sucrose is a cheap substrate for industrial fermentation that is extensively used for ethanol production ( maiorella , blanch and wilke 1984 ; della - bianca et  al.2013 ; gombert and van maris 2015 ) and can be used for a wide range of other yeast - based industrial processes ( marques et  al.2016 ; meadows et  al.2016 ) . the main sources of sucrose are sugar cane in the tropics and sugar beet in temperate regions . extraction of sucrose from feedstocks is inexpensive and no enzymatic pre - treatment is necessary prior to microbial fermentation ( maiorella , blanch and wilke 1984 ; marques et  al.2016 ) . furthermore , the use of sucrose for industrial biotechnology does not have to compete with food production . for example , in the brazilian ethanol industry , food and biofuel production can coexist ( mitchell 2008 ) . therefore , diversifying the range of products that can be produced from sucrose fermentation will have a positive impact on sustainable industrial production of fuels and chemicals ( meadows et  al.2016 ) . sucrose - based ethanol production employs bakers yeast ( saccharomyces cerevisiae ) , which efficiently consumes sugars and can withstand industrially relevant stresses such as high ethanol and acid concentrations ( della - bianca et  al.2014 ) . to convert sucrose into hexoses , s. cerevisiae secretes invertase ( -fructosidase , sucrose hydrolase ; suc ) ( winge and roberts 1952 ; hawthorne 1958 ) . invertase hydrolyses sucrose into the monomers , glucose and fructose , which subsequently enter the cell through facilitated diffusion via hexose transporters and are metabolised via the classical embden - meyerhof - parnas glycolytic pathway ( lagunas 1993 ) . alternatively , sucrose can be imported by a proton symporter ( mal11 , formerly known as agt1 ) and hydrolysed in the cytosol ( stambuk et  al.1999 ) . cytosolically localised invertase and maltases ( malx2 ) then hydrolyse sucrose intracellularly . in vitro studies have shown that isomaltases can also hydrolyse sucrose , but their contribution to in vivo sucrose metabolism remains to be addressed ( carlson and botstein 1982 ; stambuk et  al.1999 ; badotti et  al.2008 ; brown , murray and verstrepen 2010 ; voordeckers et  al.2012 ; deng et  al.2014 ) . even closely related yeast strains exhibit significant diversity in the identity and number of representatives from each of the three disaccharide hydrolase - encoding gene families ( suc , malx2 , i m a ) ( carlson and botstein 1983 ; brown , murray and verstrepen 2010 ) . for example , the genome of the haploid laboratory strain s. cerevisiae cen.pk113-7d contains five different i m a genes on subtelomeric regions : ima1 ( three copies : chriii , chrvii , chrxi ) , ima2 ( chrxv ) , ima3 ( chrix ) , ima4 ( chrx ) and ima5(chrx ) ( teste , franois and parrou 2010 ; nijkamp et  al.2012 ) , while the related strain cen.pk102-3a , which was used in this study , does not contain the ima1 copy at chrxi ( de kok et  al.2011 ) . detailed complete knowledge of sucrose transport and hydrolysis is important for both industry and academia . functional analysis of eukaryotic sucrose transporters and hydrolytic or phosphoroclastic sucrose - cleaving enzymes is often done in s. cerevisiae . an example is the development of a functional screening system for plant sucrose exporters ( zhou , grof and patrick 2014 ) . in this system , functional expression of plant sucrose exporters is analysed by monitoring conversion of glucose into sucrose and subsequent sucrose efflux from an s. cerevisiae strain that heterologously expresses sucrose - phosphate synthase ( sps ) and sucrose - phosphate phosphatase ( spp ) genes . in industry , engineering of sucrose metabolism can contribute to increased product yields on substrate . in a proof - of - principle study with a laboratory strain of s. cerevisiae , ( 2011 ) demonstrated an increase in the ethanol yield of s. cerevisiae on sucrose of 11% , when compared to the reference strain , by deleting the secretion signal sequence from the invertase coding gene ( suc2 ) . this modification resulted in a predominant intracellular localisation of invertase activity , thereby creating a necessity for sucrose uptake via proton symport . to avoid intracellular acidification , protons entering the cell via the sucrose - proton symporter have to be expelled by the plasma - membrane h - atpase ( pma1 ) . this export mechanism costs one atp per proton , thus reducing the atp yield of alcoholic fermentation from 4 to 3 atp per sucrose molecule . this change in energy coupling was shown to lead to a decrease of the biomass yield and an increase of the ethanol yield of the engineered strain on sucrose . in metabolic engineering , incomplete knowledge of the targeted pathway can delay strain construction due to the activity of competing pathways or activation of such pathways by suppressor mutations . although mutants that are unable to grow on sucrose have been reported ( carlson , osmond and botstein 1981 ) , it has not yet been investigated whether these strains can regain the ability to grow on sucrose after laboratory evolution ( marques et  al.2016 ) , for instance through recruitment of any of the numerous glycoside hydrolases in s. cerevisiae ( yamamoto et  al.2004 ; naumoff and naumov 2010 ; naumoff 2011 ) . to address this question , s. cerevisiae strains in which either the genes encoding known sucrose transporters or those encoding disaccharide - hydrolysing enzymes were inactivated were subjected to laboratory evolution and mutations underlying the acquired phenotypes were identified by whole - genome resequencing . saccharomyces cerevisiae strains used in this work ( table  1 ) belong to the cen.pk family ( entian and ktter 2007 ; nijkamp et  al.2012 ) . to prepare stock cultures , yeast strains were grown in 50 ml shake flasks containing 10 ml of either yeast - peptone - dextrose ( ypd ) medium or , for strains carrying plasmids with auxotrophic marker genes , synthetic medium ( sm ) . ypd contained 10 g l bacto yeast extract , 20 g l bacto peptone and 20 g l glucose in demineralised water . sm , supplemented with vitamins , trace elements and 20 g l glucose , was prepared according to verduyn et  al . glycerol was added to growing cultures ( final concentration 30% v / v ) and 1 ml aliquots were stored at 80c . the abbreviation mal indicates mal11-mal12::loxp mal21-mal22::loxp mal31 - 32::loxp and mph indicates mph2/3::loxp mph2/3::loxp - hphnt1-loxp . diagnostic pcr was performed using dreamtaq dna polymerase ( thermo fisher scientific , waltham , ma , usa ) . pcr amplification for cloning and sequencing purposes was performed with phusion high - fidelity dna polymerase ( thermo fisher scientific ) . primers and oligonucleotides were purchased from sigma - aldrich ( st . louis , mo , usa ) . dna purification from pcr reaction mixtures was done using genelute pcr clean - up kit ( sigma - aldrich ) . separation of dna fragments was performed in 1% ( w / v ) agarose gel ( thermo fisher scientific ) in tae buffer ( 40 mm tris - acetate ph 8.0 and 1 mm edta ) . dna fragments were purified from gels using the zymoclean gel dna recovery kit ( zymo research , irvine , ca , usa ) . plasmid assembly was done with the gibson assembly master mix ( new england biolabs , beverly , ma ) according to the manufacturer 's protocol . restriction endonucleases ( thermo fisher scientific ) and t4 dna ligase ( promega corporation , madison , wi , usa ) were used according to the manufacturer 's instructions . plasmids were isolated from escherichia coli using genelute hp plasmid miniprep kit ( sigma - aldrich ) according to the provided protocol . plasmid pudc156 ( table  2 ) was assembled by in vivo homologous recombination of a plasmid backbone and an insert fragment containing a yeast expression cassette for the cas9 gene from streptococcus pyogenes ( kuijpers et  al.2013 ) . the plasmid backbone was amplified from mb4917 ( zelle et  al.2010 ) with primers 7833 and 4697 and the cas9-expression cassette was amplified from p414-tef1p - cas9-cyc1 t ( dicarlo et  al.2013 ) with primers 1768 and 7236 ( table s1 , supporting information ) . both fragments contain 60 bp overlaps with each other and were assembled by co - transformation to s. cerevisiae strain imx469 resulting in imz571 . 2 ) contains two grna cassettes : one for deletion of ima5 ( grna - ima5 ) and one targeting sequence shared by ima1 , ima2 , ima3 and ima4 ( grna - imas ) . this plasmid was constructed via gibson assembly of three fragments : two grna cassettes overlapping with each other in the 2  m replicon and a plasmid backbone . the plasmid backbone was obtained via pcr amplification , using pros11 ( mans et  al.2015 ) as template with primer 6005 ( table s1 ) . the grna cassette grna - ima5 was obtained via pcr from pros11 with primers 8761 and 5975 , and cassette grna - imas was obtained via pcr from pros11 with primers 8759 and 5974 ( table  s1 ) . plasmid pudi084 was made by removal of the mal11 coding sequence from pudi035 ( de kok et  al.2011 ) using spei and nhei restriction sites and subsequent recircularisation of the plasmid using t4 dna ligase ( table  2 ) . similarly , pude260 was constructed by removal of pgm from pude063 ( de kok et  al.2011 ) using pvuii sites and subsequent recircularisation of the plasmid ( table  2 ) . the ima - reinsertion plasmids ( pude427 to pude430 ) were made by gibson assembly of an i m a expression cassette and a plasmid backbone . the latter was obtained via pcr with p426-gpd ( mumberg , mller and funk 1995 ) as a template , using primer pairs 7823 and 5975 and 5974 and 7812 , which amplify the backbone in two parts to minimise chances of plasmid recircularisation ( table s1 ) . templates for the i m a expression cassettes were pcr amplified using genomic dna from strain imu048 with primers that are specific to each i m a ( primers 8607 and 8611 for ima1 ; 8608 and 8612 for ima2 ; 8609 and 8613 for ima3,4 ; and 8610 and 8614 for ima5 ) . from these templates , the coding sequence of each i m a could be individually amplified using primer pairs 9302 and 9305 ( ima1 ) , 9303 and 9306 ( ima2 and ima3,4 ) and 9304 and 9307 ( ima5 ) . plasmids were transformed to e. coli for storage and amplification , where necessary after isolation of in vivo assembled plasmids from yeast cultures . strategy for deletion of the i m a genes using a shared cas9 target site . grna - imas ( in red ) with a target sequence to cleave all i m a genes except ima5 and  grna - ima5 , in purple . 2  m : origin of replication in yeast ; pmb1 : origin of replication in bacteria ; amp : cassette for ampicillin resistance in e. coli ; amdsym : dominant marker that allows use of acetamide as nitrogen source . ( b ) illustration of the shared cas9 target sequences among the i m a genes . the coding sequence of each i m a is represented by black lines . the red boxes correspond to the shared target sequence . the dark- and light - grey fragments are the homology regions ( 60 bp ) up- and downstream of the target site where the repair fragment ( 120 bp ) can recombine . in view of its sequence divergence from the other i m a genes , an exclusive target sequence , in purple , and repair sites , in dark and light green , were chosen for inactivation of ima5 . saccharomyces cerevisiae transformations were carried out according to gietz and woods ( 2002 ) using 1  g dna , unless specified otherwise below . g l ; leucine 0.5 g l. cells expressing the amdsym marker were selected on plates according to solis - escalante et  al . strain imk289 ( de kok et  al.2011 ) was transformed with a deletion cassette containing loxp - kanmx4-loxp marker . this cassette was amplified from plasmid pug6 using primers 1482 and 1483 that has homology to the sequence outside the suc2 open reading frame . to construct mal11-expressing strains , vector pudi035 ( table  2 ) was linearised with bsteii ( restriction site at leu2 marker ) and transformed into imk291 , resulting in strain imx469 ( table  1 ) . linearisation of pudi084 ( table  2 ) with bsteii and transformation into imk291 resulted in imx470 . imk700 was made via co - transformation of imz571 with 1  g of plasmid pudr127 together with 4  g of each dsdna repair fragment : one for ima5 and another one for all the other i m a genes ( fig .  1 ) . repair fragments were obtained by annealing two complementary page - purified single - stranded oligonucleotides according to mans et  al . ( 2015 ) : 8592 and 8593 for ima1 - 4 and 8763 and 8764 for ima5 ( table  2 ) . in order to delete i m a genes from strains evolved on sucrose ims422 , ims423 and ims424pude260 was cured from these strains by cultivation on sm plates with 20  g  l glucose and 1 g l 5-fluoroorotic acid ( boeke , la croute and fink 1984 ) , resulting in strains ims517 , ims518 and ims519 , respectively . transformation of pudc156 into these three strains yielded strains ims604 , ims605 and ims606 , respectively . finally , transformation of pudr127 and repair fragments for ima5 and ima1 - 4 ( as mentioned above ) into these three strains yielded strains imk717 , imk718 and imk719 . for re - insertion of the i m a genes on multicopy plasmids , pudc156 was cured from imk700 resulting in imk743 . then , each i m a overexpression plasmid ( named pude427-pude430 , table  2 ) was transformed into imk743 resulting in strains imz620 - 623 . shake - flask cultures were performed in 500 ml shake flasks containing 100 ml of sm with 20 g l sucrose , in an innova incubator shaker ( new brunswick scientific , edison , nj , usa ) set at 200 rpm and at 30c under an air atmosphere . for growth rate determinations , cells were inoculated in sm with 20 g l glucose from a frozen stock culture . after reaching stationary phase , the culture was transferred to sm with 20 g l sucrose ( initial od660 nm = 0.2 ) and incubated until exponential growth was observed . exponentially growing cultures were then transferred to fresh medium ( initial od660 nm = 0.2 ) and samples were taken hourly until stationary phase was reached . optical density at 660 nm was measured with a libra s11 spectrophotometer ( biochrom , cambridge , uk ) . sequential batch cultivation of strain imu048 was performed in 10 ml sm ( verduyn et  al.1992 ) with 20 g l sucrose , in 50 ml polypropylene tubes ( greiner bio - one , frickenhausen , bw ) in an innova incubator shaker ( new brunswick scientific ) , at 200 rpm and at 30c . at the end of each cultivation cycle ( od660 nm , 34.2 ) , 0.1 ml of culture was transferred to fresh medium to start a next cycle . in total , 30 transfers were carried out in 70 days , which accounts for 180 generations . one single - colony isolate from each evolution line was obtained by restreaking three times on non - selective medium ( ypd ) plates . concentrations of sucrose and glucose in culture supernatants were analysed by high - performance liquid chromatography ( agilent 1100 hplc , agilent technologies , santa clara , ca ) , using an aminex hpx-87h ion exchange column ( biorad , richmond , ca ) coupled to a refractive - index detector and eluted with 0.5 mm h2so4 at 0.8 ml min and at 40c . this temperature was used instead of the regularly applied temperature of 60c to avoid sucrose hydrolysis during analysis . for enzyme activity assays , shake - flask cultures on sm with 20  g  l sucrose imk700 , imk716 , imk717 and imk718 , 20 g l ethanol was used as carbon source plus 20 g l sucrose as inducer . samples were harvested , washed and prepared for sonication according to postma et  al . cell extracts were prepared by sonication with 0.7 mm glass beads at 0c for 2 min at 0.5 min intervals with an mse sonicator ( 150 w output ; 8  m peak - to - peak amplitude ) . unbroken cells and debris were removed by centrifugation ( 4c , 20  min , 47  000  g ) . the supernatant was used for enzyme activity assays . for measurement of extracellular enzyme activity , 100 ml of exponentially growing cells ( od660 nm  = 34 ) was centrifuged ( 4c , 10 min , 5000  g ) , the supernatant was concentrated up to 200 times with a vivaspin 20 filter with a 10 000 mw cut - off ( sartorius stedim , aubagne , france ) and dialysed overnight against 10 mm potassium - phosphate buffer ( ph 7.5 ) at 4c . protein levels in extracellular samples and cell extracts were determined with the lowry assay ( lowry et  al.1951 ) . sucrose or isomaltose hydrolytic activity was measured at 30c by monitoring the reduction of nadp at 340 nm in a 1 ml reaction mixture containing 50 mm imidazole - hcl ( ph 7.0 ) , 1 mm nadp , 12.5 mm mgcl2 , 1 mm atp , 3.5 units hexokinase , 3.5 units glucose-6-phosphate dehydrogenase and 1040  l cell extract . exponentially growing cultures ( od660 nm = 34 ) in 100 ml sm ( verduyn et  al.1992 ) with 20 g l of sucrose were harvested as described previously ( piper et  al.2002 ) . rna was extracted using the hot - phenol method ( schmitt , brown and trumpower 1990 ) , and rna quality was assayed by electrophoresis using an agilent bioanalyzer 2100 ( agilent technologies ) . genomic dna elimination was performed from 2  g of total rna in a 28  l reaction using the quantitec reverse transcription kit ( qiagen , dsseldorf , germany ) . reagents for cdna synthesis from the same kit were added up to a final volume of 40  l following the manufacturer 's instructions . a portion of 6  l of cdna solution diluted 150 times was used in 20  l qpcr mix that additionally included 10  l of rotor - gene sybr green pcr master mix ( qiagen ) and forward and reverse primers ( 1  m each ) ( table s1 ) . a rotor - gene q ( qiagen ) was used with the following sequence : denaturation at 95c for 5 min followed by 40 cycles of denaturation at 95c for 5 s , annealing at 60c for 10 s and extension at 72c for 20 s. a melting curve up from 60c to 95c was performed to verify primer specificity . pcr efficiency of each primer was determined by a dilution series using a pool of cdna from different samples . transcript levels of the  housekeeping gene ubc6 ( teste et  al.2009 ) were used for data normalisation . threshold cycles ( ct ) were exported from rotor - gene q software ( version 2.0.2 ) and analysed using the rest-2009 algorithm ( pfaffl , horgan and dempfle 2002 ) . one of the two biological replicates of cen.pk113-7d was chosen as reference condition ( expression ratio = 1 ) . sanger sequencing of pcr products of each i m a gene was performed at baseclear bv ( leiden , the netherlands ) . the pcr products were obtained with genomic dna extracted from strains ims422 , ims423 and ims424 and following primers : 8607 and 8611 ( ima1 ) , 8608 and 8612 for ( ima2 ) , 8609 and 86013 for ( ima3,4 ) and 8610 and 8614 ( ima5 ) ( table s1 ) . ima3 and ima4 were treated as a single gene ( ima3,4 ) since their coding sequences are identical ( teste , franois and parrou 2010 ) . dna for whole genome sequencing was extracted using qiagen 100/g kit following the manufacturer 's protocol ( qiagen , hilden , germany ) . a library of 350-bp genomic fragments was created and sequenced paired end ( 150-bp reads ) . a minimum data quantity of 4000 mb was generated per strain , representing a minimum 330-fold coverage . the sequencing data of the parental strain , imu048 , and of the three evolved isolates , ims422 , ims423 and ims424 , were deposited at ncbi under the bioproject i d : prjna353914 . although sucrose is generally stable when dissolved in water , under certain conditions it can be hydrolysed into fructose and glucose , such as for instance by acid catalysis in low ph solutions ( krieble 1935 ; wolfenden and yuan 2008 ) . to investigate possible artefacts created by such non - biological hydrolysis of sucrose , strain imu051 ( mal mph suc2 ) , which can not grow on sucrose , but can grow on hexoses , was incubated in sm shake flasks at ph values between 2 and 7 ( fig .  2 ) . at ph values of 57 , 2a ) , which was consistent with the occurrence of sucrose hydrolysis in a sterile culture at this acidic ph ( fig . s2 , supporting information ) , no growth was observed at this ph value , since saccharomyces cerevisiae cen.pk113-7d can not grow at this low ph ( della - bianca et  al.2014 ) . to avoid a contribution of non - biological hydrolysis of sucrose to the initiation of growth , all further growth experiments in this study were done in cultures with an initial ph of 6 . for those cultures that did show growth however , since this drop only occurs towards the end of the exponential growth , this did not influence the initiation of growth . ( a ) growth of s. cerevisiae strain imu051 ( mal mph suc2 ) in sm with 20 g l sucrose as sole carbon source with initial ph set at different values : ph 2 , filled squares ; ph 3 , filled triangles ; ph 4 , inverted filled triangles ; ph 5 , open squares ; ph 6 ,  crosses and ph 7 ,  plus. the symbols filled square , open square , cross and plus are overlapping . ( b ) sugar concentrations in flasks with sm at an initial ph of 3 . closed symbols : glucose ( filled diamonds ) and sucrose ( filled circles ) concentrations in flasks inoculated with imu051 . open symbols ; glucose ( open diamonds ) and sucrose ( open circles ) concentrations in flasks without inoculum . at day 0.00 and 1.06 : filled circles overlapping with open circles and filled diamonds overlapping with open diamonds . data were not corrected for evaporation to keep the experimental setup identical to that used for growth studies . the experiment was conducted with two independent replicates , of which one representative replicate is shown . 500 ml shake flasks containing 100 ml sm with sucrose 20 g l were incubated at 30c and at 200 rpm . to investigate what is needed to completely abolish both sucrose transport and hydrolysis in s. cerevisiae cen.pk102-3a , a strain was constructed in which the following genes were deleted : suc2 ( encoding invertase ) ; mal12 , mal22 and mal32 ( encoding maltases ) ; and mal11 , mal21 , mal31 , mph2 , mph3 ( encoding transporters ) . the resulting strain ( imu051 ; mal mph suc2 ) did not show any growth over a period of up to 2 months in sm with sucrose as the sole carbon source ( table  3 ) . however , for future metabolic engineering strategies concerning sucrose metabolism , it is important to understand whether this inability to grow is caused by lack of transport or lack of sucrose hydrolysis . to investigate this , three new strains were constructed : one expressing the transporter mal11 ( imu048 ; mal mph suc2 mal11 ) , one expressing the -glucosidase mal12 ( imu054 ; mal mph suc2 mal12 ) and a control strain expressing both genes ( imu055 ; mal mph suc2 mal11 mal12 ) . the maltase mal12 was chosen for intracellular cleavage of sucrose rather than the invertase suc2 , since previous work has shown that extracellular suc2 activity can be detected even if suc2 is expressed without the secretion signal sequence ( basso et  al.2011 ) . maximum specific growth rates of s. cerevisiae strains grown in shake flasks containing sm ( initial ph 6.0 ) with 20 g l sucrose as sole carbon source . for enzyme - activity assays , strains were grown in sm with 20  g  l sucrose as sole carbon source . for enzymatic - activity determination , imu054 and imu055 were grown in sm with 20 g l ethanol as sole carbon source . strain imk700 was grown in sm with 20 g l ethanol as sole carbon source plus 20 g l sucrose as inducer . averages and mean deviations were obtained from duplicate experiments . : below detection limit , i.e. < 0.03  mol mg protein min . combined expression of mal11 and mal12 resulted in a maximum specific growth rate of 0.19 h for the control strain imu055 ( table  3 ) . even after prolonged incubation of up to 2 months , expression of only the hydrolysing enzyme mal12 ( imu054 ) did not enable growth in sm with sucrose as the sole carbon source , indicating that there is no residual sucrose transport activity sufficient to allow growth and that this phenotype is stable . in contrast , the strain that only expressed the transporter mal11 ( imu048 ; mal mph suc2 mal11 ) grew at a maximum specific growth rate of 0.08 h in sm with sucrose , after a lag phase of about 4 days ( table  3 ) . all shake - flask cultivations were performed under an air atmosphere . in line with this observation , an activity of sucrose hydrolysis of 0.48  0.03  mol mg protein min was detected in cell extracts of this strain . although lower than the activity of 1.67  0.08  mol mg protein min observed in the reference strain imu055 , this activity was sufficient to sustain growth on sucrose ( table  3 ) . to investigate which genes encode the enzymes responsible for residual sucrose hydrolysis activity in strain imu048 ( mal mph suc2 mal11 ) , this strain , which grows slowly in a sucrose - based medium (  = 0.08 h , table  3 ) , was subjected to sequential batch cultivation in sm with sucrose 2% ( w / w ) as sole carbon source . after 30 transfers ( 70 days , corresponding to 180 generations ) , single - colony isolates ( ims422 , ims423 and ims424 ) were obtained from three independent evolution experiments . in addition to a 3-fold increase in maximum specific growth rate ( from 0.08 to 0.250.26 h , table  3 ) , the lag phase of these strains was shortened from 4 days to 1 day . enzyme - activity assays were conducted to investigate whether the increased growth rate of the evolved strains on sucrose correlated with sucrose hydrolytic activity . indeed , intracellular sucrose hydrolysis activities in the evolved strains were 2- to 3-fold higher ( 1.101.57  mol mg protein min ) than in the non - evolved parental strain imu048 ( 0.48  mol mg protein min ) . in contrast , in cultures of the reference strain cen.pk113-7d , extracellular sucrose activity was 5.40  mol mg protein min while intracellular activity was only 0.43  mol mg protein min ( table  3 ) . activity of isomaltases , which are known to also hydrolyse sucrose ( deng et  al.2014 ) , was measured to investigate a possible contribution of these enzymes to the observed evolved phenotype . indeed , isomaltase activity was about 1.52 times higher in the evolved strains ( 1.592.31  mol mg protein min ) compared to imu048 ( 1.05  mol mg protein min ) ( table  3 ) . in cen.pk113-7d , isomaltase activity was only 0.13  mol mg protein min ( table  3 ) . since isomaltases can not hydrolyse maltose ( deng et  al.2014 ) , it was checked if the evolved strains could consume this sugar . quantitative real - time pcr was performed to investigate if the differences in sucrose and isomaltose hydrolysis observed among reference , unevolved and sucrose - evolved strains could be explained by differences in gene expression . expression of ima2 , ima3 and ima4 was analysed with a single primer pair since the sequences of these genes are highly similar . no difference in expression ratio was observed for the pool consisting of ima2 , ima3 and ima4 transcripts among the strains tested ( fig . in contrast , ima1 and ima5 expression was significantly higher in imu048 ( mal mph suc2 mal11 ) than in cen.pk113-7d . expression of these genes in the evolved strains was even 3- to 5-fold higher than in the unevolved strain imu48 ( fig . the relative expression level of ima5 was approximately 510 times higher than that of ima1 ( fig . relative expression of ima1 - 5 in s. cerevisiae strains evolved for faster growth on sm with sucrose as the sole carbon source . relative expression of ima5(a ) ; ima1(b ) ; and ima2 , ima3 and ima4(c ) . expression of ima2 , ima3 and ima4 was assayed by a single primer pair since these genes are highly similar in nucleotide sequence . cen.pk113-7d : reference strain ( imax malxx mphx suc2 ) ; imu048 : unevolved parental strain ( genotype : mal mph suc2 mal11 ) ; and three independently evolved strains ( ims422 , ima423 and ims424 ) . averages and standard errors were obtained from independent duplicate biological experiments and three technical replicates . cells were harvested at mid - exponential phase ( od660 nm = 3 ) from shake - flask cultures on sm with sucrose as sole carbon source . based on the results shown above , we tested whether deletion of the i m a genes would be sufficient to abolish sucrose consumption in the evolved strains ( ims422 , ims423 and ims424 ) and in an unevolved strain ( imz571 ; mal mph suc2 mal11-leu2 cas9-ura3 ) ( table  1 ) . all six copies of i m a genes present in strains imz571 and ims422 - 424 , all derived from strain cen.pk102-3a ( table  1 ) ( de kok et  al.2011 ; nijkamp et  al.2012 ) , were deleted in a single transformation step using only two grnas cloned in a single plasmid . one of these grnas targeted ima5 while the other simultaneously targeted all other i m a genes . none of the resulting strains ( named imk716 , imk717 , imk718 and imk700 , respectively ) grew in sm with sucrose as sole carbon source after 10 days of incubation ( table  4 ) or after a further 2 months of incubation . both the sucrose and isomaltose hydrolysis activities in the resulting strains were below the detection limit ( table  4 ) . maximum specific growth rates of s. cerevisiae strains grown in shake flasks containing sm ( initial ph 6.0 ) with 20 g l sucrose as sole carbon source . for enzymatic activity determination , strains were grown in sm with 20 g l ethanol as sole carbon source ( sucrose was added besides ethanol to the culture of strains imk716 - 18 as an inducer ) . averages and mean deviations were obtained from duplicate experiments . 
 incubation period : 10 days . sanger sequencing of the i m a genes including their promoter regions did not reveal any mutations ( i m a genes from the evolved strains in comparison to that of the parental ) . whole genome sequencing of the parental strain imu048 revealed that , upon sequential cycle of deletions using loxp sites , the strain became aneuploid for chriii and chrvii . regarding chriii , the parental strain gained an extra copy after deletions using loxp sites , truncating the majority of the left arm . sequencing of three independently evolved isolates indicated a further increase in copy number of the right arm of chriii ( fig . relative to the left arm of the same chromosome ( until approximately position 90  000 ) , the region between positions 90  000 and 150  000 , which includes the centromere , showed an approximately 2-fold increase in read depth . the right - arm distal end ( beyond position 150  000 ) even showed a 3-fold higher increase in read depth in all three evolved strains relative to the distal end of the left arm ( fig . retrotransposons ( e.g. yclw15 and ycrc6 ) located near positions 90  000 and 150  000 may have contributed to translocation / duplication events at these loci . the duplicated chriii region in the parental strain imu048 and triplicated in the evolved isolates harboured ima1 and mal23-c . additionally , a region from chrvii located between retrotransposons ( from position 70  700 to 82  000 ) was found duplicated already in the parental strain and , in the evolved strains , this duplication was lost . depth of coverage analysis of chromosome iii from unevolved strain imu048 ( a ) and the evolved strain ims422 ( b ) . sequencing reads from the imu048 and ims422 have been mapped onto the cen.pk113-7d genome sequence ( nijkamp et  al.2012 ) . read count data represent the average coverage of non - overlapping 500 bp window . these data are representative of the other two evolved strains ( ims423 and ims424 ) . to investigate the impact of an increased expression of i m a genes during growth on sucrose , they were individually overexpressed in a strain in which all known sucrose - hydrolysing activities were inactivated ( imk743 ; mal mph suc2 mal11 ima ) ( table  1 ) . all four constructed strains grew in a sucrose - based medium : strains imz620 and imz621 ( overexpression of ima1 and ima2 , respectively ) both grew at specific growth rate of 0.19 h. accordingly , sucrose and isomaltose hydrolysis activities in these strains were ca . strain imz622 , which overexpressed ima3/4 , grew at 0.17 h , while imz623 ( ima5 overexpressed ) grew at 0.04 h. sucrose hydrolysis activities in cell extracts of strains imz622 and imz623 were 0.92 and 0.17  mol mg protein min , respectively ( table  4 ) . this study confirms the power of laboratory evolution in identifying suppressor mutations of seemingly essential genes ( liu et  al.2015 ) . prolonged incubation of the mal mph suc2 mal11 ima strain for up to 2 months demonstrated that deletion of the i m a genes is both required and sufficient to eliminate growth on sucrose in a strain lacking invertase and maltase activities . deletion of the disaccharide transporter genes mal11 , mal21 , mal31 , mph2 and mph3 similarly resulted in a sucrose - negative phenotype that was stable for over 2 months of incubation in a sucrose - containing medium . the phenotypic stability of the sucrose - negative strains generated in this study makes them a suitable platform for use in diverse applications : ( i ) screening heterologous disaccharide transporters ( e.g. characterisation of plant sucrose transporters in yeast ; study maltose / maltotriose transporters relevant for beer brewing ( alves et  al.2008 ) ) ; ( ii ) screening and characterisation of disaccharide hydrolases and phosphorylases ; ( iii ) metabolic engineering of disaccharide metabolism for improving the production of biofuels and other bio - based chemicals . before laboratory evolution for improved growth on sucrose , strain imu048 ( mal mph suc2 mal11 ) already showed a much higher expression of ima1 and ima5 compared to cen.pk113-7d . a main difference between imu048 and cen.pk113-7d is the location of sucrose hydrolysis . in the reference strain cen.pk113-7d , sucrose is predominantly hydrolysed extracellularly ( basso et  al.2011 ) , whereas in imu048 sucrose is imported via mal11 ( proton symporter ) and hydrolysed in the cytosol . the presence of intracellular sucrose could possibly activate transcription factors such as the mal activators malx3 ( alame et  al.2003 ; weinhandl et  al.2014 ) . it is important to clarify that the deletions carried out in this study included maltases and mal transporters but not the mal activators ( mal13 , mal23-c and mal33 ) . mal23-c ( known as mal2 - 8 ) is constitutively expressed ( gibson et  al.1997 ) but might undergo posttranslational inhibition by chaperones , as has been shown for mal63 , for which the inhibition is relieved when maltose is present ( bali et  al.2003 ; ran , bali and michels 2008 ) . teste , franois and parrou ( 2010 ) showed that the ima1 and ima5 promoter regions contain one and three binding sites for malx3 , respectively . the other i m a promoters do not contain binding sites for malx3 ( teste , franois and parrou 2010 ) . the same authors showed that maltose induces higher expression levels of ima5 than of ima1 , and has no effect on the expression levels of ima2 , ima3 and ima4 . the strongly increased transcript levels of ima1 ( 110-fold ) and ima5 ( 950-fold ) in imu048 , relative to those in cen.pk113-7d ( fig .  3a and b ) , but not of ima2,3,4 ( fig . 3c ) , is in line with the hypothesis that sucrose activates the mal - activators . extracellular glucose and fructose released in cultures of the strain cen.pk113-7d by the action of suc2 might further contribute to the repression of the mal activators ( hu et  al.1995 ; hork 2013 ) in this reference strain . as mentioned above , the complement of genes belonging to the disaccharide hydrolase gene families varies greatly among saccharomyces cerevisiae strains . with even closely related strains showing a high degree of genetic variation , it is important to have access to the genome sequence of the immediate parental strain that is used as a platform on which a metabolic engineering strategy will be implemented . the importance of strain - dependent gene contents is exemplified by a literature debate on the number of binding sites for malx3 in the promoters of ima1 and ima5 . in contrast to teste , franois and parrou ( 2010 ) , pougach et  al . ( 2014 ) stated that the ima5 promoter only has a single binding site for malx3 . in our study , sanger sequencing of the promoter region of ima5 confirmed the presence of three malx3 binding sites previously identified by teste , franois and parrou ( 2010 ) : the motif cggn{9}cgg was found at positions 141 and 543 and motif mgcn{9}mgs at position 516 ( sirenko , ni and needleman 1995 ) . this confirmation does not exclude the possibility that the data of pougach et  al . ( 2014 ) are correct , since they used a different s. cerevisiae strain ( kv5000 , which originates from by4741 , an s288c - derived strain ) whilst teste , franois and parrou ( 2010 ) used the cen.pk113-7d strain . after laboratory evolution , expression of both ima1 and ima5 increased by 3-fold compared to the unevolved imu048 . whole - genome sequencing showed that the strain subjected to evolution ( imu048 ) already contains a duplication of a large part of chriii , which starts before the centromere and extends until the right end of the chromosome ( fig . , chrvii of imu048 contains a duplicated region between retrotransposon sites that was lost after the evolution . although such aneuploidies often go undetected , this mechanism has also been previously identified in a fraction of deletion mutants generated by the saccharomyces genome deletion consortium ( winzeler et  al.1999 ; hughes et  al.2000 ) . several cases reported that the extra chromosome harboured a close homologue of the gene deleted . in our study , deletion of mal31 and mal32 may have led to the aneuploidy of chriii that harbours the mal2 locus . additionally both unevolved ( imu048 ) and evolved strains ( ims422 - 4 ) carried structural variant of the chriii . such rearrangement , especially at chromosome 3 , has been extensively reported in the literature as result of recombination between retrotransposons located around positions 90  000 and 150  000 ( mieczkowski , lemoine and petes 2006 ; de kok et  al.2012 ) . in all the three evolved strains obtained in this study , a third copy of the mentioned region was found ( fig . if , as discussed above , sucrose indeed activates mal23-c , ima1 expression level could be increased by increase in copy number of the gene encoding its activator , mal23-c . while in this study overexpression of ima1 was shown to result in faster growth on sucrose , ima5 was shown to be predominantly an isomaltase and overexpression resulted in only very slow growth and low intracellular sucrose hydrolysis activity ( table  4 ) ( deng et  al.2014 ) . it therefore seems likely that the increased expression of ima5 is collateral to the upregulation of mal23-c . in addition to the scientific value of the sucrose - negative strain platform , this study demonstrates how a single crispr targeting sequence can be used to simultaneously delete multiple genes in s. cerevisiae . this strategy allows the deletion and/or modification of entire gene families in a single transformation event . wesley leoricy marques , robert mans , jack t. pronk and antonius j. a. van maris were supported by the be - basic r&d program , which was granted an fes subsidy from the dutch ministry of economic affairs , agriculture and innovation ( el&i ) . in addition , wesley leoricy marques was supported by the fundao de amparo  pesquisa do estado de so paulo ( fapesp , so paulo , brazil ) , grant numbers 2012/05548 - 1 and 2012/16630 - 0 . this work was carried out as part of a dual degree phd project under the agreement between unicamp and delft university of technology .
abstractmany relevant options to improve efficacy and kinetics of sucrose metabolism in saccharomyces cerevisiae and , thereby , the economics of sucrose - based processes remain to be investigated . 
 an essential first step is to identify all native sucrose - hydrolysing enzymes and sucrose transporters in this yeast , including those that can be activated by suppressor mutations in sucrose - negative strains . 
 a strain in which all known sucrose - transporter genes ( mal11 , mal21 , mal31 , mph2 , mph3 ) were deleted did not grow on sucrose after 2 months of incubation . 
 in contrast , a strain with deletions in genes encoding sucrose - hydrolysing enzymes ( suc2 , mal12 , mal22 , mal32 ) still grew on sucrose . its specific growth rate increased from 0.08 to 0.25 h1 after sequential batch cultivation . 
 this increase was accompanied by a 3-fold increase of in vitro sucrose - hydrolysis and isomaltase activities , as well as by a 3- to 5-fold upregulation of the isomaltase - encoding genes ima1 and ima5 . 
 one - step cas9-mediated deletion of all isomaltase - encoding genes ( ima1 - 5 ) completely abolished sucrose hydrolysis . 
 even after 2 months of incubation , the resulting strain did not grow on sucrose . 
 this sucrose - negative strain can be used as a platform to test metabolic engineering strategies and for fundamental studies into sucrose hydrolysis or transport .