Patent Application: US-201314431225-A

Abstract:
the disclosure relates to the use of a mutant ssk1 gene encoding a truncated ssk1 protein for the construction of a mutant yeast strain with decreased glycerol production , when compared to the wild - type strain . it relates further to the use of such strains for high - yield bioethanol production , especially in high osmotic media , or on cellulosic hydrolysates , where normal yeast strains do produce a significant amount of glycerol .

Description:
all s . cerevisiae strains used are listed in table 1 . strain cbs6412 was originally indicated as sake yeast kyokai no . 7 in the cbs collection , but comparison of the genome sequence revealed that this indication was erroneous . e . coli strain dh5αtm ( invitrogen corp ., carlsbad ) was used for amplification of plasmids . the strain was grown in luria - bertani ( lb ) medium containing 0 . 5 % wv yeast extract , 1 % wv bacto tryptone , 1 % wv nacl , ( ph 7 . 5 ) at 37 ° c . e . coli transformation and isolation of plasmid dna was carried out using standard techniques ( sambrook et al ., 1989 ). mating , sporulation and dissection of asci were carried out according to standard procedures ( sherman and hicks , 1991 ). mating type of segregants was determined by diagnostic pcr for the mat locus ( huxley et al ., 1990 ). a selection of 52 s . cerevisiae wild - type strains was screened in 250 ml oxygen limited and stirred fermentations containing 1 % wv yeast extract , 2 % wv peptone and 12 % wv glucose . screening of the selected parent strains and the segregants was performed in 15 ml falcon tubes containing 5 ml minimal medium containing 1 . 9 g 1 - 1 yeast nitrogen base ( difco ), 5 g 1 - 1 ammonium sulphate , 250 mg 1 - 1 leucine , 50 mg 1 - 1 uracil , 100 mg 1 - 1 histidine , 30 mg 1 - 1 lysine , 20 mg 1 - 1 methionine and 50 g 1 - 1 glucose . fermentations were inoculated with an initial od of 1 and their progress followed by weight loss . selected segregants were also tested in 100 ml oxygen - limited stirred fermentations . all fermentations were carried out at 30 ° c . high gravity fermentations were carried out in fermentation tubes containing 250 ml of yp and 33 % wv glucose . precultures used as inoculum were first grown on yp 2 % wv glucose for 24 hours and then on yp 10 % wv glucose up to an od600 of 1 . the fermentations were inoculated with 5 . 107 cells / ml and kept at 25 ° c . stirring was applied for the first 4 h ( 120 rpm ). when the weight loss was stable for 2 consecutive days , the fermentation was considered to be finished . shf ( separate hydrolysis and fermentation ) fermentations were carried out with wheat liquefact ( 24 . 5 % dry mass content ) acquired from a local ethanol plant . after adjustment of the ph to 4 . 5 with sulfuric acid , it was treated with dextrozyme ® ( novozyme , denmark ) for 24 h at 60 ° c . to obtain hydrolysate . the latter was boiled at 100 ° c . for 20 min and then cooled . oxygen - limited fermentations were carried out with 100 ml of this medium inoculated with 5 ml of yeast suspension . the fermentations were performed at 30 ° c . and were continuously stirred at 200 rpm . assessment of osmotolerance was performed in fermentations containing minimal medium with or without 0 . 7m or 1 . 4m nacl , or 1m or 2m sorbitol . the fermentations were continuously stirred at 200 rpm . in all fermentations weight loss was used to follow the progress of the fermentation . glucose , glycerol and ethanol in the medium were determined by hplc ( waters ® isocratic breeze ™ hplc , ion exchange column wat010290 ). column temperature was 75 ° c ., 5 mm h2so4 was used as eluent with a flow rate of 1 ml min - 1 and refractive index detection was used ( waters , 2414 ri detector ). biomass was determined by od600 at the beginning and the end of fermentation and yeast dry mass also at the end . the product yield was calculated from the final product concentration ( g . 1 - 1 ) and the difference in glucose concentration at the start and end of fermentation ( consumed glucose in g . 1 - 1 ). the product yields in the ssf were based on the final product concentrations and the equivalent initial glucose concentration ( the latter was measured in a completely hydrolyzed sample of wheat liquefact ). yeast genomic dna was extracted with phenol / chloroform / isoamyl - alcohol ( 25 : 24 : 1 ) ( hoffman and winston , 1987 ) and further purified with diethyl - ether extraction or ethanol precipitation if required . pcr was performed with high - fidelity polymerases phusion ® ( finnzymes ) or extaq ™ ( takara ) for cloning , amplification of deletion or insertion cassettes , and sequencing purposes . sequencing was carried out using the dideoxy chain - termination method ( sanger and coulson , 1975 ) at the vib genetic service facility ( antwerp ). the sequences were analyzed with geneious ( geneious basic 5 . 3 . 4 ), seqman ® ( lasergene coresuite 8 ) or clc dna workbench ( clc bio ) software . after crossing the two parent strains cbs4c and er7a , the 20 most superior segregants ( lowest glycerol production ) were assembled in the “ selected pool ” while 20 random segregants were used to assemble the “ unselected pool .” the two pools were made by combining equal amounts of cells based on od600 . high molecular weight dna ( 3 μg , ˜ 20 kb fragments ) was isolated from the pools and parent strains according to johnston and aust ( 1994 ). the purity of the dna sample was estimated from uv measurement ( 260280 = 1 . 7 - 2 . 0 ). the dna samples were provided to gatc biotech ag ( konstanz , germany ) and bgi ( hong kong , china ) for whole genome sequence analysis by illumina technology . qtl analysis based on the distribution of snp variant frequency over the length of the chromosomes was carried out as described by swinnen et al ., ( 2012a ). the short read sequences obtained from the parental strains and the pools were mapped against the known s288c reference sequence using the mapping software bfast ( homer et al ., 2009 ). after pairing , unique alignments for the cbs4c strain were selected and homozygous variants , i . e ., snps and small indels , were called using snvq ( duitama et al ., 2012 ). in addition , regions with coverage below 0 . 5 or above 1 . 5 of the average coverage were identified and snps of those regions were filtered out . for each polymorphic position the variant calls in the aligned reads for the er7a strain were then extracted and variants were filtered out for which the coverage of the reference variant was too small (& lt ; 20 ×) or too large (& gt ; 150 ×) or snps of both parents coincided but were different from the reference . finally , the number of calls to the reference and the alternative variant of each selected polymorphic position was determined from the set of aligned reads corresponding with the segregant pools . the snp variant frequencies were calculated by dividing the number of the alternative variant by the total number of aligned reads . a very high or a very low frequency was a sign of a one - sided snp segregation preferentially coming from one parent , indicating a genetic linkage to the trait of interest . genetic linkage was statistically confirmed using the methods described earlier ( swinnen et al ., 2012a ). individual snps were scored by pcr . the forward and reverse primer contained the nucleotide of er7a or cbs4c as the 3 ′ terminal nucleotide . the annealing temperature was optimized using dna extracts of er7a and cbs4c so as to allow only hybridization with primers containing a complete match . for rha analysis ( steinmetz et al ., 2002 ), two diploid strains were constructed by crossing cbs4c and er7a wild - type or ssk1δ strains , so that the resulting diploids only contained a single ssk1 allele , either cbs4c derived ssk1 e330n . . . k356n or er7a derived ssk1 . deletion cassettes were constructed essentially as described by gueldner et al ., ( 2002 ) with the phleomycin resistance marker bler and ssk1 gene deletion was confirmed by pcr . the selection marker was removed using the creloxp system . the removal of the selection marker was verified by phleomycin sensitivity as well as by pcr . rha was performed with three independent isolates of all tested diploids . the repeat region h1 was pcr amplified with the primers a - 6101 and a - 6103 using genomic dna of cbs4c and er7a as template . the resulting pcr fragment was digested with kpni and sali and purified from an agarose gel . ssk1 was pcr amplified from genomic dna of cbs4c and er7a and the primers a - 6100 and a27 6102 . the obtained product of around 2800bp was digested with sali and xmai . the cloning vector pbluescriptii sk (+) ( fermentas ) was digested with kpni and xmai and ligated with the repeat region h1 and the ssk1 allele of the respective strain . the construct was verified using sanger sequencing . the two selectable and counter - selectable systems , amd1 and nat1 - gin11 , were used to introduce the insertion cassette . during counter - selection , the marker genes spontaneously looped out via the h1 repeat region , leaving no scars of non - s . cerevisiae dna in the genome . the amd1 marker of zygosaccharomyces rouxii was cut out of the plasmid pf6a - amd1 - mx6 using saci and bglii ( shepherd and piper , 2010 ). the fragment was gel purified and ligated with pug66 , which was also digested with the same two enzymes . the resulting plasmid pug - amd was used for pcr amplification of the amd1 marker using the primers a - 5166 and a - 6770 . the pcr product as well as the h1 - ssk1 plasmids were digested with sali and ligated , resulting in plasmids pbluescriptii_amd1_ssk1 e330n . . . k356n and pbluescriptii_amd1_ssk1 . the selection marker nat1 was amplified from pag25 using primers a - 7116 and a - 7117 . the gin11 counter - selection marker ( akada et al ., 2002 ) was amplified from pg119 using primers a - 7118 and a - 7119 . both fragments were sequentially digested with draiii and sali and ligated with the h1 - ssk1 plasmid , which was previously digested with sali resulting in the plasmids pbluescriptii_nat1 _gin11_ssk1 e330n . . . k356n and pbluescriptii_nat1_gin11_ssk1 . the insertion cassette h1 - loxp - amd1 - loxp - ssk1 and h1 - loxp - amd - loxp - ssk1 e330n . . . k356n were amplified from pbluescriptii_amd1_ssk1 and pbluescriptii_amd1_ssk1 e330n . . . k356n using the outside flanking primers matching the m13 primer binding sites of the plasmid pbluescriptii sk (+). the pcr product was purified and used for transformation . cassettes with h1 - nat1 - gin11 - ssk or h1 - nat1 - gin11_ssk1 e330n . . . k356n were digested with bsphi and digestion products were used for transformation . yeast was transformed with the liac / peg method ( gietz et al ., 1992 ). site - directed modification of the cbs4c and er7a ssk1 locus was carried out using a two step - method . in the first step , the ssk1 insertion cassettes ( see above ) were transferred to the ssk1 deletion strains , cbs4c ssk1δ and er7a ssk14 . after transformation , positive clones were selected on yd agar plates containing 200 μg / ml clonnat . the presence of the insertion cassette was verified by pcr using the primers a - 5168 and a - 7301 o in the second step , the marker genes were removed by selection of spontaneous loop - outs on galactose - containing medium after induction of the counter - selectable marker gin11 ( akada et al ., 2002 ; akada et al ., 1999 ; olesen et al ., 2000 ). positive looped - out clones were identified by clonnat sensitivity and verified by pcr using the forward primer a - 5168 and the ssk1 allele specific reverse primers a - 5126 and a - 5127 . the inserted ssk1 alleles were verified by sanger sequencing . the ssk1 e330n . . . k356n allele of cbs4c was inserted twice in the ethanol red derivative , hg5 ( see table 1 ), which had both ssk1 alleles deleted . the latter strain was constructed by introducing a disruption cassette flanked with loxp sites using homologous recombination ( gueldener et al ., 2002 ; kotaka et al ., 2009 ). the disruption cassette was constructed with homologous sequences ( h1 / h2 ) corresponding to the 5 ′ and 3 ′ end of the ssk1 orf surrounding the phleomycin resistance - gene bler used as selectable marker . the selectable marker , bler , was removed by cre recombinase . a second disruption cassette was constructed with the recombination sites h1 * and h2 *, which were located inside the first homologous integration sites , h1 and h2 , enabling specific recombination into the 2nd ssk1 allele of the diploid strain . gene disruption was verified by pcr . the bler marker gene was again removed using the creloxp system . the double deletion was confirmed by pcr using primers located outside the integration site . the two ssk1 e330n . . . k356n insertion cassettes , h1 - loxp - amd1 - loxp - ssk1 e330n . . . k356n and h1 - nat1 - gin11 - ssk1 e330n . . . k356n , were successively transformed . after the 1 st transformation of the h1 - loxp - amd1 - loxp - ssk1 e330n . . . k356n cassette , transformants were selected based on hydrolysis by amd1 of acetamide used as sole nitrogen source . the correct integration of the insertion cassette was verified by pcr using the primers a - 5168 and a - 5894 inside the amd gene . in the 2nd transformation , the h1 - nat1 - gin11 - ssk1 e330n . . . k356n was transferred . positive transformants were selected on acetamide medium , containing 200 μg / ml clonnat . correct integration in the 2 nd chromosome was verified by pcr using the primers a - 5168 and a - 7301 to test the presence of the insertion cassette as well as the primers a - 5168 and a - 5169 to verify the disappearance of the two orf deletions of the ethanol red ssk1δ / δ . counter - selection was simultaneously applied for both marker systems using medium with 100 mm fluoroacetamide and 0 . 04 % galactose ( induction of gin11 ). we have evaluated 52 diploid s . cerevisiae strains from diverse origins for the ratio between the amount of glycerol and ethanol produced in small - scale ( 250 ml ) fermentations with complex medium containing 12 % glucose . a continuous and normal distribution of the trait was observed ( fig1 ). the cbs6412 strain showed the lowest glycerol yield ( 0 . 043 g . g - 1 ) of all strains tested , which was about 63 % of that of the reference industrial strain ethanol red ( 0 . 068 g . g - 1 ) ( fig2 a ), an industrial strain commonly used for bioethanol production with corn and wheat starch hydrolysate . as it had both a low glycerol / ethanol ratio and a low glycerol yield , cbs6412 was chosen as the superior strain and ethanol red was used as the inferior strain . in order to obtain haploid strains for genetic mapping analysis , the two diploid strains were sporulated and segregants were tested in small - scale fermentations . glycerol yields of the segregants were normally distributed around those of the diploid parents ( fig2 b ), indicating a highly heritable phenotype . the cbs6412 segregant , cbs4c , had an even lower glycerol yield than its parental diploid ( fig2 a ), indicating acquirement of one or more beneficial , recessive alleles present in heterozygous form in the diploid strain . cbs4c was selected as the superior parent strain for the genetic mapping . the ethanol red segregant er7a had a glycerol yield closest to its parental diploid and served as inferior parent strain . construction of the cbs4c / er7a hybrid and selection of superior segregants with low glycerol yield the cbs4c and er7a haploid strains were crossed with each other and 257 segregants were isolated and first characterized for glycerol and ethanol yield in 5 ml fermentations with 5 % glucose in minimal medium . fig2 c shows a histogram of the glycerol yield in the segregant population in comparison with that of the cbs4c and er7a haploid parents . the glycerol yield showed a normal distribution and most segregants had a glycerol yield close to the average ( 0 . 063 g . g - 1 , ± 142 % of the cbs4c glycerol yield ). we re - tested the 48 segregants with a glycerol yield below 120 % of the cbs4c parent in 100 ml small - scale fermentations ; 44 segregants showed the same low glycerol yield also under these conditions . among these , the 20 segregants showing the lowest glycerol yield (≦ 0 . 054 g g - 1 ) were selected for qtl mapping with pooled - segregant whole - genome sequence analysis . the 24 remaining segregants were used for subsequent validation of the results as described below . a second pool with 20 randomly selected segregants was also subjected to pooled - segregant whole - genome sequence analysis and used as control . qtl mapping using pooled - segregant whole - genome sequence analysis . the genomic dna of the selected and random pools , as well as the two parent strains , was extracted and submitted to custom sequence analysis using illumina hiseq 2000 technology ( gatc biotech ag , konstanz , germany ; bgi , hong kong , china ). the sequence reads of the cbs4c and er7a parent strains were aligned with the s288c standard sequence , which allowed to identify 21 , 818 snps between cbs4c and er7a . the snps were filtered as described previously ( duitama et al ., 2012 ). the variant frequency of the quality - selected snps in the dna of the two pools was then plotted against the snp position on the chromosome . the scattered raw data were smoothened by fitting smoothing splines in the generalized linear mixed model framework as previously described ( swinnen et al ., 2012a ). the results are shown in fig3 . a prominent qtl with strong linkage was present on chromosome xii ( between 135 , 000 and 200 , 000 bp ) and is shown in more detail in fig4 b . individual snps from that region , as well as from the qtl with lower linkage on chromosome ii ( fig4 a ), were scored by pcr detection in the 20 individual segregants of the selected pool ( fig4 a and 4b ). the precise snp variant frequency determined in this way was used to verify the linkage of the two regions on chromosome ii and xii , respectively . this revealed a very strong linkage with low glycerol yield for the qtl on chromosome xii with the minimal p - value being 1 . 45 . 10 - 4 , while the p - values for the qtl on chromosome ii only dropped just below the 0 . 05 threshold for significance ( 0 . 009 ). the same snps were also scored in the 24 remaining segregants with a glycerol yield below 120 % of the cbs4c parent . calculation of the p - values for the whole group of 44 segregants no longer revealed significant linkage for the qtl on chromosome ii . on the other hand , the p - values for the qtl on chromosome xii dropped to 9 . 10 - 11 , strongly increasing significance of the linkage . hence , we concentrated the further analysis on the qtl of chromosome xii . identification of ssk1 as a causative gene in the qtl on chromosome xii the 20 , 000 bp region with the strongest linkage in the qtl on chromosome xii contained 13 genes , of which four genes contained non - synonymous mutations in the orf ( fig4 b ). one of those four genes , ssk1 , was located in the center of the qtl , which had a slightly stronger linkage . ssk1 has a known function in the hog pathway . sequence comparison of the ssk1 alleles of the parental strains cbs4c and er7a with the allele of the reference strain s288c revealed ten polymorphisms between the sequence of the ssk1 orf in cbs4c and er7a . a single base pair deletion at position 162 , 907 bp of chr . xii was the most prominent mutation in the cbs4c ssk1 orf , since it caused a reading frame shift and a new stop codon at position 357 in the protein . this resulted in a new primary amino acid sequence from position 330 until 356 , while the wild - type ssk 1 protein had a total length of 712 amino acids . hence , we named the new allele ssk1 e330n . . . k356n . the dramatic change in amino acid sequence and the truncation would normally be expected to result in a completely inactive protein and , therefore , in a phenotype similar to that of the ssk1δ strain . however , this was not the case . the ssk1 e330n . . . k356n allele caused a different phenotype compared to deletion of ssk1 ( see below ). next , we evaluated ssk1 as possible causative gene using reciprocal hemizygosity analysis ( rha ) ( steinmetz et al ., 2002 ). for that purpose , two cbs4c / er7a hybrid and hemizygous diploid strains were constructed differing only in a single ssk1 allele , the other allele being deleted . the diploid strain with the single ssk1 e330n . . . k356n allele derived from cbs4c showed a significantly reduced glycerol yield and a significantly higher ethanol yield than the diploid strain with the ssk1 allele from the er7a strain ( fig5 ). this showed that ssk1 e330n . . . k356n was a causative gene in the qtl on chromosome xii . to evaluate whether the ssk1 e330n . . . k356n a llele of cbs4c behaved as a recessive allele and whether it caused the same phenotype as deletion of ssk1 , we also constructed a cbs4c / er7a hybrid diploid strain with both ssk1 alleles deleted and compared its phenotype with that of cbs4c / er7a with its original ssk1 alleles . the glycerol and ethanol yields of these strains were similar to that of the hemizygous diploid strain with the ssk1 allele from er7a or the ssk1 e330n . . . k356n allele from cbs4c , respectively , ( fig6 a ). this indicates that the ssk1 e330n . . . k356n allele from cbs4c is a recessive allele and that ssk1 e330n . . . k356n behaves as a loss of function allele , at least in the hybrid background and the fermentation conditions used ( 100 ml anaerobic fermentations in minimal medium containing 5 % glucose ). when the glycerol yield ( 0 . 043 g . g - 1 ) of the cbs4c parent strain was normalized to 100 %, the glycerol yield of er7a ( 147 %) and that of the diploids er7acbs4c ( 145 %) and er7a / cbs4c ssk1δ ( 148 %) was very similar ( fig6 a ). in contrast , the strains er7a ssk1δ / cbs4c ssk1 e330n . . . k356n and er7a ssk1δ / cbs4c ssk1δ had a glycerol yield of 119 % and 122 %, respectively , ( fig6 a ) suggesting that ssk1e330n . . . k356n was responsible for the majority of the reduction in glycerol yield in cbs4c compared to er7a . this agrees with the result of the pooled - segregant whole - genome mapping , which revealed the ssk1 locus as the only qtl with significant linkage . to confirm the importance of ssk1 in an alternative way , we reciprocally exchanged the ssk1 alleles of cbs4c and er7a by homologous recombination . introduction of ssk1 e330n . . . k356n in the er7a strain reduced its glycerol yield and enhanced its ethanol yield , while introduction of ssk1 in cbs4c enhanced its glycerol yield and reduced its ethanol yield ( fig6 b ). these results confirmed ssk1 as a causative allele for reduced glycerol and enhanced ethanol production in cbs4c . given the recessive character of the ssk1 e330n . . . k356n allele , we tested its presence in the original diploid strain cbs6412 and found it to be present in two copies . this suggests that the unusual allele may provide a selective advantage in specific environmental niches . reduction of the glycerol / ethanol ratio in an industrial bioethanol strain using ssk1 e330n . . . k356n as a novel gene tool to test the functionality of ssk1 e330n . . . k356n as a novel gene tool for reduction of glycerol yield under industrially relevant conditions , both ssk1 alleles of the industrial bioethanol production strain , ethanol red , were replaced by the ssk1 e330n . . . k356n variant using homologous recombination . in addition , an ethanol red ssk1δ / ssk1δ strain and an ethanol red ssk1 e330n . . . k356n / ssk1δ strain were constructed . these strains were tested in fermentations with minimal medium ( 5 % wv glucose ), high gravity medium ( yp with 33 % wv glucose ) and wheat hydrolysate ( shf : separate hydrolysis and fermentation ). the results are shown in fig7 a . the double deletion of ssk1 reduced the glycerol yield . interestingly , further reduction of glycerol yield was observed after introduction of one copy of ssk1 e330n . . . k356n while introduction of the second copy of ssk1 e330n . . . k356n lowered glycerol yield even more . ethanol yields clearly increased in all ethanol red mutants compared to the wild - type strain in the minimal medium . the reduction of glycerol yield under high gravity or shf conditions was generally less pronounced compared to minimal medium . thus , the concomitant increase in ethanol yield in the ethanol red mutants was less obvious . nevertheless , particularly the result obtained in minimal medium indicated that in the ethanol red diploid background the ssk1 e330n . . . k356n allele did not simply behave as a loss - of - function allele but had a stronger reducing effect on the glycerol / ethanol ratio than deletion of the ssk1 gene . these results confirm the usefulness of the ssk1 e330n . . . k356n allele as a novel gene tool for lowering glycerol production in industrial yeast strains . the novel gene tool ssk1 e330n . . . k356n retains its positive effect under high osmolarity conditions several previous studies successfully reduced glycerol yield in s . cerevisiae with a concomitant increase in ethanol yield . however , many of the resulting strains showed a significantly reduced maximal volumetric ethanol production rate and increased sensitivity against osmotic stress ( bjorkqvist et al ., 1997 ; guadalupe medina et al ., 2010 ; hubmann et al ., 2011 ; nissen et al ., 2000a ). in order to address this issue , we determined both the glycerol / ethanol ratio and the maximal volumetric ethanol production rate in the ethanol red strains containing one or two ssk1 e330n . . . k356n alleles under conditions of high osmolarity . in general , the cells produced higher levels of glycerol under hyperosmotic stress , i . e ., in the presence of 1 . 4 m and 2 m sorbitol or 0 . 7 m and 1 m nacl ( fig7 b and 7c ). in spite of this , a similar improvement in the glycerol / ethanol ratio was observed in the ethanol red strains containing one or two ssk1 e330n . . . k356n alleles . the maximal volumetric ethanol production rate dropped with increasing osmolarity but this drop was not correlated with the presence or the number of ssk1 e330n . . . k356n alleles . hence , the ssk1 e330n . . . k356n allele does not appear to cause an increase in osmosensitivity and retains its positive effect under conditions of high osmolarity . close examination of the effect of ssk1 e330n . . . k356n on glycerol production in the ethanol red background also allows to make a quantitative assessment of the contribution of this allele to the phenotype . the initial glycerol yield was 167 % of the cbs4c yield while the double insertion of ssk1 e330n . . . k356n caused a drop to 128 % of the cbs4c yield . hence , the ssk1 mutation appears to determine 50 - 60 % of the trait . this indicates that the ssk1 mutation may be combined by other mutant alleles from other genes to obtain a maximal effect . selection of a rare segregant displaying the trait of low glycerol / high ethanol production and lacking the ssk1 e330n . . . k356n allele previous work has identified the s . cerevisiae strain cbs6412 as a strain with an unusually low ratio of glycerol / ethanol production and genetic analysis identified the ssk1 e330n . . . k356n allele as a major causative gene ( hubmann et al ., 2013 ) ( fig8 a ). in order to identify the minor qtls and their causative genes responsible for determining this complex trait , we have first screened all superior segregants with a glycerol / ethanol ratio as low as the superior parent strain , for a segregant that lacked the ssk1 e330n . . . k356n allele . among the 44 superior segregants available , only a single such segregant , 26b , was present . its glycerol yield was equally low and its ethanol yield equally high as the superior parent strain cbs4c , both in minimal medium with 5 % glucose and in rich yeast extract - peptone medium with 10 % glucose ( fig8 b ). hence , 26b showed the same phenotypic difference with the inferior parent strain er7a as cbs4c ( fig8 b ). backcross of the unique superior segregant 26b with the inferior parent er7a and screening for superior segregants we subsequently switched the mating type of 26b from matα to mata ( see materials and methods ) and crossed the 26b strain with the inferior parent strain , er7a , which is a derivative of the industrial strain ethanol red , currently used worldwide in bioethanol production . the hybrid diploid er7a / 26b showed an intermediate phenotype between er7a and 26b ( fig8 b ). from this cross , 260 meiotic segregants were screened for low glycerol yield ( and corresponding higher ethanol production ) in 100 ml fermentations with yp 10 % glucose . the parent strains 26b and er7a , and the hybrid diploid , were used as controls in each batch of fermentations . glycerol and ethanol yield of the segregants in each batch were normalized to those of 26b , which were set to 100 %. er7a and the diploid 26b / er7a showed an average glycerol yield of 146 % and 124 % and a concomitantly decreased ethanol yield of 98 . 1 % and 99 . 4 % ( fig9 a ). the glycerol and ethanol yield showed a gaussian distribution in the segregants , which extended over the range of the two parental stains . in the case of low glycerol yield , this extension was only marginal . the population mean of the glycerol yield ( 123 %) and ethanol yield ( 98 . 8 %) were located closely to that of the diploid 26b / er7a . in general , glycerol and ethanol yield of the segregant population correlated inversely ( as determined with a pearson test ), meaning that low glycerol yield usually resulted in high ethanol yield . nearly all exceptions to this correlation were strains with an unusually low ethanol yield that failed to show a correspondingly higher glycerol yield . two cut - off criteria were defined , a glycerol yield lower than 120 % of 26b and an ethanol yield higher than 99 % of 26b , which resulted in the selection of a set of 34 superior segregants . these were all retested in 100 ml fermentations with yp 10 % glucose and 22 segregants showed again a low glycerol yield combined with a correspondingly higher ethanol yield ( fig9 b ). these 22 segregants were selected for qtl mapping with pooled - segregant whole - genome sequence analysis . a second pool with 22 randomly selected segregants was also subjected to pooled - segregant whole - genome sequence analysis and used as the unselected control pool ( fig9 b ). the genomic dna of the selected and unselected pools , as well as the parent strain 26b , was extracted and submitted to custom sequence analysis using illumina hiseq 2000 technology ( bgi , hong kong , china ). the parent strain er7a has been sequenced in our previous study ( data accession number sra054394 ) ( hubmann et al ., 2013 ). read mapping and snp filtering were carried out as described previously ( swinnen et al ., 2012 ; claesen et al ., 2013 ). the snp variant frequency was plotted against the snp chromosomal position ( fig1 ). of the total number of 21 , 818 snps between cbs4c and er7a , 5 , 596 snps of cbs4c were found back in 26b . these snps were used for mapping minor qtls in the genomic areas that were not identical between 26b and er7a . the other genomic areas were completely devoid of snps because they were identical between the 26b and er7a parents ( fig1 ). the scattered raw snp variant frequencies were smoothened and a confidence interval was calculated , as previously described ( swinnen et al ., 2012 ; claesen et al ., 2013 ). the hidden markow model , explora ( see materials and methods ) was used to evaluate whether candidate regions showed significant linkage to the low glycerol phenotype . explora reported six candidate qtls : on chr . i ( 3859 - 11045 ), chr . ii ( 584232 - 619637 ), chr . iv ( 316389 - 375978 and 696486 - 748140 ), and chr . xiii ( 600902 - 610995 and 634582 - 640415 ) for the selected segregants pool . the locus on chr . i was present in both the selected and unselected pool and was thus likely linked to an inadvertently selected trait , such as sporulation capacity or spore viability . it was excluded from further analysis . the locus on chr . ii was also present in the previous mapping with the two original parents , cbs4c and er7a , but in that case it was not pronounced enough to be significant ( hubmann et al ., 2013 ). the backcross has now confirmed its relevance . on chr . iv and xiii , new qtls were detected , which were not present in the mapping with the original parent strains cbs4c and er7a . explora also reported two significantly linked loci on chr . vi ( 169586 - 170209 ) and chr . vii ( 472620 - 493523 ) for the unselected pool . both loci were linked to the inferior parent , er7a . for the region on chr . vii , the linked locus with the inferior parent genome was also present in the selected pool . both loci likely represent linkage to inadvertently selected traits , such as sporulation capacity or spore viability . it is unclear why the locus on chr . vi was only present in the unselected pool . since both loci were not linked to the low glycerol phenotype they were not investigated further . the qtls on chr . ii , iv and xiii were further investigated in detail . selected individual snps were scored in the 22 individual superior segregants to determine precisely the snp variant frequency and the statistical significance of the putative linkage . however , using the binomial test previously described ( swinnen et al ., 2012 ; claesen et al ., 2013 ) none of the three loci was found to be significantly linked to the genome of the superior parent strain 26b with the number of segregants available . therefore , we screened 400 additional f1 segregants of the diploid 26b / er7a for low glycerol / high ethanol production . in addition , we performed four rounds of random inbreeding with a very large number of f1 segregants from the diploid 26b / er7a to increase the recombination frequency ( parts et al ., 2011 ) and subsequently evaluated 400 f5 segregants in small - scale fermentations for glycerol / ethanol yield . the results for the 400 f1 and 400 f5 segregants are shown in fig1 a . the glycerol and ethanol yields are expressed as percentage of that of the superior parent strain 26b . there was again a clear inverse relationship between glycerol and ethanol yield . from the 800 segregants , we selected in total 48 superior segregants , 22 f1 segregants and 26 f5 segregants ( fig1 b ). we next scored selected snps in the putative qtls on chr . ii , iv and xiii in all individual segregants , i . e ., the 22 segregants of the sequenced selected pool , the 22 additional selected f1 segregants , the total of 44 selected f1 segregants , the 26 selected f5 segregants and the total of 70 selected segregants . the mean snp variant frequency for these groups of segregants and the corresponding p - value were calculated as described previously ( swinnen et al ., 2012 ; claesen et al ., 2013 ) and are shown in fig1 c . for the three qtls we could now demonstrate significant linkage ( p - value & lt ; 0 . 05 ) to the genome of the superior parent strain 26b . for the qtls on chr . ii and iv the linkage was now very strong , while for the qtl on chr . xiii it was still weak , but significant . on the other hand , the second region on chr . iv did not show significant linkage with none of the pools . identification of causative genes in the qtls on chr . ii , iv and xiii we selected three candidate genes in the three qtls based on their known function in glycerol metabolism . smp1 , which is located in the qtl on chr . ii ( 594 , 864 to 593 , 506 bp ), encodes a putative transcription factor involved in regulating glycerol production during the response to osmostress ( de nadal et al ., 2003 ). the gene is located in the chromosomal region from 584 , 232 to 619 , 637 bp , which was predicted as most significant by the explora model . the 26b smp1 allele has two point mutations , which are changing the primary protein sequence at position 110 from arginine to glycine and at position 269 from proline to glycine . hence , we have named this allele smp1 r110q , p269q . on chr . iv , the snp with the highest linkage was located at position 411 , 831 bp ( fig1 c ), which is within the open reading frame of gpd1 ( 411 , 825 to 413 , 000 bp ). this is the structural gene for the nad +- dependent cytosolic glycerol 3 - phosphate dehydrogenase ( larsson et al ., 1993 ; albertyn et al ., 1994 ). it catalyzes the conversion of dihydroxyacetone phosphate ( dhap ) to glycerol 3 - phosphate through the oxidation of nadh . the gpd1 allele of 26b harbors a point mutation , changing leucine at position 164 into proline . this mutation was found earlier ( ddbj database data , accession number ay598965 ). the gpd1 allele of 26b was named gpd1 l164p . on chr . xiii , the snp with the highest linkage was located at position 606 , 166 by ( fig1 c ), which is within the open reading frame of hot1 ( 605 , 981 to 608 , 140 bp ). hot1 encodes a transcription factor required for the response to osmotic stress of glycerol biosynthetic genes , including gpd1 , and other hog - pathway regulated genes ( alepuz et al ., 2003 ; rep et al ., 1999 ). the 26b hot1 allele contains two non - synonymous point mutations , changing proline at position 107 to serine and histidine at position 274 to tyrosine . we have named the hot1 allele of 26b , hot1 p107s , h274y . we first investigated the effect of smp1 r110q , p269q , gpd1 l164p and hot1 p107s , h274y on the low glycerol / high ethanol phenotype using reciprocal hemizygosity analysis ( rha ) ( steinmetz et al ., 2002 ). for that purpose , we constructed for each gene a pair of hemizygous diploid 26b / er7a hybrid strains , in which each pair contained a single copy of the superior allele or the inferior allele of smp1 , gpd1 or hot1 , respectively . the three pairs of hemizygous diploids were tested in the same 100 ml yp 10 % glucose fermentations as used for the screening . the parent strains 26b and er7a and the hybrid diploid 26b / er7a were added as controls . the glycerol and ethanol yields were expressed as percentage of those of 26b , which were set at 100 %. the significance of any differences between the strains was evaluated using a two - tailed unpaired t - test with a p - value & lt ; 0 . 05 considered to indicate a significant difference . the results of the rha are shown in fig1 . they indicate that both smp1 r110q , p269q and hot1 p107s , h274y but not gpd1 l164p , derived from the superior parent 26b cause a significant drop in the glycerol / ethanol ratio compared to the alleles of the inferior parent strain er7a . for smp1 r110q , p269q only the reduction in glycerol , and not the increase in ethanol , was significant with the p - value & lt ; 0 . 05 used . these results indicate that smp1 r110q , p269q is a causative gene in the qtl on chr . ii . they do not exclude that the qtl may still contain a second causative gene , especially since smp1 r110q , p269q is not located in the region with the strongest linkage ( lowest p - value ). the rha with the gpd1 alleles failed to show any difference both for glycerol and ethanol production ( fig1 ). hence , the superior character of the gpd1 l164p allele could not be confirmed with rha . this is remarkable because the snp with the strongest linkage ( lowest p - value ) in the qtl on chr . iv was located in the open reading frame of gpd1 and showed very strong linkage to the low glycerol / high ethanol phenotype . the hot1 p107s , h274y allele of the superior strain 26b , on the other hand , caused a reduction in glycerol and an increase in ethanol production , and both changes were significant ( p - value & lt ; 0 . 05 ) ( fig1 ). hence , these results indicate that hot1 p107s , h274y is a causative allele in the qtl on chr . xiii and because it contains the snp with the strongest linkage ( lowest p - value ), it is likely the main causative allele in this qtl . the glycerol yield for the inferior parent er7a and the diploid 26b / er7a were on average 143 % and 126 % of the 26b yield ( fig1 ). ethanol yield of both strains was correspondingly reduced to 98 % and 98 % of the 26b yield , respectively . clearly , the smp1 r110q , p269q and hot1 p107s , h274y alleles can only be responsible for part of the difference in the glycerol / ethanol ratio between the parent strains . the same was found previously for the ssk1 e330n . . . k356n allele ( hubmann et al ., 2013 ). this confirms that the glycerol / ethanol ratio in yeast fermentation is a true polygenic , complex trait , determined by an interplay of multiple mutant genes . expression of the gpd1 l164p allele from 26b in haploid gpd1δ strains reveals its superior character several explanations could account for the failure to confirm the superior character of the gpd1 l164p allele from 26b in the rha test . a closely located gene may be the real causative gene , the gpd1 l164p allele may be effective only in a haploid genetic background or the effect of the gpd1 l164p allele may be suppressed through epistasis by one or both of the other two superior alleles , smp1 r110q , p269q and hot1 p107s , h274y . to distinguish between these possibilities , we amplified the gpd1l164p allele from strain cbs4c and the gpd1 allele from strain er7a by pcr ( 410 , 523 to 413 , 479 bp , including promotor , orf and terminator ). the pcr fragment was ligated in the centromeric plasmid ycplac33 , resulting in plasmids ycplac33 / gpd1 l164p - cbs4c and ycplac33 / gpd1 - er7a . both plasmids were transformed into gpd1δ strains of the two parents 26b and er7a , the hybrid diploid 26b / er7a and the lab strain by4742 ( giaever et al ., 2002 ; winzeler et al ., 1999 ). all strains were tested in 100 ml scale fermentations with yp 10 % glucose . glycerol and ethanol yield were determined after 120 h fermentation and were expressed as percentage of those of 26b . the results are shown in fig1 . the expression of the gpd1 l164p - cbs4c or gpd1 - er7a allele in the gpd1δ strains of the superior parent 26b and the hybrid diploid 26b / er7a enhanced glycerol production and reduced ethanol production to the same extent for the two alleles . on the other hand , in the gpd1δ strains of the inferior parent er7a and the lab strain by4742 , the gpd1 l164p - cbs4c allele enhanced glycerol production and reduced ethanol production significantly more than the gpd1 - er7a allele . the latter shows that the gpd1 l164p - cbs4c allele is superior compared to the gpd1 - er7a allele . the difference between the two alleles is apparently not dependent on the haploid or diploid background of the strain but seems to be related with the presence of the two other superior alleles , smp1 r110q , p269q and hot1 p107s , h274y . they are both present in the two strains , 26b and 26b / er7a , in which gpd1 l164p - cbs4c has no differential effect and absent in the two strains , er76a and by4742 , in which gpd1 l164p - cbs4c causes a differential effect . hence , the superior potency of gpd1 1164p - cbs4c is likely suppressed through epistasis by smp1 r110q , p269q and hot1 p107s , h274y . we have scored the final 70 superior segregants with a glycerol production & lt ; 120 % and an ethanol production & gt ; 99 % of that of the superior parent 26b , for the presence of the three causative alleles , smp1 r110q , p269q , gpd1 ll164p and hot1 p107s , h274y . the results are shown in fig1 a . the largest group of superior segregants contained all three mutant alleles , followed by smaller groups with only two of the three mutant alleles and finally the three smallest groups with only one mutant allele . hence , there was a clear correlation between the number of the mutant alleles and low glycerol / high ethanol yield in this group of selected segregants . on the other hand , although there was a tendency for a lower mean glycerol / ethanol production ratio with an increasing number of mutant alleles , the differences between the means of the different groups were small . we have also investigated a possible correlation between the different mutant alleles and the strength of the low glycerol / high ethanol phenotype . for that purpose , we determined the percentage of segregants with a specific mutant allele in sets of strains with a different glycerol / ethanol ratio . the results show that there is no preference between the different alleles in the strains with a higher glycerol / ethanol ratio , but in the strains with the lowest glycerol / ethanol ratio the gpd1 l164p allele is preferentially present , followed by the hot1 p107s , h274y allele , although the latter is only true in the category with the lowest glycerol / ethanol ratio ( fig1 b ). hence , the order of potency of the three alleles appears to be : gpd1 l164p & gt ; hot1 p107s , h274y ≧ smp1 r110q , p269q . there was no correlation between the variant frequency of the three alleles for low glycerol yield and high ethanol yield , indicating that other minor qtls may independently affect ethanol yield more than the three new alleles identified or that it is the combination of the alleles that is important . akada r , hirosawa i , kawahata m , hoshida h , nishizawa y ( 2002 ) sets of integrating plasmids and gene disruption cassettes containing improved counterselection markers designed for repeated use in budding yeast . yeast 19 : 393 - 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