) growth) performance) and)
glucose/galactose)selectivity.&We&compared&quantitative&growth&parameters&for&
all& the& natural& yeast& strains& included& in& this& study& with& respect& to& glucose& and&
galactose&utilization.&As&shown&in&Figure&2A,&the&difference&of&the&absolute&growth&
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rates& for& both& sugars& is& extremely& divergent& among& the& yeast& strains.& In& the&
BY4741& reference& strain,& the& growth& rate& on& galactose& slows& down& to& 60%& as&
compared&to&glucose.&S./mikatae&and&especially&S./bayanus&demonstrate&less&sugar&
discrimination.&Remarkably,&S./bayanus&cells&grow&with&indistinguishable&rates&on&
both& sugar& substrates.& This& correlates& well& with& their& better& and& more& sensitive&
GAL1&gene&induction.&On&the&lower&end,&we&identify&yeast&strains&with&a&much&more&
pronounced& sugar& discrimination,& which& grow& on& galactose& more& inefficiently.&
These& strains& a re& the& S./ cerevisiae& Y12& isolate,& S./ paradoxus& and& the& DBVPG6044&
and& NCYC110& isolates,& which& altogether& are& characterized& by& their& sub-optimal&
GAL1&induction&profiles.&These&growt h&differences&are&also&well&recapitulated&when&
we&calculated&the&lag&phase&needed&by&all&strains&to&engage&in&active&growth&when&
switched& from& glucose& to& galactose& media& (Figure& 2A).& We& next& tested& the&
performance& of& selected& yeast& strains& during& the& diauxic& shift& on& mixed& sugar&
media& with& limited& amounts& of& glucose& (Figure& 2B).& Under& these& conditions,& the&
cells&have&to&switch&from&glucose&to&galactose&utilization.&Moderate&GAL1&inducers&
such&as&BY4741&or&Y12,&show&a&characteristic&diauxic&lag&phase&separating&glucose&
and&galactose&consumption.&However,&optimized&GAL1&inducers,&S./mikatae&and&S./
bayanus,& do& not& display& a& diauxic& lag& and& grow& continuously& on& the& mixed& sugar&
media.& Interestingly,& S./ bayanus& shows& a& slight& growth& disadvant a ge& on& glucose&
media& as& compared& to& BY4741,& which& is& more& than& compensated& during& the&
glucose-galactose& switch& where& S./ bayanus& cells& are& able& to& outgrow& BY4741&
(Figure&2B).&As&expected,&poor&GAL1&inducers&DBVPG6044&and&NCYC110&show&an&
extreme&diauxic&lag&and&low&growth&rates&on&galactose&media.&These&data&indicated&
that& yeast& strains& with& an& optimized& GAL& gene& induction& acquire& a& growth&
advantage&during&mixed&sugar&or&pure&galactose&growth.&&
We&next&wanted&to&know&whether&the&inefficient&galactose&growth&performance&of&
some&selected& yeast& strains& could&be&improved& by& previous& galactose&adaptation.&
We& therefore& compared& the& growth& parameters& for& galactose& of& cells& that& were&
either& pre-grown& in& glucose& or& galactose& (Figure& 3).& For& the& best& galactose&
performers& including& S./ mikatae& and& S./ bayanus,& but& also& the& BY4741& laboratory&
strain,&galactose&pre-growth& did& not&improve&neither& the& growth& rate&nor&the& lag&
phase&on&galactose&media&(Figure&3A&and&3B).& However,&poor&galactose&performers&
such&as&S./cerevisiae&Y12,&DBVPG6044&and&NCYC110,&largely&improve&their&growth&
rates& and& cut& down& the& lag& phase& on& galactose& media& after& previous& galactose&
adaptation.& This& suggests& that& all& strains& tested& have& conserved& the& ability& to&
metabolize& galactose& as& an& alternative& energy& source,& however,& signaling& differs&
substantially&in&different&yeasts&creating&very&divergent& ga lactose&preferences.&&
Specialization) towards) highly) sensitive) galactose) consumption:) the) S.'
bayanus) case.& In& our& previous& experiments,& S./bayanus&was&characterized& as& the&
most&efficient&galactose&consumer&based&on&its&highly&sensitive&and&efficient&GAL1&
induction&profile,&equal &growth&performance&on&glucose&or&galactose&media&and&an&
optimized& transition& from& glucose& to& the& alternative& sugar& in& mixed& sugar&
environments.&A&closer&inspection&of&the&GAL1&dose&response&data&revealed&that&S./
bayanus&displays&a&remarkably&robust&gene&induction,&which&is&almost&complete&at&
very& low& galactose& concentrations& (0.03%)& (Figure& 4A).& O ne& consequence& of& this&
galactose& hypersensitivity& is& a& growth& advantage& over& other& yeasts& with& a& more&
repressed& galactose& signaling,& such& as& BY4741,& with& a& pronounced& adaptation&
phase&during&the&switch&from&one&sugar&to&the&other&(Figure&4B).&We&reasoned&that&
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S./ bayanus& was& less& dependent& on& glucose& fermentation& and& more& readily&
metabolized& galactose& on& mixed& substrates.& We& tested& this& hypothesis& by& the&
application& of& the& naturally& occurring& glucose& analogue& glucosamine& (2-amino-2-
deoxy-glucose).& Glucosamine& is& taken& up& and& phosphorylated& b y& Saccharomyces&
species,&however,&cannot&be&further&metabolized&via&glycolysis,&which&results&in&a&
strong&growth&inhibition&(37).&When&BY4741&reference&and&S./bayanus&cells&were&
offered&mixtures&of&glucosamine&and&galactose,&we&found&that&S./bayanus&was&much&
less&susceptible&to&glucosamine&growth&arrest&(Figure&4C).&These&results&confirmed&
that&the&S./bayanus&strain&had&a&superior&affinity&for&galactose&consumption,&which&
caused&the&observed&hyperresistance&to&the&glucose&analogue.&We&next&addressed&
the&question,&whether&a&previous&galactose&encounter&improved&the&transcriptional&
response&in&these&two&divergent&yeasts&(Figure&4D).&S./cerevisiae&laboratory&strains&
such& as& BY4741& are& characterized& by& a& strong& improvement& of& their& induction&
kinetics,&both&efficiency&and&sensitivity,&by&mechanisms&of&transcriptional&memory&
(28,&31).&As&expected,&a&robust&transcriptional&memory&effect&was&determined&for&
the& S./ c.& BY4741& strain.& However,& S./ bayanus& displayed& an& already& optimal& GAL1&
dose& response& in& naïve& cells,& which& was& not& at& all& improved& after& galactose& pre-
treatment& (Figure& 4E).& These& data& suggest& that& S./ bayanus& has& evolved& an&
optimized&galactose&signaling,&which&is&constitutively&active&and&not&susceptible&to&
improvement&by&p revious&galactose&consumption.&We&next&wanted&to&identify&the&
molecular&bases&for&this&galactose&specialization.&
One& key& factor& for& a& more& sensitive& galactose& recognition& and& signaling& is& the&
expression&of&the&galactose&sensor&Gal3&(19,&28).&Therefore,&we&analyzed&the&dose&
response& behavior& of& the& GAL3& upstream& control& regions& of& S./ bayanus& in&
comparison& to& the& S./ cerevisiae& BY4741& reference.& We& additionally& included& the&
GAL3& promoter& variants& from& suboptima l& gala ctose& consumers,& such& as& S./
cerevisiae& Y12& and& DBVPG6044,& in& this& study.& We& recorded& the& complete& dose-
response&profiles&of&all&GAL3&promoter&variants&by&time&elapsed&luciferase&assays&
in&the&BY4741&genetic&background&(Figure&5A).&We&first&noticed&that&GAL3p
S.bayanus/
displayed& a& significantly& higher& basal& activity& as& compared& to& all& other& GAL3&
promoters& (Figure& 5B).& Moreover,& GAL3p
S.bayanus/
was& activated& by& very& low&
galactose&concentrations&(<0.1%)&to&almost&complete&efficiency&(Figure&5C).&Thus,&
the& Gal3& expression& in& S./ bayanus& is& driven& by& a& constitutively& more& active&
promoter,& which& is& more& sensitively& activated& by& l ow& inducer& concentrations.&
Inspection& of& the& respective& nucleotide& sequences& of& the& GAL3& upstream& control&
regions&revealed&no&obvious&changes&w it hin&the&perfectly&conserved&binding&region&
for& the& Gal4& transcriptional& activator& (Figure& 5D).& However,& we& observed&
important&differences&in&the&sequences&of&two&Mig1&repressor&binding&sites&in&the&
GAL3& upstream& sequences.& The& reference& GAL3p
BY4741
& contains& two& predicted&
consensus& sequences& for& Mig1,& proximal& site& 1& between& the& Gal4
UAS
& and& the&
transcription&start&site&and&distal&site&2&just&upstream&of&Gal4
UAS
&(Figure&5D).&Site&1&
perfectly& matches& the& previously& characterized& Mig1& consensus& with& the& 3´-G/C&
box&and& the&adjacent&5´-AT-rich&flanking&region&(38).&Site&2&only& conserves&the&G/C&
box&and&might&therefore&not&be&functional.&In&any&case,&the&GAL3p
S.bayanus/
sequence,&
but& not& the& other& S./ cerevisiae& variants,& shows& several& point& mutations& in& the&
essential&G/C&box&motifs,&thereby&inactivating&both&Mig1& binding&sites&(Figure&5D).&
These& data& suggest& that & S./ bayanus& has& evolved& an& especially& sensitive& galactose&
consumption&behavior,&which&at&least&in&part,&might&be&caused&by&its&derepressed&
and&hyper-activatable&GAL3&control&lacking&Mig1&repression.&
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Specialization) towards) desensitized) galactose) consumption:) the) case) o f) S.'
cerevisiae) Y12.& Our& previous& results& showed& that& the& S./ cerevisiae& Y12& isolate&
needed&higher&galactose&concentrations&in&the&medium&to&efficiently&consume&the&
alternative& sugar.& This& behavior& is& characterized& by& a& less& sensitive& GAL1& gene&
induction,&a&pronounced&diauxic&lag&phase&and& decreased&galactose& growth&rates.&
We& investigated& in& dep t h& this& sugar& consumption& strategy& as& an& example& of& a&
naturally& evolved& enhanced&sugar&discrimination.&We&first& looked& in& detail& at& the&
GAL1& induction& capacity& of& Y12& and& found& that& it& was& impaired& only& at& very& low&
galactose& concentrations& showing& a& significant& response& delay& (Figure& 6A).& High&
galactose&concentrations,&however,&caused&a&timely&and&even&more&dynamic&GAL1&
activation& in& Y12& as& compared& to& BY4741.& This& more& discriminate& galactose&
behavior& was& perfectly& recapitulated& by& the& absolute& induction& velocities,& which&
were&lower&than&BY4741& in&a&range&up&to&0.02%& galactose&and&actually&higher&than&
BY4741&for&galactose& >0.02%& (Figure&6B).& We&next&tested&whether&this&apparent&
less&affinity&for&low&galactose&doses&had&consequences&for&the&susceptibility&to&the&
toxic& glucose& analogue& GlcN.& Indeed,& we& found& a& pronounced& sensitivity& of& S./
cerevisiae/Y12&to&low&GlcN&doses,&indicating&a&higher&preference&of&Y12&for&glucose&
consumption&in& the&presence&of& alternative&galactose& (Figure&6C).& At&low&inducer&
concentrations,&the&yeast&GAL&response&can&be&bimodal&with&only&a&fraction&of&the&
cells&in&the&population&actively&engaged&in&gene&expression.&In&order&to&determine&
the& fraction&of&actively&responding&cells&during&the&transition&from&the&off&to&the&on&
state,&we&measured&the&GAL1p-driven&GFP&expression&upon&low&and&high&gal a ctose&
induction& by& flow& cytometry& (Figure& 6D).& We& found& that& high& galactose&
concentrations& caused& a& rapid& and& comparable& transition& to& GFP& positive& cells& in&
BY4741&and&Y12.&As&expected,&the&transition&from&the&off&to&the&on&state&occurred&
more&slowly&in&BY4741&upon&low&galactose&induction&(<0.02%).&However,&the&Y12&
isolate&showed&a&severe&induction&delay&upon&those&inducer&concentrations&(Figure&
6D).& The& comparison& of& the& induction& levels& in& b oth& yeast& strains& again&
demonstrated& that& Y12& cells& discriminate& much& more& low& and& high& galactose&
concentrations& in& the& medium& (Figure& 6D).& We& finall y& confirmed& that,& like& the&
BY4741& reference& strain,& Y12& cells& possess& a& strong& positive& memory& upon&
repeated& galactose& encounters& (Figure& 6E).& However,& even& in& galactose&
experienced& Y12& cells,& it& seemed& that& low& galactose& stimulation& still& caused& a n&
inefficient& response& as& compared& to& the& very& much& improved& induction& in&
experienced& BY4741& cells& (Figure& 6E).& This& observation& suggested& that& Y12& cells&
have&a&l ess&sensitive&Gal3&sensor,&which&needs&higher&galactose&level s&to&efficiently&
induce& GAL& gene& expression.& In& order& to& test& this& hypothesis,& we& expressed& the&
Gal3
BY4741
& and& Gal3
Y12
& sensor& variant s& from& the& strong& GAL1
BY4741
& promoter& and&
compared&their&dose&dependent&activat ion& of&a&GAL1p-luciferase&reporter&(Figure&
7A,& B).& Tra nsa ctivation& of& both& Gal3& sensors& was& indistinguishable& at& high&
galactose& concentrations,& while& low& galactose& doses& triggered& a& more& efficient&
GAL1&induction&through&Ga l 3
BY4741
&as&compared&to&Gal3
Y12
&(Figure&7C).&These&data&
demonstrate&that&S./cerevisiae& Y12&possesses&a&less&sensitive&Gal3&sensor.&Indeed,&
the&concentration&dependent&Gal3
Y12
&activation&profile&in&BY4741&cells&was&similar&
to&the&one&observed&in&Y12&galactose&experienced&cells&(Figure&7D),&indicating&that&
the& desensit ized& galactose& signaling& of& the& Y12& strain& stems& mainly& from& its& less&
sensitive&Gal3&sensor&protein.&&&
Extremely)insensitive)ga lactose)consumption:)the)case)of )the)West)African)S.'
cerevisiae) DBVPG6044) and) NCYC110.& The& West& African& yeast& isolates&
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investigated& here& displayed& an& extraordinarily& long& lag& phase& and& poor,& but& not&
absent,&growth&rates&on&galactose&media.&Accordingly,&no&GAL1&gene&induction&was&
detected& in& the& first& hours& of& galactose& stimulation.& Additionally,& short& term&
memory& effects,& such& as& previously& found& here& for& S./ cerevisiae& BY4741& or& Y12,&
were&completely&absent&(data&not&shown).&The&DBVPG6044&and&NCYC110&isolates&
required&a&previous&galactose&pre-incubat ion&for&several&days&in&order&to&improve&
galactose&growth&parameters&(Figure&3)&or&GAL1&gene&induction&(data&not&shown).&
We& re-sequenced& the& GAL3
DBVPG6044
& allele& and& found& a& single& nucleotide&
substitution,&C
456
&to&A
456
,&which&created&a&premature&stop& codon&after&aa
151&
(Figure&
8A).& This& mutation& has& been& previously& reported& for& some& West& African& yeast&
isolates&including&DBVPG6044&an d&NCYC110&(39).&However,&if&this&inactivation&of&
Gal3& function& was& the& only& defect& in& the& GAL& system& in& these& isolates,& their&
galactose&consumption&should& b e&restored&by&re-introducing&a&functional&Gal3&wild&
type&copy.&Indeed,&when&we&transformed&DBVPG6044&or&NCYC110&yeast&cells&with&
a&GAL3
BY4741
&allele,&both&strains&recovered&robust&growth&on&pure&galactose&media&
(Figure&8B).& These&data&indicated& that& the&West& African& isolates&DBVPG6044&and&
NCYC110& have& adopted& a& slow& and& insensitive& galactose& response& by& genetic&
interruption&of&their&GAL3&sugar&sensor.&
Discussion)
The& natural& habitats& of& microorganisms& often& experience& fluctuations& in& the&
nutrient& composition.& These& changes& trigger& the& induction& of& the& metabolic&
pathways& needed& to& efficiently& utilize& the& available& nutrients& (2,& 40).& Related&
microbial& organisms& have& evolved& divergent& strategies,& both& in& terms& of& the&
nutrients& recognized& and& of& the& efficiency& of& metabolizing& them& (39,& 41).& In&
general,& the& decision& to& utilize& a& particular& alternative& nutrient& is& conditioned& by&
the&cost&to&maintain&the&metabolic&enzymes&even&w hen&the&nutrient&is&scarce&and&
the&benefit&for&cellular&energy&metabolism&by&efficiently&consuming&the&metab olite&
(20,& 42,& 43).& Here,& we& reasoned& that& in& the& case& of& galactose& utilization& in& yeast,&
different& sugar& preferences& should& be& quantifiable& by& comparing& the& dose-
response&induction&profile&of& its&principal&metabolic&enzyme&Gal1.&The&destabilized&
luciferase& technology& applied& by& us& faithfully& identifies& such& changes& in& the& GAL&
dose-response& across& different& S./ cerevisiae& natural& isolates& and& related&
Saccharomyces/species.&&
In& all& yeasts& analyzed& here,& GAL1& gene& expression& is& repressed& in& the& absence& of&
galactose.& Thus,& divergent& galactose& preferences& should& arise& from& a& different&
facility&to&overcome&this&repression&and&efficiently&transcribe&GAL1.&&The&key&GAL&
inducer&is&the&GAL3&sensor,&which&arose&from&the&original&GAL1&galactokinase&gene&
by&gene& duplication.&Further& specialization&of& GAL3& through&evolution& turned&the&
protein& from& a& galactose& phosphorylating& enzyme& to& a& highly& sensitive& galactose&
sensor& needed& to& switch& the& Gal4-Gal80& complex& to& an& efficient& transcriptional&
activator& of& GAL1(44–46).& Importantly,& GAL3& expression& itself& is& galactose&
dependent& (47),& thus& the& sensitivity& of& the& Saccharomyces& GAL& switch& could& be&
either& modulated& by& the& expression& dynamics& or& the& intrinsic& activity& of& a& single&
sugar&sensor,&Gal3.&Indeed,&the&dominant&role&of&polymorphisms&in&the&GAL3&locus&
in& the& diversification& of& galactose& responses& in& different& S./ cerevisiae& isolates& has&
been& recently& demonstrated& (19).& Here& w e& identify& very& divergent & galactose&
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consumption& strategies,& which& at& least& in& great& part,& can& be& explained& by&
differential&expression&or&altered&sensitivities&of&the&Gal3& sensor.&&
The& galactose& behavior& of& S./ bayanus& is& an& ext reme& adaptation& towards& highly&
efficient&galactose&consumption,&while&keeping&the&GAL&system&still&under&control&
of& galactose& induction.& This& induction,& however,& is& very& much& improved,& so& that&
maximal&GAL&gene&induction&is&achieved&in&this&yeast&at&5&to&10-fold&less&galact ose&
concentrations& as& compared& to& its& S./ cerevisiae& relatives.& Consequently,& this&
hypersensitive& GAL& switch& enables& a& continuous& proliferation& in& habitats& where&
glucose&is&exhausted&in&the&presence&of&galactose.&Here,&S./bayanus&outgrows&other&
yeast&species&with&a&diauxic&lag&phase.&Thus,&the&S./bayanus&galactose&consumption&
strategy& might& be& advantageous& in& rapidly& changing& mixed& sugar& environments.&
One& key& event& in& creating& this& extra& sensitive& GAL& switch& is& the& here& reported&
modification& of& the& GAL3& upstream& control& region.& An& increased& basal& level& and&
more&sensitive&galactose&induction&of&GAL3&in&S./bayanus&seems&to&create&the&hyper&
sensitive& GAL& response.& It& is& likely& that& the& Gal3& levels& in& S./bayanus& are& not& rate&
limiting& even& in& the& absence& of& the& alternative& sugar.& This& would& explain& the&
complete& lack& of& transcriptional& memory& in& this& yeast.& Improvement& of& the& GAL&
switch&after&repeated&galactose&exposures&depends&on&higher&Gal3&(and&to&a&lesser&
extent& Gal1)& protein& levels& (31),& and& even& slight& overexpression& of& Gal3& strongly&
improves& the& sensitivity& of& GAL& induction& in& laboratory& yeast& strains& (28,& 48).&
Consistent& with& our& finding,& it& has& been& reported& previously& that& GAL& up stream&
control&regions&are&the&key&elements&that&confer&delayed&galactose&consumption&in&
S./ cerevisiae& as& compared& to& the& long-diverged& S./ bayanus& (49).& Specifically,& the&
single& su b stitution& of& the& GAL3& promoter& in& S./ bayanus& with& the& S ./ cerevisiae&
counterpart& restored& an& evident& diauxic& lag& (49),& which& confirms& that& Gal3&
expression&levels&are&key&to&GAL&switch&sensitivity&and&galactose&select ivit y.&&
Diversification&of&the&GAL&switch&among&different&S./cerevisiae&isolates&dominantly&
depends&on&evolutionary&differences&in&the&GA L3&locus& (19).&However,&how&specific&
mutations&in& the&Gal3&protein&confer&different&GAL&switch&characteristics&remained&
unknown.&The&S./cerevisiae/Y12&behavior&is&characterized&here&as&an&intra-species&
adaptation& towards& higher& galactose& discrimination.& Here,& the& basis& is& not& an&
altered&Gal3& expression,&as&the&GAL3
Y12&
promoter&is&equally&active&in&the&absence&or&
presence& of& low& galactose& concentrations.& The& Gal3
Y12
& sensor& needs& higher&
galactose& concentrations& to& be& fully& active& and& operate& the& GAL& switch& through&
Gal80& inactivation.& Several& polymorphisms& are& present& in& the& Gal3
Y12
& coding&
sequence:& K
122
R,& I
135
V,& P
137
L,& Q
149
L,& L
302
P,& H
352
D,& and& L
370
P.& The& reversible& Gal3&
interaction& with& the& Gal80& repressor& requires& binding& of& ATP& and& galactose& (16,&
18).&Thus,& some&amino&acid&substitutions&in&Gal3
Y12
&may&have&specific&effects&on&its&
galactose/ATP& or& Gal80& binding& efficiency.& The& Gal3
Y12
& sensor& is& an& interesting&
candidate&for&further&characterization&by&biochemical&experiments.&Less&sensitive&
galactose&recognition&might&be&advantageous&in&environments,&where&galactose&is&
rarely&available& or&at&con cen trations,&which& do& not& sustain& cell& proliferation.& Y12&
cells&have&tuned&down&the&Gal3&sensitivity&and&thus&raised&the&galactose&threshold&
needed&to&activate&the&GAL&switch&and&to&metabolize&the&alternative&sugar&only&at&
higher&concentrations.&A&further&adaptation&towards&even&less&sensitive&galactose&
consumption& has& occurred& in& some& West& African& S./ cerevisiae& isolates& by& a&
premature& stop& in& the& GAL3& coding& region.& Those& yeast& isolates& have& been&
previously& categorized& as& galactose& non-fermenters& (19,& 39).& However,& we& show&
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here&that&these& yeasts&have&not&given&up&metabolizing&galactose,&instead&they&have&
evolved&an&extremely&insensitive&GAL&sw itch&by&inactivating&their&Gal3&sensor.&This&
likely&adaptation&to&very&infrequent&galactose&availability&still&enables&to&consume&
the& alternat ive& sugar& at& very& low& rates& and& to& display& a& positive& memory& after& a&
prolonged& galactose& encounter& presumably& via& the& remaining& Gal1& galactokina se&
(24,&30).&In&summary,&the&function&of&a&dedicated&and&inducible&nutrient&sensor&in&
the&GAL&switch&has&enabled&the&evolution&of&very&divergent&galactose&consumption&
strategies&in&yeast.&&&
Funding)
)
This& work& was& funded& by& a& grant& from& Ministerio& de& Ciencia,& Innovación& y&
Universidades&PID2019-104214RB-I00&to&AP-A&and&MP.&&
)
Acknowledgments&
&
The&authors&thank&Jun-Yi&Leu&for&the&kind&gift&of&&S./cerevisiae&natural&isolates&and&
Saccharomyces/ bayanus,& Saccharomyces/ mikatae& and& Saccharomyces/ paradoxus&
strains.&&
&
Figure)legends:)
&
Fig.) 1& Dose-dependent) GAL1) expression) across) natural) yeast) isolates.& (A)&
Yeast& strains& and& natural& isolates& from& different& geographical& origin& used& in& this&
study.& (B)& Time-elapsed& study& of& the& dose& dependent& induction& of& GAL1& in& the&
indicated&yeast&strains.&A&single&copy&GAL1-lucCP
+
&reporter&was&used&in&continuous&
live&cell&luciferase&a ssays.&Results&are&depicted&from&at&least&3&b iological&replicates.&
Light&emission&at&time&point&0&was&set&to&1&for&all&induction&curves.&(C)&Comparison&
of&t he&sensitivity&of&GAL1&induction.&Representation&of&the&maximal&light&increment&
(v
max
)&versus&the&galactose&inducer&concentration&for&each&yeast&strain.&Left&panel:&
Absolute&v
max
&values&plotted&against&gal&concentrations,&right&panel:&v
max
&values&set&
to&100%&for&the&inducer&concentration,&which&leads&to&maximal&induction.&
&
Fig.) 2&Glu cose/galacto se) growth) performance) across) natural) yeast) isolat es.&
(A)& Left& panel:& The& growt h& rates& on& 2%& glucose& or& galactose& media& were&
determined& for& the& indicated& yeast& strains& pregrown& on& glucose& medium.& The&
velocity& on& glucose& medium& was& set& to& 100%& for& each& strain.& Right& panel:&
Representation&of&the&lag&time&needed&for&each&strain&to&reach&50%&growth&on&the&
indicated& media.& The& lag& time& for& glu cose& growth& w a s& set& to& 1& for& each& strain.&
Represented&are&the&mean&values&(n&=&3)&+/-&SD.&(B)&Continuous&growth&curves&for&
the& indicated& yeast& strains& on& pure& glucose& or& galactose& (2%)& media,& or& on& a&
mixture&of&glucose/galactose&(0.25%/1.75%).&Data&are&mean&values&(n&=&3)&+/-&SD.&
&
Fig.) 3 ) Growth) performance) after) adaptation) to) galactose) medium.& The&
indicated& yeast& strains& were& pre-grown& in& glucose& (Glucose,& Galactose)& or&
galactose&(Gal-Memory)&media&and&then&dilut ed&in&the&indicated&growth&media.&(A)&
Absolute&growth&rates&and&(B)&the& l ag&times&were&compared&by&setting&the&glucose&
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growth&rate&and&la g&time&for&each&strain&to&100%&or&1,&respectively.&Data&are&mean&
values&(n&=&3)&+/-&SD.&
&
Fig.) 4) Features) of) highly) sensitive) galactose) consumption) in) Saccharomyces'
bayanus.& (A)& Optimized& gene& expression& induction& b y& very& low& galactose&
concentrations.&The&relative&light&increment&(v
max
)&of&a&GAL1-luciferase&reporter&is&
compared&between& S./cerevisiae&BY4741& and& S./bayanus& for& the& indicated&inducer&
concentrations.&Data&are&taken&from&the&experiment&described&in&Figure&1B,&C.&(B)&
Lack& of& diauxic& growth& delay.& Yeast& st ra ins& were& grown& in& a& glucose/ga lactose&
mixture& (0.25%/1.75%).& (C)& Resistance& to& the& glu cose& analogue& glucosamine.&
Yeast& cells& were& grown& in& pure& galactose& medium& (1%)& with& or& without& the&
addition&of&the&indicated&amounts&of&glucosamine&(GlcN).&(D)&Experimental&setup&
for&the&determination&of&transcriptional&memory&upon&galactose&pre-treatment.&(E)&
S./ bayanus& lacks& a& transcriptional& galactose& memory.& Comparison& of& the& GAL1-
luciferase& induction& profiles& of& the& indicated& yeast& strains& after& a& previous&
galactose&encounter&(+&memory)&or&in&naïve&cells&(-&memory).&In&all&experiments,&at&
least&3&independent&biological&replicates&were&analyzed.&&
Fig.)5)S.'bayanus)contains)a)constitutively)active)and)galactose)hypersensitive)
GAL3) promoter.& (A)& Dose-response& profiles& of& different& S./ cerevisiae& and& S./
bayanus& GAL3& variant-luciferase& reporters.& Live& cell& luciferase& reporter& assays&
were&performed& in& S./cerevisiae&BY4741&containing& the& indicated& GAL3p& variants.&
Results& are& depicted& as& medium& values& from& at& least& 3& b iological& replicates.& (B)&
Representation&of&the&basal&GAL3&p romoter&activity&of&the&indicated&yeast&strains.&
Data&are&mean& (n=3)&+/-&SD.& Statistically&significant&differences& were&determined&
with&the&unpaired&Student´s&t -test&(**&p<0.01).&(C)&Comparison&of&the&sensitivity&of&
GAL3&induction&across&the&different&variants.&Representation& of&the&maximal&light&
increment&(v
max
)&versus&the&galactose&inducer&concentration&for&each&yeast&strain.&
v
max
& values& were& set& to& 100%& for& the& inducer& concentration,& which& leads& to&
maximal&induction.&(D)&GAL3&promoter&sequence&comparison.&Highlighted&are&the&
unique&Gal4&UAS&motif&and&the&proximal&(1)&and&distal&(2)&Mig1&repressor&binding&
sites.& Nucleotide& substitutions& in& S./bayanus,& which& inactivate& the& Mig1& sites,& are&
indicated&in&red.&&
Fig.) 6) Features) of) a) desensitized) galactose) consumption) strategy) in)
Saccharomyces'cerevisiae) Y12.&(A)&The&relative&luciferase&expression&of&a&GAL1-
luciferase& reporter& is& compared& between& S./ cerevisiae& BY4741& and& Y12& for& the&
indicated&inducer&concentrations.&Data&are&taken&from&the&experiment& described&in&
Figure&1B,&C.&(B)&The&relative&light&increment&(v
max
)&of&a&GAL1-luciferase&reporter&is&
compared& between& S./ cerevisiae& BY4741& and& Y12& for& the& indicated& inducer&
concentrations.&Data&are&taken&from&the&experiment&described&in&Figure&1B,&C.&(C)&
Resistance& to& the& glucose& analogue& glucosamine.& Yeast& cells& were& grown& in& pure&
galactose&medium&(1%)&wit h&or&without&the&addition&of&the&indicated&amounts&of&
glucosamine& (GlcN).& (D)& Flow& cytometry& analysis& of& t he& GAL1-GFP& induction&
process& in& S./ cerevisiae& BY4741& and& Y12& upon& high& (0.5%)& and& low& (0.015%)&
inducer& concentrations.& Left& panel:& Comparison& of& t he& fraction& of& GFP& positive&
cells;&right&panel:&comparison&of&the&induction&levels&of&the&GFP&positive&cells.&(E)&S./
cerevisiae/Y12&shows&a&transcriptional&galactose&memory.&Comparison&of&the& GAL1 -
luciferase& induction& profiles& of& the& indicated& yeast& strains& after& a& previous&
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted January 16, 2023. ; https://doi.org/10.1101/2023.01.12.523712doi: bioRxiv preprint
galactose& encounter& (+& memory)& or& in& naïve& cells& (-& memory)& as& described& in&
Figure& 4D.& In& all& experiments,& at& least& 3& independent& biological& replicates& were&
analyzed.&&
Fig.)7)S.'cerevisiae)Y12)harbors)a)less)sensitive)Gal3 )sensor.&(A)&Experimental&
strategy&to&quantitatively&compare&the&trans-activation&capacities&of&the&galactose&
sensors& Gal3
BY4741
& and& Gal3
Y12
.& (B)& Dose& response& profiles& of& GAL1p-luciferase&
through&Gal3
BY4741
&and&Gal3
Y12
.&(C)&Comparison&of&the&sensitivity&of&GAL1&induction.&
Representation&of&the&maximal&light&increment&(v
max
)&versus&the&galactose&inducer&
concentration&for&each&Gal3&variant.&V
max
&values&were&set&to&100%&for&the&inducer&
concentration,&which&leads&to&maximal&induction.&(D)&Gal 3
Y12
&confers&less&sensitive&
GAL&gene&induction&even&after&previous&galactose&encounter.&Data&are&taken&from&
the& experiment& described& in& Figure& 6E& and& represented& as& in& (C).& In& all&
experiments,&at&least&3&independent&biological&replicates&were&analyzed.&
&
Fig.)8)West)African)S.'cerevisiae)isolates)DBVPG 6044 ) and)NCYC110)are)poor)
galactose) consumers) due) to) a n) inactivated) GAL3) allele.) (A)& Sequence&
comparison&of&GAL3&of&the&indicated&S./cerevisiae&isolates.&(B)&Re-introduction&of&a&
Gal3
BY4741
&copy&restores&efficient&galactose&growth&of&S./cerevisiae&DBVPG6044&and&
NCYC110.&Cells& harboring&the& empty&plasmid& control&(EV)& or&the&GPD-GAL3
BY4741
&
expression&plasmid&were&grown&in&synthetic&glucose&or&galactose&media.&Data&are&
mean&(n=3)&+/-&SD.&
&
&
& &
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted January 16, 2023. ; https://doi.org/10.1101/2023.01.12.523712doi: bioRxiv preprint
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preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted January 16, 2023. ; https://doi.org/10.1101/2023.01.12.523712doi: bioRxiv preprint
Figure 1
A
B
C
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The copyright holder for thisthis version posted January 16, 2023. ; https://doi.org/10.1101/2023.01.12.523712doi: bioRxiv preprint
Figure 2
A
B
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Figure 3
A B
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Figure 4
A B
C
E
1% Gal Gluc
120min 60min
mock Gluc
120min 60min
Gal$
Gal$
Raf
90min
90min
Raf
Raf
GAL1-lucCP
+
+ memory
- memory
D
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted January 16, 2023. ; https://doi.org/10.1101/2023.01.12.523712doi: bioRxiv preprint
Figure 5
GAL3
Gal4
Mig1
Mig1
Galactose
Glucose
1
2
G A A T A C C T G G G G BY4741, Y12, DBVPG6044 (-72/-83)
T A A T A C T T G G A A S. bayanus (-79/-90)
C C G A A C G T G G G G BY4741, Y12, DBVPG6044 (-286/-297)
C G A A A C G T C G G G S. bayanus (-289/-300)
Mig1 consensus
T T C G G N(11) C C G A A Gal4 consensus
T T C G G N(11) C C G A A BY4741, Y12, DBVPG6044 (-290/-270)
T T C G G N(11) C C G A A S. bayanus (-292/-272)
1
2
B C
D
A
S.c.$
BY4741$
S.c.$
BY4741$
S.c.$
DBVPG6044$
S.c.$
Y12$
S.bayanus$
lucCP
+
GAL3p
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The copyright holder for thisthis version posted January 16, 2023. ; https://doi.org/10.1101/2023.01.12.523712doi: bioRxiv preprint
Figure 6
A
B
D
E
C
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted January 16, 2023. ; https://doi.org/10.1101/2023.01.12.523712doi: bioRxiv preprint
Figure 7
A
B
S.c.$BY4741$
S.c.$BY4741$
S.c.$Y12$
GAL1p
BY4741
GAL1p
BY4741
GAL3
GAL3
lucCP
+
GAL1p
BY4741
Δ
gal3
S.c. BY4741 S.c. Y12
C D
Figure 8
A
B
...ATC TTT TGC CAG AGC... GAL3
BY4741
...ATC TTT TGA CAG AGC... GAL3
DBVPG6044;NCYC110
...I
150
F
151
* Q
153
S
154
... Gal3
DBVPG6044;NCYC110
...I
150
F
151
C
152
Q
153
S
154
... Gal3
BY4741
Table&1:!Yeast!strains!used!in!this!study!
!
Strain
Genotype
Source
S. cerevisiae
BY4741
MATa; his3∆1; leu2∆0; met15∆0; ura3∆0
EUROSCARF
S. paradoxus
ho::HPH; ura3; lys2; his3∆::3xHA;
leu2∆::3xHA
Jun-Yi Leu
S. mikatae
ho::HPH; ura3; lys2; his3∆::3xHA;
leu2∆::3xHA
Jun-Yi Leu
S. bayanus
ho::HPH; ura3; his3∆::3xHA; leu2∆::3xHA
Jun-Yi Leu
S. cerevisiae
UWOPS05-217.3
ho::HGB; ura3::G418-barcode;
his3::clonat
Jun-Yi Leu
S. cerevisiae
NCYC110
ho::HGB; ura3::G418-barcode;
his3::clonat
Jun-Yi Leu
S. cerevisiae
DBVPG6044
ho::HGB; ura3::G418-barcode;
his3::clonat
Jun-Yi Leu
S. cerevisiae Y12
ho::HGB; ura3::G418-barcode;
his3::clonat
Jun-Yi Leu
S. cerevisiae
YJM981
ho::HGB; ura3::G418-barcode;
his3::clonat
Jun-Yi Leu
S. cerevisiae
L1374
ho::HGB; ura3::G418-barcode;
his3::clonat
Jun-Yi Leu
S. cerevisiae
YPS128
ho::HGB; ura3::G418-barcode;
his3::clonat
Jun-Yi Leu
S. cerevisiae
YPS606
ho::HGB; ura3::G418-barcode;
his3::clonat
Jun-Yi Leu
BY4741 GAL1-
lucCP
+
BY4741 with plasmid pAG413-GAL1-
lucCP
+
(HIS3)
This study
S. paradoxus
GAL1-lucCP
+
S. paradoxus with plasmid pAG413-
GAL1-lucCP
+
(HIS3)
This study
S. mikatae GAL1-
lucCP
+
S. mikatae with plasmid pAG413-GAL1-
lucCP
+
(HIS3)
This study
S. bayanus GAL1-
lucCP
+
S. bayanus with plasmid pAG413-GAL1-
lucCP
+
(HIS3)
This study
UWOPS05-217.3
GAL1-lucCP
+
UWOPS05-217.3 with plasmid pAG413-
GAL1-lucCP
+
(HIS3)
This study
NCYC110 GAL1-
lucCP
+
NCYC110 with plasmid pAG413-GAL1-
lucCP
+
(HIS3)
This study
DBVPG6044
GAL1-lucCP
+
DBVPG6044 with plasmid pAG413-
GAL1-lucCP
+
(HIS3)
This study
Y12 GAL1-lucCP
+
Y12 with plasmid pAG413-GAL1-lucCP
+
(HIS3)
This study
YJM981 GAL1-
lucCP
+
YJM981 with plasmid pAG413-GAL1-
lucCP
+
(HIS3)
This study
L1374 GAL1-
lucCP
+
L1374 with plasmid pAG413-GAL1-
lucCP
+
(HIS3)
This study
YPS128 GAL1-
lucCP
+
YPS128 with plasmid pAG413-GAL1-
lucCP
+
(HIS3)
This study
YPS606 GAL1-
lucCP
+
YPS606 with plasmid pAG413-GAL1-
lucCP
+
(HIS3)
This study
BY4741
GAL3
BY4741
-lucCP
+
BY4741 with plasmid pAG423-
GAL3
BY4741
-lucCP
+
(HIS3)
This study
BY4741
GAL3
S.bayanus
-
lucCP
+
BY4741 with plasmid pAG423-
GAL3
S.bayanus
-lucCP
+
(HIS3)
This study
BY4741
GAL3
DBVPG6044
-
lucCP
+
BY4741 with plasmid pAG423-
GAL3
DBVPG6044
-lucCP
+
(HIS3)
This study
BY4741 GAL3
Y12
-
lucCP
+
BY4741 with plasmid pAG423-GAL3
Y12
-
lucCP
+
(HIS3)
This study
BY4741 GAL1-
GFP
BY4741 with plasmid pRS416-
GAL1
BY4741
-eGFP (URA3)
This study
Y12 GAL1-GFP
Y12 with plasmid pRS416-GAL1
BY4741
-
eGFP (URA3)
This study
BY4741 gal3∆
GAL1-lucCP
+
BY4741 gal3::KAN with plasmid pAG413
GAL1
BY4741
-lucCP
+
(HIS3)
This study
Gal3
BY4741
GAL1-
lucCP
+
BY4741 gal3::KAN GAL1
BY4741
-lucCP
+
with plasmid pAG416-GAL1p-GAL3
BY4741
(URA3)
This study
Gal3
Y12
GAL1-
lucCP
+
BY4741 gal3::KAN GAL1
BY4741
-lucCP
+
with plasmid pAG416-GAL1p-GAL3
Y12
(URA3)
This study
DBVPG6044 GPD
DBVPG6044 with plasmid pAG416-GPD-
ccdB (URA3)
This study
NCYC110 GPD
NCYC110 with plasmid pAG416-GPD-
ccdB (URA3)
This study
DBVPG6044
Gal3
BY4741
DBVPG6044 with plasmid pAG416-GPD-
GAL3
BY4741
(URA3)
This study
NCYC110
Gal3
BY4741
NCYC110 with plasmid pAG416-GPD-
GAL3
BY4741
(URA3)
This study
Table&2:!Oligonucleotide!primers!used!in!this!study!
!
5´-3´Sequence
Application
S.c. GAL3-896SacI
GCCGAGCTCATTTAAGTATTG
TTTGTGCACTTG
subclone S. cerevisiae
GAL3 promoter in
pAG423-lucCP
+
S.c. GAL3-1SmaI
TCCCCCGGGACTATGTGTTG
CCCTACCTTT
subclone S. cerevisiae
GAL3 promoter in
pAG423-lucCP
+
S.bay GAL3-900Kpn2I
ATCGTCCGGAGCACGTTGCC
TACAAAGC
subclone S. bayanus
GAL3 promoter in
pAG423-lucCP
+
S.bay GAL3-1SmaI
TCCCCCGGGTTATGTGTTGT
GTCCTATTTCG
subclone S. bayanus
GAL3 promoter in
pAG423-lucCP
+
pGAL1XbaI
CCGGTCTAGATTCACCTACAG
CCTTTAAAC
subclone GAL1
BY4741
promoter in pRS416-GFP
pGAL1XhoI
CCGGCTCGAGTTTTTTCTCCT
TGACGTT
subclone GAL1
BY4741
promoter in pRS416-GFP
GAL3GWfw
CGATCCCGGGAACTATGACA
GTTTAATAATTATTTATTG
subclone S. cerevisiae
GAL3 ORF in pDONR221
and pAG416-GAL1-ccdB
GAL3GWrev
GGGGACCACTTTGTACAAGA
AAGCTGGGTTATTGTTCGTAC
AAACAAGTACCC
subclone S. cerevisiae
GAL3 ORF in pDONR221
and pAG416-GAL1-ccdB