BACKGROUND
the chinese liquor-making process is a unique and complicated spontaneous fermentation process under solid state conditions  <cit> .
chinese liquor is produced by the technique of simultaneous saccharification and fermentation  using grains, such as sorghum, wheat and barley.
the cost-effective ssf combines the enzymatic hydrolysis of starch with the simultaneous fermentation of sugars, resulting in an outpouring carbon source  <cit> .
in addition, chinese liquor is fermented using combinations of microorganisms, including yeast, bacteria and filamentous fungi  <cit> .
the complex filamentous fungal community produces diversified hydrolases to degrade starch into various sugars, including glucose, galactose, maltose and melibiose  <cit> .
high temperatures  at the starter are generated by the metabolic activities of the microorganisms and the low thermal conductivities of the solid matrices  <cit> .
in addition, there are two other prominent pressures: acidity stress  and ethanol stress  in the alcoholic fermentation stage  <cit> .
such a severe environment during the ssf process leads to a distinctive community of microorganisms with specific physiological properties and performances  <cit> , in particular, distinctive heat- and acid-resistant properties  <cit> .
although the microorganisms in the chinese maotai-flavored liquor have been investigated since the 1960s  <cit> , there is little information on the functional genomics of the yeast involved in the process.
this has inhibited our understanding of the fermentation mechanism and the development of maotai-flavored liquor.
functional genomics approaches, such as whole-genome sequencing, are powerful tools for the analysis of specific traits on a genomic scale and provide a method of studying cellular physiology from micro-perspective.
since the first complete genome of strain s288c was sequenced in  <dig>  <cit> , it has served as the reference for the saccharomyces cerevisiae species and has promoted the development of whole-genome approaches  <cit> .
meanwhile, the genomes of several s. cerevisiae strains have been sequenced and compared with the s288c reference genome, including laboratory strains  <cit>  and industrial strains, such as sake yeast k <dig>  wine yeasts, beer yeasts and bioethanol yeasts .
the phenotypic diversity among yeast isolates is significant, and variation is apparent among the surveyed strains at different levels.
ecological factors and geographical locations, as the main reasons for biodiversity, have important effects on the domestication of microbial physiological characteristics  <cit> .
among the  <dig> strains with sequenced genomes in the ncbi database, there are only three strains from china  <cit> .
however, strains involved in the ssf process of chinese liquor are poorly understood.
theoretically, the genomic analysis of strains with different backgrounds should help identify the sequence changes that play important roles in specific physiological characteristics.
comparisons of the publicly available s. cerevisiae genome sequences have revealed the clear signatures  in the different strains  <cit> .
compared with laboratory strains, industrial strains generally show a higher adaptability to specific environments.
however, the genetic basis for their improved characteristics is not well understood.
further studies are needed to explore how the genetic variations confer the specific phenotypes.
the comprehensive identification of polymorphisms among individuals within a species is essential for studying the genetic basis of phenotypic differences and for elucidating the evolutionary history of the species.
in this study, s. cerevisiae mt <dig> was isolated from the fermentation environment of chinese maotai-flavored liquor.
its physiological characteristics were evaluated in several phenotypic assays.
meanwhile, the mt <dig> genome was sequenced and compared with the type strain s288c.
in addition, we elucidated how genetic variations of mt <dig> were correlated with specified traits using rna-seq and revealed the molecular mechanisms related to the adaptation of yeast in chinese liquor-making.
this whole-genome analysis of the s. cerevisiae strain used in chinese liquor-making will supplement the genomic and phylogenetic knowledge of yeast and provide a guide for the construction of strains with desired traits.
RESULTS
phenotypic characterization
several physiological and biochemical characteristics of the s. cerevisiae strain mt <dig>  isolated during the process chinese maotai-flavored liquor-making, were compared with those of the reference strain s288c and other industrial strains .
in addition to the sake yeast k <dig>  other strains had high tolerance to 38 °c which is consistent with previous studies that it was widely believed that s. cerevisiae had a thermal tolerance of 25–37 °c or 39 °c  <cit> .
while a higher thermal resistance was found for mt <dig> when temperature was higher than 38 °c compared with not only s288c but also other industrial yeasts.
population of mt <dig> was twice that of other strains  in 16 % ethanol.
it indicated that mt <dig> had a higher ethanol tolerance.
in addition, mt <dig> had a growth advantage compared with other strains at ph  <dig> – <dig> .
in particular, the population of mt <dig> is almost three times of that of s288c at ph  <dig> .
this suggested that mt <dig> has a much higher resistance to acidity than s288c and other industrial strains, as most industrial strains could barely grow at ph  <dig>   <cit> .fig.
<dig>
a effects of temperature, ethanol and acidity on cell growth of several yest strains.
s, saccharomyces cerevisiae type strain s288c; m, chinese maotai-flavored liquor yeast mt1; k, sake yeast strain k7; h, hangjiu  yeast cicc30394; w, wine yeast strain cicc31089; b, beer yeast strain atcc <dig>  * represents the significant difference with p-value <  <dig> , compared with mt <dig>  calculated using a one-way analysis of variance in spss.
b growth differences between mt <dig> and s288c with various carbon sources.
left: oxidation tests that detect carbon utilization via a redox color change in the organism’s suspension; right: assimilation reaction tests that are based on turbidity increases due to carbon utilization of the organisms.
<dig>  monosaccharides; , disaccharides; , trisaccharides; , oligosaccharides; , acids; , alcohols; and , others
carbon source utilization exerts a strong influence on the classification of isolates and may be a useful and functional measure for the characterization and classification of different strains.
unfortunately, there is limited research on the simultaneous fermentation of various carbon sources to produce ethanol.
the primary reason is that not many strains can ferment ethanol from various sugars.
presently, there is some research on a strain that could simultaneous utilize glucose and xylose, or arabinose and galactose  <cit> .
mt1s ability to uptake and assimilate sugars was also investigated .
among the monosaccharides, glucose could be used for growth and fermentation by both mt <dig> and s288c, with od <dig> and od <dig> values exceeding  <dig> .
when galactose was the sole carbon source, the od <dig> and od <dig> of mt <dig> reached  <dig>  and  <dig> , respectively, while those of s288c were less than  <dig> .
this indicated that mt <dig> could uniquely utilize galactose.
the other carbon sources that mt <dig> could uniquely uptake and assimilate were mainly disaccharides, such as melibiose, maltose, trehalose and turhanose.
in addition, raffinose, a trisaccharide constituted by galactose, fructose and glucose, as well as methyl-a-d-glucopyranoside, are well utilized by mt <dig>  with od <dig> values reaching  <dig>  and  <dig> , respectively.
these results suggested that mt <dig> was a strain appropriate for multi-carbon fermentation, having the ability to utilize various carbon sources.
mt <dig> genome structure
the mt <dig> genome assembly resulted in  <dig> contigs  with an n <dig> value of  <dig> .
then,  <dig> scaffolds  were obtained with an n <dig> value of  <dig> .
the assembled data were evaluated, and the detailed statistical data are shown in table  <dig>  finally, the mt <dig> assembled genome was  <dig>  mbp with a gc% of  <dig>  %.
the genome size of mt <dig> is smaller than most of the s. cerevisiae genomes deposited in the ncbi database .
a total of  <dig>  genes were predicted with an average length of  <dig>  kb, occupying  <dig>  % of the whole genome .table  <dig> chinese maotai-flavored liquor yeast mt <dig> library assembly statistics
n50a
n <dig> and n <dig> were obtained in the same way
an50: computed by sorting all contigs from largest to smallest and by determining the minimum set of contigs whose sizes total 50 % of the entire genome
within the aligned regions of the mt <dig> and s288c genomes, we identified  <dig>  high-confidence snps , including  <dig>  missense variants and  <dig>  synonymous variants .
remarkably, the level of nucleotide polymorphism observed between mt <dig> and s288c is very similar to that of yjm <dig> , a strain of s. cerevisiae isolated from the lung of an aids patient, and the wine yeast strain awri <dig>   <cit> , far higher than the biofuel producing industrial strain yjs <dig>  which was also isolated from china  <cit>  and the commercial wine yeast strain ec <dig>   <cit> , but far lower than that of the sake yeast strain k <dig>   <cit> , and the biofuel producing industrial strain jay <dig>   <cit> .
in addition, there were  <dig>  small indels , including  <dig>  homozygous indels and  <dig>  heterozygous indels .
within the annotated indels, there were  <dig> frameshift variants that resulted in feature elongation, and  <dig> frameshift variants that resulted in feature truncation .
the densities of snps and indels were far from constant across either the whole genome  or individual chromosomes .
this phenomenon mostly appears in genomes of industrial strains, such as the genome of k <dig>  awri <dig> and yjs <dig>  it is more likely that a complex history of out- and/or back-crossings of the ancestral strain in the industrial environments by environmental domestication and artificial breeding could have caused this pattern.
in addition,  <dig> copy number variations were obtained using cnvnator  <cit> .
they were comprised of  <dig> duplication regions and  <dig> deletion regions .
all of these results indicated that mt <dig> had a genome distinct from that of s288c.
aimed at detecting the evolutionary origin of mt <dig>  a neighbor-joining tree was constructed on the basis of whole genome and protein sequences.
the tree included mt <dig> and  <dig> other representative s. cerevisiae strains from library, clinic and sake, bioethanol, wine and beer production.
as shown in fig.
2b, mt <dig> is located far from the wine and beer strain clusters but adjacent to the sake yeast strain k <dig>  and they were both isolated in asia.fig.
<dig>
a genome-wide distribution of heterozygosity in the chinese maotai-flavored liquor yeast mt <dig> genome.
the frequency of the extracted heterozygosity was plotted using 10-kb windows of the chromosomal coordinates, where black vertical lines represent the division between two chromosomes.
heterozygous snps or indels in mt <dig> are shown in red and blue, respectively.
b phylogenetic tree generated by mega <dig>  <cit>  using the whole genome sequence of mt <dig> and the available saccharomyces cerevisiae genomes in ncbi.
different strains are represented in the diagrams by different shapes and colors.
red triangles, the typical chinese liquor fermentation strain mt1; yellow triangles, sake yeast strain; red rhombuses, clinical strains; white circles, library strains; aqua circles, bioethanol strain; blue circles, wine strains from europe; pink circles, wine strains from italy; and dark cyan circles, beer strains.
c functional classification of the specific genes in mt <dig>  d functional classification of the genes lost from mt1
among the  <dig>  genes of mt <dig>  less than  <dig> genes displayed similarity levels below 95 % when compared with s288c .
using an all-vs-all blastp and clustering analysis with orthomcl ,  <dig> mt1-specific genes were identified, 90 % of which had near-perfect-match hits to the nucleotide sequence of s288c, and likely reflect orf annotation differences, whereas others had nonreciprocal-best-hit homologs in s288c.
the functions of these genes fell within stress response , carbohydrate metabolic process , organic substance transport , biosynthetic process , organic substance metabolic process , and others .
in addition,  <dig> genes were missing from mt <dig> compared with s288c.
among them, there were  <dig> genes and  <dig> genes in the location of centromere and telomeres, respectively, where assemble errors happen frequently.
these genes are mic17p, vma <dig>  taf <dig>  tir <dig>  tir <dig>  prp <dig>  cox <dig>  cox <dig>  shu <dig>  mnn <dig>  rfa <dig>  puf <dig>  avt <dig>  ten <dig>  gat <dig>  cms <dig>  gat <dig>  sin <dig>  pfa <dig>  mcd <dig>  psf <dig>  pdr <dig>  rer <dig>  sgf <dig>  pau <dig>  pau <dig>  pau <dig>  pau <dig>  pau <dig>  pau <dig>  pau <dig>  pau <dig>  pau <dig>  pau <dig>  pau <dig>  pau <dig>  pau <dig>  pau <dig>  pau <dig>  rds <dig>  hsp <dig>  yps <dig>  aif <dig>  bds <dig> and enb <dig>  60 % of  <dig> missing genes were hypothetical or uncharacterized orfs encoding putative proteins of unknown functions as reported in the saccharomyces genome database.
the other 40 % of the genes were mainly involved in ion transport, regulation of transcription and translation, dna and rna binding and cellular component synthesis, such as membrane components, organelle organization and cellular protein components .
while there were only about  <dig> genes listed in s288c as essential, including dad <dig>  dad <dig>  dad <dig>  brr <dig>  rrp <dig>  esf <dig>  fap <dig>  uso <dig>  mcd <dig> and spc <dig>  function of each genes was listed in additional file 1: table s <dig>  as indicated by the functional classification of the specific and lost genes in mt <dig>  there is a speculation that mt <dig> may have a special genomic system.
it weakened transcriptional, translational and cytoplasmic synthesis.
on the other hand, it strengthened carbohydrate transport and metabolism, energy metabolism and informational processing  metabolism.
transcriptomic comparison using rna-seq
the transcriptomic comparison between mt <dig> and s288c was carried out in sorghum extract , which is the raw material for liquor-making.
the expression levels of genes related to carbohydrate and energy metabolism were significantly up-regulated in mt <dig> compared with in s288c .
within the carbohydrate metabolism, the most significant responsive pathway was the tricarboxylic acid cycle  with  <dig> up-regulated genes.
in addition, five genes in the pentose phosphate pathway  and four genes in galactose metabolism  were more highly expressed than in s288c.
in energy metabolism, oxidative phosphorylation  had the largest number of up-regulated genes.
nucleotide metabolism, translation, and cell growth and death were the responsive pathways that had a large number of down-regulated genes compared with in s288c .
ribosome  had the largest number of markedly lower expressed genes .
based on the analyses of the transcriptome, we predicted that, compared with other yeast strains, mt <dig> tends to make higher investments in carbon and energy metabolism than transcription and translation.
this is consistent with the corresponding speculation indicated by the functional classification of the specific and lost genes in mt <dig> table  <dig> main differences in the metabolic pathways of the chinese maotai-flavored liquor yeast mt <dig> compared with the saccharomyces cerevisiae type strain s288c when grown in sorghum juice medium, ph  <dig>
although there was no difference between the growth of mt <dig> and s288c at ph  <dig> , mt <dig> had a gradually increasing growth advantage as the ph decreased to  <dig>  .
s288c could not grow when the ph was below  <dig> , thus a ph level of  <dig>  was selected as the control condition, versus ph  <dig>  for transcriptomic comparisons, to identify the genetic mechanisms of acid tolerance.
then, the two strains were compared at ph levels between  <dig>  and  <dig> .
when the ph was reduced to  <dig>  from  <dig> , s288c genes in seven pathways were up-regulated , including nucleotide metabolism , amino acid metabolism , translation  and folding, sorting and degradation .
within energy metabolism in s288c, three genes were up-regulated, while two genes were down-regulated.
in addition, five carbohydrate metabolism genes were down-regulated.
however, there were  <dig> responsive pathways in the mt <dig> transcriptome, indicating that mt <dig> had a stronger response to acid than s288c.
the seven responsive pathways in s288c were also identified in mt <dig> but involved many more responsive genes.
in particular, the translational pathway in mt <dig> had  <dig> up-regulated genes.
the other responsive pathways that had up-regulated genes in mt <dig> were involved in transcription, replication and repair, environmental information processing, transport and catabolism, cell growth and death, and cell communication .
in addition, several metabolic pathways of mt <dig> had genes up-regulated, such as terpenoid and polyketide metabolism, xenobiotic biodegradation and metabolism, and carbohydrate metabolism.
in particular, terpenoids and polyketides are the important healthy active materials in chinese liquor.
as indicated by the transcriptomic comparison, the transcription- and translation-related genes of mt <dig>  which were inactive compared with those of s288c under normal conditions, were more active under acidic condition and achieved the same level of expression as in s288c.fig.
<dig> transcriptionally changed pathways of the saccharomyces cerevisiae type strain s288c  and the chinese maotai-flavored liquor yeast mt <dig>  when the ph of the sorghum juice medium was reduced to  <dig>  from  <dig>
under normal conditions , there were not only less genes related to transcription and translation in mt <dig> but they were also expressed at far lower levels than in s288c; however, mt <dig> could attain a greater biomass than s288c.
when the ph was reduced from  <dig>  to  <dig> , genes with functions related to transcription, replication and repair and, especially, translation in mt <dig> were strongly up-regulated.
within these, several genes are essential to acid ph resistance, such as cik <dig>  hsp <dig>  rps27b, rpl31a, rpl <dig> and aro <dig>  there may be two reasons for this strange phenomenon.
one is that mt <dig> requires less proteins and nucleotides for growth than s288c.
the other may be that genes involved in transcription, translation, replication and repair in mt <dig> are more efficient than those in s288c.
there is no need to mobilize a large number of related genes under suitable conditions.
once threatened, the cell will increase its investment to survive.
genetic mechanism of multi-carbon co-utilization
mt <dig> can uniquely assimilate several disaccharides, such as melibiose and maltose, and some monosaccharides, such as galactose.
however, the mt <dig> and s288c contain the same genes related to galactose utilization and its regulatory mechanism, including gal <dig>  gal <dig>  gal <dig>  gal <dig>  gal <dig> and others .
therefore, a transcriptomic comparison was carried out between the two strains.
it was discovered that several genes involved in galactose metabolism, such as yhr104w, yil162w, ima <dig> and others, in mt <dig> were up-regulated compared with those in s288c.
the enzyme of yhr104w, akr <dig> , is responsible for the transformation between d-galactose and galactitol, while yil162w encodes the enzyme saca  which is involved in the degradation of raffinose into melibiose or galactose.
the metabolic networks of melibiose and galactose are only different in the initial step, the intracellular breakdown of melibiose into glucose and galactose  <cit> .
this step involves an alpha-galactosidase encoded by mel <dig>  this important gene was identified in contig  <dig> of mt <dig> but does not exist in s288c.
it is most likely the reason for mt1’s specific ability to utilize melibiose.
this gene was identified in the genome of the laboratory strain, s. cerevisiae cen.pk113-7d  <cit> , a model strain for industrial biotechnology.
the enzyme system of maltose is a typical inducible system in yeast cells, which includes the maltose permease and maltase  <cit> .
the mal loci is an important gene in maltose metabolism and is composed of three genes: mals, malt and malr .
the malr genes encode maltose fermentation regulatory proteins that induce the expression of mals, encoding the maltase , and malt, encoding a high-affinity maltose transporter, maltose permease  <cit> .
these three kinds of genes were identified in the mt <dig> genome.
the two malt genes  and one mals gene  also exist and are functional in the genomic reference strain s288c.
the differences mainly exist in the regulatory protein encoding genes: mal <dig>  mal <dig> and mal <dig>  notably, mal <dig> is absent in the reference strain s288c, while mal <dig> and mal <dig> exist but are non-functional.
in addition, there were no differences in the expression levels of mal <dig> and mal <dig> between mt <dig> and s288c.
this suggests that mal <dig> might be the important gene of the mt1’s maltose metabolism pathway.
in contrast to s288c, strains from the cen.pk lineage are able to grow on maltose when it is the sole carbon source.
however, unlike mt <dig>  the mal <dig> mutant allele known as mal2-8c is responsible for the utilization of maltose in cen.pk strains  <cit> .fig.
<dig>
a metabolic pathways and gene regulatory mechanisms of galactose, melibiose and maltose.
the red parts of the pathways are specific to the chinese maotai-flavored liquor yeast mt <dig> strain.
b dynamic curves of fermentation parameters.
ypd medium ;  modified ypd medium ;  modified ypd medium .
the open figures represent the saccharomyces cerevisiae type strain s288c; the solid figures represent mt1
the transport of sugars across the cell membrane is important for growth and fermentation.
our analyses suggest that mt <dig> possesses different sugar uptake and assimilation properties than s288c.
mt <dig> contains  <dig> hxt family genes, encoding hexose transporter proteins.
among these genes, hxt <dig>  hxt <dig>  hxt <dig>  hxt <dig>  hxt <dig>  hxt <dig> and hxt <dig> in the mt <dig> genome were identified by local blast searches and confirmed by pcr testing.
the products of these genes show different glucose affinities and different expression levels to coordinately control glucose uptake in environments containing a broad range of glucose concentrations  <cit> .
when cells were collected for a transcriptomic analysis, we found that the expression levels of hxt <dig> and hxt <dig> in mt <dig> were much higher than in s288c, even though the glucose concentration in the sorghum extract adopted for the transcriptomic assay reached 70 g/l .
this differs from a conventional report  <cit>  that suggested that the two genes were repressed by high levels of glucose.
in particular, as the glucose concentration rose from  <dig>  to 40 %, the expression level of hxt <dig> decreased by 80 %.
the high expression levels of hxt <dig> and hxt <dig> indicate that mt <dig> may have no glucose repression and is able to simultaneously utilize multiple sugars.table  <dig> comparison of the expression levels of hxt genes between the chinese maotai-flavored liquor yeast mt <dig> and the saccharomyces cerevisiae type strain s288c when grown in sorghum juice medium
hxt1
hxt2
hxt 3
hxt5
hxt8
hxt9
hxt10
hxt13
hxt14
to verify the multi-carbon co-utilization without glucose repression, which was indicated by the genomic and transcriptomic analyses, cell growth, sugar consumption and ethanol production were compared between mt <dig> and s288c in media containing mixed sugars.
as shown in fig.
4b, when compared with s288c in 2 % glucose, mt <dig> grew faster but showed no difference in the glucose consumption and ethanol production.
however, in 1 % glucose plus 1 % maltose, mt <dig> could use maltose to grow and ferment, and consequently its biomass and ethanol yield were twice that of s288c.
this was also true in 1 % glucose plus 1 % galactose.
as indicated by the dynamic curves of sugar consumption , mt <dig> could utilize maltose and galactose in the early stages of the logarithmic phase without the phenomenon of secondary growth.
this was confirmed by the lack of a “lag” period in the growth of mt <dig> in a medium containing mixed sugars .
these results indicate that the high expression levels of hxt <dig> and hxt <dig> without glucose repression may contribute to the multi-carbon co-utilization.
other unique genes lead to unique phenotypes
khr <dig> is another unique gene of mt <dig>  encoding a heat-resistant killer toxin.
this gene is located in a 2-kb fragment inserted into mt <dig> scaffold  <dig>  which can be mapped to s288c chromosome ix.
this orf has been identified at the same location in the genome of yjm <dig>  <cit> .
the killer toxin provides mt <dig> with a stronger survival rate compared with other microorganisms in the chinese liquor fermentation environment.
in addition, two specific bio genes were found on contig <dig>  resulting in mt <dig> having biotin prototrophy.
the two bio genes share 100 % identity with bio <dig> and bio <dig>  which encode pimeloyl-coa synthase and kapa synthase  <cit> , respectively.
they are also conserved  in several other s. cerevisiae strains, such as sigma1278b , yjm <dig> and uc <dig> .
as shown in additional file 1: figure s <dig>  bio <dig> is located between esmt <dig> and mcm <dig>  while bio <dig> lies between axl <dig> and rev <dig>  compared with s288c, mt <dig> could grow in the medium without biotin .
this confirmed the biotin prototroph of mt <dig>
CONCLUSIONS
the physiological and biochemical characteristic diversity of microbes is very important for industrial applications.
our analysis indicated that mt <dig> not only had higher tolerances to acidity and ethanol but was also capable of simultaneously producing ethanol from various carbon sources without glucose repression.
these abilities could be applied to cellulose ethanol to eliminate the bottleneck during multi-carbon source fermentation.
in addition, mt <dig> is a biotin prototroph, a phenotypic trait with potential in industrial applications.
biotin  is an essential nutrient for all organisms, being a cofactor of many enzymes involved in carboxylation reactions, such as gluconeogenesis, amino acid metabolism, fatty acid biosynthesis and energy metabolism  <cit> .
these physiological and biochemical characteristics place mt <dig> in a special category of yeast desirable for industry.
revealing the genetic basis of these traits would provide a platform for not only understanding chinese liquor but also for developing an economically advanced biofuel process.
it is necessary to analyze the evolutionary mechanisms of such special phenotypic and physiological characteristics.
we assume that these variances may be induced by the raw materials and environmental stresses in the process of chinese liquor-making.
the ssf technique used in making chinese liquor combines the enzymatic hydrolysis of starch with the simultaneous fermentation of sugars.
diverse hydrolases produced by the complex filamentous fungal community generate various sugars.
this results in various carbohydrates that can be utilized by cells to grow and produce ethanol.
in addition, chinese maotai-flavored liquor is produced by a complicated ssf process in an extremely severe environment, with high temperatures, as well as acidic and ethanol stresses.
in the environment, temperatures could reach 50 °c, the ph could be as low as  <dig> , and the ethanol concentration could be as high as  <dig>  %.
over time, this specific environment has produced specific physiological properties and performances.
environmental pressure is the external induction force, while the specific genome contains the internal underlying determinants.
the genomic structural analysis indicated that mt <dig> retained a smaller genome than the reference strain s288c and some other industrial strains .
the genome of mt <dig> has lost several segments mostly comprised of non-coding protein genes, such as the large missing fragment of 25 kb on chromosome i , which was verified by pcr .
in s288c, this segment contains only one functional gene, flo <dig>  mt <dig> also lost hundreds of genes whose functions fell within transcription, translation, dna and rna binding, and cellular component synthesis.
in addition, mt <dig> gained several specific genes, such as mel <dig> and mal <dig>  related to its multi-carbon co-utilization, and khr <dig>  associated with its competitiveness.
these genomic adaptations embody the economy of the mt <dig> strain: removing some redundant genes, retaining necessary genes for growth and fermentation, and adapting to the complex fermentation environment of chinese liquor.
consequently, these alterations make mt <dig> a naturally ascendant strain with a minimal genome, containing special functional genes/clusters  <cit> .
the small genome has a higher economic efficiency.
it may have been beneficial to reduce the metabolic redundancy and then improve the metabolic efficiency  <cit> .
therefore, mt <dig> has the advantages of a short growth cycle, high production efficiency, high conversion rate, and strain stability in the fermentation process.
various mechanisms are known to be involved in the adaptive evolution of yeast to the fermentation process, such as gene duplication, polyploidy, chromosomal rearrangements, interspecific hybridization and introgression  <cit> .
on the basis of diverse examples drawn from both mammalian and microbial genetics, maynard v. olson proposed the “less is more” hypothesis that states that genetic loss may be an important engine of evolutionary change  <cit> .
aravind et al.
<cit>  and braun et al.
<cit>  revealed that s. cerevisiae may have lost at least  <dig> genes, and another  <dig> or so genes have become highly diverged, since their split from their common ancestor with saccharomyces pombe.
a notable feature of the lost genes is the co-elimination of functionally connected groups of proteins, such as the signalosome and the spliceosome components.
the other missing genes are involved in basic cellular processes, translation and ion homeostasis.
however, intriguingly, some of the strains in which genes had been disrupted grew better than wild-type strains on a rich medium  <cit> .
the counterintuitive idea that “less is more”, is well-reflected in the evolution of mt <dig>  the gene loss in mt <dig> has resulted in no obvious disadvantages in growth and fermentation, but provide mt <dig> the advantages of a short growth cycle and strain stability.
furthermore, a speculative hypothesis is proposed that mt <dig> gained the genomic space to obtain special functional genes  by reducing the gene redundancy.
this strategy of evolution by gene loss is an important aspect of yeast diversification and may play a major role in their adaptation to the chinese liquor fermentation ecosystem.
by revealing the genetic basis of specific phenotypes and elucidating the evolutionary history of the chinese maotai-flavored liquor yeast mt <dig>  this work enriches our knowledge of s. cerevisiae, especially with regard to minimal genomic research.
furthermore, this functional genomics study of industrial microorganisms could be beneficial in improving breeding strategies to obtain the desired production traits in industrial microorganisms.

 the chinese liquor-making process is a unique and complicated spontaneous fermentation process under solid state conditions. chinese liquor is produced by the technique of simultaneous saccharification and fermentation using grains, such as sorghum, wheat and barley. the cost-effective ssf combines the enzymatic hydrolysis of starch with the simultaneous fermentation of sugars, resulting in an outpouring carbon source. in addition, chinese liquor is fermented using combinations of microorganisms, including yeast, bacteria and filamentous fungi. the complex filamentous fungal community produces diversified hydrolases to degrade starch into various sugars, including glucose, galactose, maltose and melibiose. high temperatures at the starter are generated by the metabolic activities of the microorganisms and the low thermal conductivities of the solid matrices. in addition, there are two other prominent pressures: acidity stress and ethanol stress in the alcoholic fermentation stage. such a severe environment during the ssf process leads to a distinctive community of microorganisms with specific physiological properties and performances, in particular, distinctive heat- and acid-resistant properties. although the microorganisms in the chinese maotai-flavored liquor have been investigated since the 1960s, there is little information on the functional genomics of the yeast involved in the process. this has inhibited our understanding of the fermentation mechanism and the development of maotai-flavored liquor. functional genomics approaches, such as whole-genome sequencing, are powerful tools for the analysis of specific traits on a genomic scale and provide a method of studying cellular physiology from micro-perspective. since the first complete genome of strain s288c was sequenced in, it has served as the reference for the saccharomyces cerevisiae species and has promoted the development of whole-genome approaches. meanwhile, the genomes of several s. cerevisiae strains have been sequenced and compared with the s288c reference genome, including laboratory strains and industrial strains, such as sake yeast k wine yeasts, beer yeasts and bioethanol yeasts. the phenotypic diversity among yeast isolates is significant, and variation is apparent among the surveyed strains at different levels. ecological factors and geographical locations, as the main reasons for biodiversity, have important effects on the domestication of microbial physiological characteristics. among the strains with sequenced genomes in the ncbi database, there are only three strains from china. however, strains involved in the ssf process of chinese liquor are poorly understood. theoretically, the genomic analysis of strains with different backgrounds should help identify the sequence changes that play important roles in specific physiological characteristics. comparisons of the publicly available s. cerevisiae genome sequences have revealed the clear signatures in the different strains. compared with laboratory strains, industrial strains generally show a higher adaptability to specific environments. however, the genetic basis for their improved characteristics is not well understood. further studies are needed to explore how the genetic variations confer the specific phenotypes. the comprehensive identification of polymorphisms among individuals within a species is essential for studying the genetic basis of phenotypic differences and for elucidating the evolutionary history of the species. in this study, s. cerevisiae mt was isolated from the fermentation environment of chinese maotai-flavored liquor. its physiological characteristics were evaluated in several phenotypic assays. meanwhile, the mt genome was sequenced and compared with the type strain s288c. in addition, we elucidated how genetic variations of mt were correlated with specified traits using rna-seq and revealed the molecular mechanisms related to the adaptation of yeast in chinese liquor-making. this whole-genome analysis of the s. cerevisiae strain used in chinese liquor-making will supplement the genomic and phylogenetic knowledge of yeast and provide a guide for the construction of strains with desired traits.  phenotypic characterization several physiological and biochemical characteristics of the s. cerevisiae strain mt isolated during the process chinese maotai-flavored liquor-making, were compared with those of the reference strain s288c and other industrial strains. in addition to the sake yeast k other strains had high tolerance to 38 °c which is consistent with previous studies that it was widely believed that s. cerevisiae had a thermal tolerance of 25–37 °c or 39 °c. while a higher thermal resistance was found for mt when temperature was higher than 38 °c compared with not only s288c but also other industrial yeasts. population of mt was twice that of other strains in 16 % ethanol. it indicated that mt had a higher ethanol tolerance. in addition, mt had a growth advantage compared with other strains at ph  –. in particular, the population of mt is almost three times of that of s288c at ph . this suggested that mt has a much higher resistance to acidity than s288c and other industrial strains, as most industrial strains could barely grow at ph .fig. a effects of temperature, ethanol and acidity on cell growth of several yest strains. s, saccharomyces cerevisiae type strain s288c; m, chinese maotai-flavored liquor yeast mt1; k, sake yeast strain k7; h, hangjiu yeast cicc30394; w, wine yeast strain cicc31089; b, beer yeast strain atcc * represents the significant difference with p-value < , compared with mt calculated using a one-way analysis of variance in spss. b growth differences between mt and s288c with various carbon sources. left: oxidation tests that detect carbon utilization via a redox color change in the organism’s suspension; right: assimilation reaction tests that are based on turbidity increases due to carbon utilization of the organisms. monosaccharides;, disaccharides;, trisaccharides;, oligosaccharides;, acids;, alcohols; and, others carbon source utilization exerts a strong influence on the classification of isolates and may be a useful and functional measure for the characterization and classification of different strains. unfortunately, there is limited research on the simultaneous fermentation of various carbon sources to produce ethanol. the primary reason is that not many strains can ferment ethanol from various sugars. presently, there is some research on a strain that could simultaneous utilize glucose and xylose, or arabinose and galactose. mt1s ability to uptake and assimilate sugars was also investigated. among the monosaccharides, glucose could be used for growth and fermentation by both mt and s288c, with od and od values exceeding. when galactose was the sole carbon source, the od and od of mt reached and, respectively, while those of s288c were less than. this indicated that mt could uniquely utilize galactose. the other carbon sources that mt could uniquely uptake and assimilate were mainly disaccharides, such as melibiose, maltose, trehalose and turhanose. in addition, raffinose, a trisaccharide constituted by galactose, fructose and glucose, as well as methyl-a-d-glucopyranoside, are well utilized by mt with od values reaching and, respectively. these results suggested that mt was a strain appropriate for multi-carbon fermentation, having the ability to utilize various carbon sources. mt genome structure the mt genome assembly resulted in contigs with an n value of. then, scaffolds were obtained with an n value of. the assembled data were evaluated, and the detailed statistical data are shown in table  finally, the mt assembled genome was mbp with a gc% of  %. the genome size of mt is smaller than most of the s. cerevisiae genomes deposited in the ncbi database. a total of genes were predicted with an average length of  kb, occupying  % of the whole genome.table chinese maotai-flavored liquor yeast mt library assembly statistics n50a n and n were obtained in the same way an50: computed by sorting all contigs from largest to smallest and by determining the minimum set of contigs whose sizes total 50 % of the entire genome within the aligned regions of the mt and s288c genomes, we identified high-confidence snps, including missense variants and synonymous variants. remarkably, the level of nucleotide polymorphism observed between mt and s288c is very similar to that of yjm, a strain of s. cerevisiae isolated from the lung of an aids patient, and the wine yeast strain awri, far higher than the biofuel producing industrial strain yjs which was also isolated from china and the commercial wine yeast strain ec, but far lower than that of the sake yeast strain k, and the biofuel producing industrial strain jay. in addition, there were small indels, including homozygous indels and heterozygous indels. within the annotated indels, there were frameshift variants that resulted in feature elongation, and frameshift variants that resulted in feature truncation. the densities of snps and indels were far from constant across either the whole genome or individual chromosomes. this phenomenon mostly appears in genomes of industrial strains, such as the genome of k awri and yjs it is more likely that a complex history of out- and/or back-crossings of the ancestral strain in the industrial environments by environmental domestication and artificial breeding could have caused this pattern. in addition, copy number variations were obtained using cnvnator. they were comprised of duplication regions and deletion regions. all of these results indicated that mt had a genome distinct from that of s288c. aimed at detecting the evolutionary origin of mt a neighbor-joining tree was constructed on the basis of whole genome and protein sequences. the tree included mt and other representative s. cerevisiae strains from library, clinic and sake, bioethanol, wine and beer production. as shown in fig. 2b, mt is located far from the wine and beer strain clusters but adjacent to the sake yeast strain k and they were both isolated in asia.fig. a genome-wide distribution of heterozygosity in the chinese maotai-flavored liquor yeast mt genome. the frequency of the extracted heterozygosity was plotted using 10-kb windows of the chromosomal coordinates, where black vertical lines represent the division between two chromosomes. heterozygous snps or indels in mt are shown in red and blue, respectively. b phylogenetic tree generated by mega using the whole genome sequence of mt and the available saccharomyces cerevisiae genomes in ncbi. different strains are represented in the diagrams by different shapes and colors. red triangles, the typical chinese liquor fermentation strain mt1; yellow triangles, sake yeast strain; red rhombuses, clinical strains; white circles, library strains; aqua circles, bioethanol strain; blue circles, wine strains from europe; pink circles, wine strains from italy; and dark cyan circles, beer strains. c functional classification of the specific genes in mt d functional classification of the genes lost from mt1 among the genes of mt less than genes displayed similarity levels below 95 % when compared with s288c. using an all-vs-all blastp and clustering analysis with orthomcl, mt1-specific genes were identified, 90 % of which had near-perfect-match hits to the nucleotide sequence of s288c, and likely reflect orf annotation differences, whereas others had nonreciprocal-best-hit homologs in s288c. the functions of these genes fell within stress response, carbohydrate metabolic process, organic substance transport, biosynthetic process, organic substance metabolic process, and others. in addition, genes were missing from mt compared with s288c. among them, there were genes and genes in the location of centromere and telomeres, respectively, where assemble errors happen frequently. these genes are mic17p, vma taf tir tir prp cox cox shu mnn rfa puf avt ten gat cms gat sin pfa mcd psf pdr rer sgf pau pau pau pau pau pau pau pau pau pau pau pau pau pau pau rds hsp yps aif bds and enb 60 % of missing genes were hypothetical or uncharacterized orfs encoding putative proteins of unknown functions as reported in the saccharomyces genome database. the other 40 % of the genes were mainly involved in ion transport, regulation of transcription and translation, dna and rna binding and cellular component synthesis, such as membrane components, organelle organization and cellular protein components. while there were only about genes listed in s288c as essential, including dad dad dad brr rrp esf fap uso mcd and spc function of each genes was listed in additional file 1: table s as indicated by the functional classification of the specific and lost genes in mt there is a speculation that mt may have a special genomic system. it weakened transcriptional, translational and cytoplasmic synthesis. on the other hand, it strengthened carbohydrate transport and metabolism, energy metabolism and informational processing metabolism. transcriptomic comparison using rna-seq the transcriptomic comparison between mt and s288c was carried out in sorghum extract, which is the raw material for liquor-making. the expression levels of genes related to carbohydrate and energy metabolism were significantly up-regulated in mt compared with in s288c. within the carbohydrate metabolism, the most significant responsive pathway was the tricarboxylic acid cycle with up-regulated genes. in addition, five genes in the pentose phosphate pathway and four genes in galactose metabolism were more highly expressed than in s288c. in energy metabolism, oxidative phosphorylation had the largest number of up-regulated genes. nucleotide metabolism, translation, and cell growth and death were the responsive pathways that had a large number of down-regulated genes compared with in s288c. ribosome had the largest number of markedly lower expressed genes. based on the analyses of the transcriptome, we predicted that, compared with other yeast strains, mt tends to make higher investments in carbon and energy metabolism than transcription and translation. this is consistent with the corresponding speculation indicated by the functional classification of the specific and lost genes in mt table main differences in the metabolic pathways of the chinese maotai-flavored liquor yeast mt compared with the saccharomyces cerevisiae type strain s288c when grown in sorghum juice medium, ph  although there was no difference between the growth of mt and s288c at ph , mt had a gradually increasing growth advantage as the ph decreased to. s288c could not grow when the ph was below, thus a ph level of was selected as the control condition, versus ph  for transcriptomic comparisons, to identify the genetic mechanisms of acid tolerance. then, the two strains were compared at ph levels between and. when the ph was reduced to from, s288c genes in seven pathways were up-regulated, including nucleotide metabolism, amino acid metabolism, translation and folding, sorting and degradation. within energy metabolism in s288c, three genes were up-regulated, while two genes were down-regulated. in addition, five carbohydrate metabolism genes were down-regulated. however, there were responsive pathways in the mt transcriptome, indicating that mt had a stronger response to acid than s288c. the seven responsive pathways in s288c were also identified in mt but involved many more responsive genes. in particular, the translational pathway in mt had up-regulated genes. the other responsive pathways that had up-regulated genes in mt were involved in transcription, replication and repair, environmental information processing, transport and catabolism, cell growth and death, and cell communication. in addition, several metabolic pathways of mt had genes up-regulated, such as terpenoid and polyketide metabolism, xenobiotic biodegradation and metabolism, and carbohydrate metabolism. in particular, terpenoids and polyketides are the important healthy active materials in chinese liquor. as indicated by the transcriptomic comparison, the transcription- and translation-related genes of mt which were inactive compared with those of s288c under normal conditions, were more active under acidic condition and achieved the same level of expression as in s288c.fig. transcriptionally changed pathways of the saccharomyces cerevisiae type strain s288c and the chinese maotai-flavored liquor yeast mt when the ph of the sorghum juice medium was reduced to from under normal conditions, there were not only less genes related to transcription and translation in mt but they were also expressed at far lower levels than in s288c; however, mt could attain a greater biomass than s288c. when the ph was reduced from to, genes with functions related to transcription, replication and repair and, especially, translation in mt were strongly up-regulated. within these, several genes are essential to acid ph resistance, such as cik hsp rps27b, rpl31a, rpl and aro there may be two reasons for this strange phenomenon. one is that mt requires less proteins and nucleotides for growth than s288c. the other may be that genes involved in transcription, translation, replication and repair in mt are more efficient than those in s288c. there is no need to mobilize a large number of related genes under suitable conditions. once threatened, the cell will increase its investment to survive. genetic mechanism of multi-carbon co-utilization mt can uniquely assimilate several disaccharides, such as melibiose and maltose, and some monosaccharides, such as galactose. however, the mt and s288c contain the same genes related to galactose utilization and its regulatory mechanism, including gal gal gal gal gal and others. therefore, a transcriptomic comparison was carried out between the two strains. it was discovered that several genes involved in galactose metabolism, such as yhr104w, yil162w, ima and others, in mt were up-regulated compared with those in s288c. the enzyme of yhr104w, akr, is responsible for the transformation between d-galactose and galactitol, while yil162w encodes the enzyme saca which is involved in the degradation of raffinose into melibiose or galactose. the metabolic networks of melibiose and galactose are only different in the initial step, the intracellular breakdown of melibiose into glucose and galactose. this step involves an alpha-galactosidase encoded by mel this important gene was identified in contig of mt but does not exist in s288c. it is most likely the reason for mt1’s specific ability to utilize melibiose. this gene was identified in the genome of the laboratory strain, s. cerevisiae cen.pk113-7d, a model strain for industrial biotechnology. the enzyme system of maltose is a typical inducible system in yeast cells, which includes the maltose permease and maltase. the mal loci is an important gene in maltose metabolism and is composed of three genes: mals, malt and malr. the malr genes encode maltose fermentation regulatory proteins that induce the expression of mals, encoding the maltase, and malt, encoding a high-affinity maltose transporter, maltose permease. these three kinds of genes were identified in the mt genome. the two malt genes and one mals gene also exist and are functional in the genomic reference strain s288c. the differences mainly exist in the regulatory protein encoding genes: mal mal and mal notably, mal is absent in the reference strain s288c, while mal and mal exist but are non-functional. in addition, there were no differences in the expression levels of mal and mal between mt and s288c. this suggests that mal might be the important gene of the mt1’s maltose metabolism pathway. in contrast to s288c, strains from the cen.pk lineage are able to grow on maltose when it is the sole carbon source. however, unlike mt the mal mutant allele known as mal2-8c is responsible for the utilization of maltose in cen.pk strains.fig. a metabolic pathways and gene regulatory mechanisms of galactose, melibiose and maltose. the red parts of the pathways are specific to the chinese maotai-flavored liquor yeast mt strain. b dynamic curves of fermentation parameters. ypd medium; modified ypd medium; modified ypd medium. the open figures represent the saccharomyces cerevisiae type strain s288c; the solid figures represent mt1 the transport of sugars across the cell membrane is important for growth and fermentation. our analyses suggest that mt possesses different sugar uptake and assimilation properties than s288c. mt contains hxt family genes, encoding hexose transporter proteins. among these genes, hxt hxt hxt hxt hxt hxt and hxt in the mt genome were identified by local blast searches and confirmed by pcr testing. the products of these genes show different glucose affinities and different expression levels to coordinately control glucose uptake in environments containing a broad range of glucose concentrations. when cells were collected for a transcriptomic analysis, we found that the expression levels of hxt and hxt in mt were much higher than in s288c, even though the glucose concentration in the sorghum extract adopted for the transcriptomic assay reached 70 g/l. this differs from a conventional report that suggested that the two genes were repressed by high levels of glucose. in particular, as the glucose concentration rose from to 40 %, the expression level of hxt decreased by 80 %. the high expression levels of hxt and hxt indicate that mt may have no glucose repression and is able to simultaneously utilize multiple sugars.table comparison of the expression levels of hxt genes between the chinese maotai-flavored liquor yeast mt and the saccharomyces cerevisiae type strain s288c when grown in sorghum juice medium hxt1 hxt2 hxt 3 hxt5 hxt8 hxt9 hxt10 hxt13 hxt14 to verify the multi-carbon co-utilization without glucose repression, which was indicated by the genomic and transcriptomic analyses, cell growth, sugar consumption and ethanol production were compared between mt and s288c in media containing mixed sugars. as shown in fig. 4b, when compared with s288c in 2 % glucose, mt grew faster but showed no difference in the glucose consumption and ethanol production. however, in 1 % glucose plus 1 % maltose, mt could use maltose to grow and ferment, and consequently its biomass and ethanol yield were twice that of s288c. this was also true in 1 % glucose plus 1 % galactose. as indicated by the dynamic curves of sugar consumption, mt could utilize maltose and galactose in the early stages of the logarithmic phase without the phenomenon of secondary growth. this was confirmed by the lack of a “lag” period in the growth of mt in a medium containing mixed sugars. these results indicate that the high expression levels of hxt and hxt without glucose repression may contribute to the multi-carbon co-utilization. other unique genes lead to unique phenotypes khr is another unique gene of mt encoding a heat-resistant killer toxin. this gene is located in a 2-kb fragment inserted into mt scaffold which can be mapped to s288c chromosome ix. this orf has been identified at the same location in the genome of yjm. the killer toxin provides mt with a stronger survival rate compared with other microorganisms in the chinese liquor fermentation environment. in addition, two specific bio genes were found on contig resulting in mt having biotin prototrophy. the two bio genes share 100 % identity with bio and bio which encode pimeloyl-coa synthase and kapa synthase, respectively. they are also conserved in several other s. cerevisiae strains, such as sigma1278b, yjm and uc. as shown in additional file 1: figure s bio is located between esmt and mcm while bio lies between axl and rev compared with s288c, mt could grow in the medium without biotin. this confirmed the biotin prototroph of mt  the physiological and biochemical characteristic diversity of microbes is very important for industrial applications. our analysis indicated that mt not only had higher tolerances to acidity and ethanol but was also capable of simultaneously producing ethanol from various carbon sources without glucose repression. these abilities could be applied to cellulose ethanol to eliminate the bottleneck during multi-carbon source fermentation. in addition, mt is a biotin prototroph, a phenotypic trait with potential in industrial applications. biotin is an essential nutrient for all organisms, being a cofactor of many enzymes involved in carboxylation reactions, such as gluconeogenesis, amino acid metabolism, fatty acid biosynthesis and energy metabolism. these physiological and biochemical characteristics place mt in a special category of yeast desirable for industry. revealing the genetic basis of these traits would provide a platform for not only understanding chinese liquor but also for developing an economically advanced biofuel process. it is necessary to analyze the evolutionary mechanisms of such special phenotypic and physiological characteristics. we assume that these variances may be induced by the raw materials and environmental stresses in the process of chinese liquor-making. the ssf technique used in making chinese liquor combines the enzymatic hydrolysis of starch with the simultaneous fermentation of sugars. diverse hydrolases produced by the complex filamentous fungal community generate various sugars. this results in various carbohydrates that can be utilized by cells to grow and produce ethanol. in addition, chinese maotai-flavored liquor is produced by a complicated ssf process in an extremely severe environment, with high temperatures, as well as acidic and ethanol stresses. in the environment, temperatures could reach 50 °c, the ph could be as low as, and the ethanol concentration could be as high as  %. over time, this specific environment has produced specific physiological properties and performances. environmental pressure is the external induction force, while the specific genome contains the internal underlying determinants. the genomic structural analysis indicated that mt retained a smaller genome than the reference strain s288c and some other industrial strains. the genome of mt has lost several segments mostly comprised of non-coding protein genes, such as the large missing fragment of 25 kb on chromosome i, which was verified by pcr. in s288c, this segment contains only one functional gene, flo mt also lost hundreds of genes whose functions fell within transcription, translation, dna and rna binding, and cellular component synthesis. in addition, mt gained several specific genes, such as mel and mal related to its multi-carbon co-utilization, and khr associated with its competitiveness. these genomic adaptations embody the economy of the mt strain: removing some redundant genes, retaining necessary genes for growth and fermentation, and adapting to the complex fermentation environment of chinese liquor. consequently, these alterations make mt a naturally ascendant strain with a minimal genome, containing special functional genes/clusters. the small genome has a higher economic efficiency. it may have been beneficial to reduce the metabolic redundancy and then improve the metabolic efficiency. therefore, mt has the advantages of a short growth cycle, high production efficiency, high conversion rate, and strain stability in the fermentation process. various mechanisms are known to be involved in the adaptive evolution of yeast to the fermentation process, such as gene duplication, polyploidy, chromosomal rearrangements, interspecific hybridization and introgression. on the basis of diverse examples drawn from both mammalian and microbial genetics, maynard v. olson proposed the “less is more” hypothesis that states that genetic loss may be an important engine of evolutionary change. aravind et al. and braun et al. revealed that s. cerevisiae may have lost at least genes, and another or so genes have become highly diverged, since their split from their common ancestor with saccharomyces pombe. a notable feature of the lost genes is the co-elimination of functionally connected groups of proteins, such as the signalosome and the spliceosome components. the other missing genes are involved in basic cellular processes, translation and ion homeostasis. however, intriguingly, some of the strains in which genes had been disrupted grew better than wild-type strains on a rich medium. the counterintuitive idea that “less is more”, is well-reflected in the evolution of mt the gene loss in mt has resulted in no obvious disadvantages in growth and fermentation, but provide mt the advantages of a short growth cycle and strain stability. furthermore, a speculative hypothesis is proposed that mt gained the genomic space to obtain special functional genes by reducing the gene redundancy. this strategy of evolution by gene loss is an important aspect of yeast diversification and may play a major role in their adaptation to the chinese liquor fermentation ecosystem. by revealing the genetic basis of specific phenotypes and elucidating the evolutionary history of the chinese maotai-flavored liquor yeast mt this work enriches our knowledge of s. cerevisiae, especially with regard to minimal genomic research. furthermore, this functional genomics study of industrial microorganisms could be beneficial in improving breeding strategies to obtain the desired production traits in industrial microorganisms. 
based on a functional genomics analysis, we revealed the genetic basis and evolutionary mechanisms underlying the traits of the chinese maotai-flavored yeast mt this work provides new insights for the genetic breeding of yeast and also enriches the genetic resources of yeast. compared with the type strain s288c, mt can tolerate high acidity, high ethanol levels and high temperatures. revealing genetic mechanisms behind specific physiological characteristics of saccharomyces cerevisiae from specific environments is important for industrial applications and requires precise understanding. maotai strain mt was isolated from the complicated chinese maotai-flavored liquor-making environment with extremely high temperatures, and acidic and ethanol stresses. in addition, mt can simultaneously utilize various sugars, including glucose, sucrose, galactose, maltose, melibiose, trehalose, raffinose and turanose.
 revealing genetic mechanisms behind specific physiological characteristics of saccharomyces cerevisiae from specific environments is important for industrial applications and requires precise understanding.  maotai strain mt was isolated from the complicated chinese maotai-flavored liquor-making environment with extremely high temperatures, and acidic and ethanol stresses. compared with the type strain s288c, mt can tolerate high acidity, high ethanol levels and high temperatures. in addition, mt can simultaneously utilize various sugars, including glucose, sucrose, galactose, maltose, melibiose, trehalose, raffinose and turanose. genomic comparisons identified a distinct mt genome,  mb smaller than that of s288c. there are mt1-specific genes that are not in s288c, including mel mal khr bio and bio a transcriptional comparison indicated that hxt and hxt which are theoretically repressed by glucose, were no longer inhibited in mt and were highly expressed even in a medium containing 70 g/l glucose. thus, other sugars may be co-utilized with glucose by mt without diauxic growth.  based on a functional genomics analysis, we revealed the genetic basis and evolutionary mechanisms underlying the traits of the chinese maotai-flavored yeast mt this work provides new insights for the genetic breeding of yeast and also enriches the genetic resources of yeast. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords saccharomyces cerevisiaegenometranscriptomemaotai-flavour liquornational high technology research and development program of china2013aa102108; 2012aa021301wu qun national natural science foundation of china31371822; 31271921wu qun china postdoctoral science foundation2014m550265wu qun priority academic program development of jiangsu higher education institutions, the projectno. 111-2-06wu qun issue-copyright-statement© the author 2015 