archaea and bacteria were also believed to synthesize lysine through diaminopimelate until it was reported that the extremely thermophilic bacterium thermus thermophilus synthesizes lysine through -aminoadipate [ 58 ] . during lysine biosynthesis in the budding yeast saccharomyces
cerevisiae , -aminoadipate is synthesized from 2-oxoglutarate and acetyl - coa by the enzymes lys20 or lys21 ( homocitrate synthase [ hcs ] ) , lys4 ( homoaconitase ) , lys12 ( homoisocitrate dehydrogenase ) , and -aminoadipate aminotransferase .
lysine is synthesized from -aminoadipate by the enzymes lys2 ( aminoadipate reductase ) , lys5 ( phosphopantetheinyl transferase which posttranslationally modifies lys2 ) , lys9 ( saccharopine dehydrogenase , glutamate forming ) , and lys1 ( saccharopine dehydrogenase , lysine forming ) [ 1 , 4 ] .
it has been unclear why s. cerevisiae has 2 hcss ( lys20 and lys21 ) .
for example , homocitrate is mainly synthesized through lys21 during growth on ethanol , while under fermentative metabolism , lys20 and lys21 play redundant roles .
it was recently reported that lys20 of s. cerevisiae functions in nuclear activities involving chromatin regulation that are distinct from its previously established role in lysine synthesis .
lys20 of s. cerevisiae is linked to the dna damage repair process via the histone acetyltransferase esa1 and the h2a.z histone variant .
i selected 71 hcss ( 31 from saccharomycotina species , 30 from pezizomycotina species , 2 from taphrinomycotina species , and 8 from basidiomycota species ) based on blastp results in the fungal genome database at ncbi ( http://www.ncbi.nlm.nih.gov/projects/genome/guide/fungi/ ) .
multiple alignments were generated with clustal w. a maximum likelihood tree was reconstructed using mega version 5 .
the -distributed rate was considered , and the number of discrete gamma categories was 3 .
the nucleosome position profiles were compared between the promoter ( 1000 bases upstream of the translational start site ) and coding regions ( between the translational start and end site ) of the hcs genes , according to a previously described method .
similarity between the two nucleosome position profiles was estimated using the spearman 's rank correlation coefficient .
the hcs phylogenetic tree ( figure 1 ) indicates that the hcs gene has been duplicated multiple times in the course of ascomycete evolution .
the 31 hcss of the saccharomycotina species ( ascomycetous yeasts ) are encoded in 17 organisms .
in contrast , the 30 hcss of the pezizomycotina species ( filamentous ascomycetes ) are encoded in 28 organisms .
thus , 14 of the 17 saccharomycotina species and 2 of the 28 pezizomycotina species have 2 hcss ( figure 1 ) .
gene duplication is not found in lys1 , lys2 , lys5 , lys9 , and their homologues .
in addition , no duplication was found in lys4 , lys12 , and their homologues ( data not shown ) .
therefore , among the fungal lysine biosynthesis - related genes , only the hcs gene has been duplicated .
phylogenetic analysis of hcss in ascomycetous yeasts showed that the s. cerevisiae hcss ( lys20 and lys21 ) are most closely related to each other ( figure 1 ) , suggesting that hcs gene duplication occurred during evolution of the genus saccharomyces . on the other hand ,
all saccharomycotina species except ashbya gossypii , vanderwaltozyma polyspora , and yarrowia lipolytica have duplicated hcs genes ( figure 1 ) .
thus , hcs gene duplication may be related to genome duplication events in saccharomycotina [ 1618 ] .
in addition to the phylogenetic analysis based on hcs amino acid sequences , i compared the nucleosome positioning of lys20 and lys21 .
interestingly , nucleosomes were mapped to the hcs gene promoters more often than to the coding regions ( figure 2 ) .
nucleosome position profiles in the coding regions were highly correlated ( spearman 's rank correlation coefficient = 0.833 ) between lys20 and lys21 .
on the other hand , those in the gene promoter regions were poorly correlated ( spearman 's rank correlation coefficient = 0.396 ) .
this result suggests that these 2 hcs genes have different regulatory systems . on the other hand ,
lys20 expression is most similar to lys21 expression , and lys21 is most similar to lys20 expression , based on the spell version 2.0.2 .
in addition , recent comparative analyses of orthologous genes in evolutionarily close yeasts indicated that divergence of nucleosome positioning is not correlated with divergence of gene expression [ 20 , 21 ] . although hcs ( lys20 and lys21 ) is located in the nucleus of s. cerevisiae , hcs is located in the cytoplasm of penicillium chrysogenum [ 23 , 24 ] .
the phylogenetic tree ( figure 1 ) showed that gene duplication is not found in basidiomycota and taphrinomycotina .
in addition , gene duplication has occurred rarely in pezizomycotina , suggesting that a common ancestor of the dikarya lacked the nuclear function of chromatin regulation .
considering that duplication of the hcs gene occurred in a limited number of ascomycetes , it may not be an essential event in the evolution of dikarya .
i hypothesize that after divergence of the phyla ascomycota and basidiomycota , s. cerevisiae obtained hcs bifunctionality .
 most ascomycetous yeasts have 2 homocitrate synthases ( hcss ) . among the fungal lysine 
 biosynthesis - related genes , only the hcs gene was duplicated in the course of evolution . 
 it was recently reported that hcs of saccharomyces cerevisiae has an additional function in nuclear activities involving chromatin regulation related to dna damage repair , which is not related to lysine biosynthesis . 
 thus , it is possible that the bifunctionality is associated with hcs gene duplication . 
 phylogenetic analysis showed that duplication has occurred multiple times during evolution of the ascomycetous yeasts . 
 it is likely that the hcs gene duplication in s. cerevisiae occurred in the course of saccharomyces evolution . although the nucleosome position profiles of the two s. cerevisiae hcs genes were similar in the coding regions , they were different in the promoter regions , suggesting that they are subject to different regulatory controls . 
 s. cerevisiae has maintained hcs activity for lysine biosynthesis and has obtained bifunctionality . 
1. Introduction
2. Materials and Methods
3. Results and Discussion