Ș̚ĥʯƊ̵Ȃ Moniliella megachiliensis % 9 VaVƆʃ"tG†€Ɋɋ

Ș̚ĥʯƊ̵Ȃ Moniliella megachiliensis % 9 VaVƆʃ"tG†€Ɋɋ

2013

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ʁ 1ɾɾ Moniliella megachiliensis%% 99EEVVaa††€€ɊɊƓƓ̵̵͈͈̟̟ʔʔ Erythrose reductasḙ̭««ĿĿ''ǷǷʳʳˣˣǟǟ""EEVVaa††€€ɊɊƓƓnjnj'' Ⱦ

ȾŅŅ 10-46

ʁ1ʆ ʡ˥ 11-13 ʁ2ʆ ņͷǚƾ 6*ǂȐ 14-27 1 ņͷǚƾ 6*ˮˏ…ǷĠ 14-15 2 Moniliella megachiliensis'ˇ²Īͱ 15-16

3 Moniliella megachiliensisOgv^Lm?†U~Bn~†7'VL†

d„M"Erythrose reductasḙ«Ŀ'̲Üˣǟ 16-21 4 ˇ²ÒtG†€'ƜÙ"ȥŅȐ 21-22 5 Moniliella megachiliensis7'RNAƜÙ"cDNAčƓ 22-23 6 Real time PCRȐ%69ɔɅ̽ˣǟ 24-25 7 Saccharomyces cerevisiae%69Erythrose reductasḙ«Ŀ'ɒɵɔɅ

25-27

ʁ3ʆ ʚǢ 28-32

1 Moniliella megachiliensisOgv^Lm?†U~Bn~†7'VL†

d„M"Erythrose reductasḙ«Ŀ'̲Üˣǟ 28-29 2 VaVƆʃ%69tG†€ˌɸ̽"Erythrose reductasḙ«Ŀ'ɔɅ

̽ǟ 29

3 EVa†€ɊƓnj'ȾŅ"Erythrose reductasḙ«Ŀ'ɔɅ̽ǟ 29-31 4 ͸Ș̚ĥǜ¦Š% 9tG†€ɊƓ̽'ȥŅ 31 5 Saccharomyces cerevisiae=ɌErythrose reductasḙ«Ŀ'ɒɵɔɅ 31-32 ʁ4ʆ ʭŌ 33-37

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€''ƫƫɳɳ 47-68 ʁ1ʆ ʡ˥ 48-50 ʁ2ʆ ņͷǚƾ 6*ǂȐ 51-54 1 ņͷǚƾ 6*ˮˏ…ǷĠ 51 2 Moniliella megachiliensis'ˇ²Īͱ 51-52 3 ˇ²ÒtG†€'ƜÙ"ȥŅȐ 52-53 4 Moniliella megachiliensis7'RNAƜÙ"cDNAčƓ 55 5 Moniliella megachiliensisb~maOgwT†N„V7'Ǯʕ 53 6 semi-quantitative RT-PCRȐ%69ɔɅ̽ˣǟ 54

ʁ3ʆ ʚǢ 55-58 1 ̃ˎʎ'ǮÙ"ɊƓnj'ȾŅ 55 2 ̃ˎʎ7EVa†€+'ƫɳ 55-56 3 ̃ˎʎˌɸnj"ǖˌɸnj'źͣ 56 4 ̃ˎʎčƓ 6*Úˣ͈̟̭«Ŀ'Ʃʕ 56-57 5 ̃ˎʎčƓ 6*Úˣ͈̟̭«Ŀ'ɔɅ̽ˣǟ 57-58 ʁ4ʆ ʭŌ 59-62

ʁ

ʁ 3ɾɾ ǁǁ$$ɌɌ̛̛%%đđ Moniliella megachiliensis%%6699 erythritol''ɔɔ ̵̵ɊɊɋɋ

69-84 ʁ1ʆ ʡ˥ 70-75 ʁ2ʆ ņͷǚƾ 6*ǂȐ 73-81 1 ņͷǚƾ 6*ˮˏ…ǷĠ 73 2 Moniliella megachiliensis'ˇ²Īͱ 73-74 3 tG†€'ȥŅ 74-75 ʁ3ʆ ʚǢ 76-77 1 M megachiliensis'̅óʳ 76 2 Ųglycerol%69Ɋʱ'źͣ 76 3 glycerolȮů%69źͣ 76-77 4 glycerol7erythritol+'IJƭîɃ 77 ʁ4ʆ ʭŌ 78-80

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ʠơơ 85-88

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ŴɌɌƼƼɁɁ 89-105

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˺̒̒ 106

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ʎ(ͩ%Ǒ3ʹǤ0țķȳȽ̇'Ɋ²óčȽ!8$ ($7$

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`kbīkb‚JTx]€ī%$ 93'=ʎ@€P†€"ɲ9ʎ

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€!8ƃ̽!(9̹Ȏȟ̳]†W$#'ɔ̵ͫěͫɌ'ͩ

6*Ǣņͩ%Ē/: 8Ċ7ʼȳ$Ź!Džŧɖ%ͫ:

Ƚ̇!9 (1, 2, 3, 4, 5)ɜŎɉėů(sucrose"Ȅ, ʐ75%!8$7 ɊɇɖEf€K†Ã(0.2 kcal/g"°H‚†'Ƚ̇!9/‡ʿ%

ʎ@€P†€(͜șóƊ!8Ķ̽%ưćĭč‡ʿ'ʎͩ68‡̦Ɗ 'Šɓ=̈4:%Ŏerythritol (ưć̽' 90%¤‰Œʸ!̝

4%ēĆ:Ɋ²Ò!£˺:%Ƨȏ:Ǿ8' 10%¤Šȯ̷IV

" ƧÙ:9"7ʣŠƊ!8'µɌ(Ŷ (6)Ķ̱Úؐ

%ƧÙ:92ʶʻ+'́ƝƒƇ:9öˏěp€'ȃƊˮͷ%

3ʘʥɒŧ(ˡŌ: $ (7)7%ƚ̷óµɌ4ƚͻ˓µɌ3

Ǔ 9 (8, 9)'6$erythritol'ȾƊ7ù‰ɉėƾ4ͮƾˈĿͩ

́æ$#ŭͫɌ%àɌ: 9 (10)ͫě¤ij%3ĉʷȓȖæóʌě'

¾Ȧæ$#dz$Ɍ̛9̖ŪiBGo~V]^L$#óƓě'Āƾ

" 'àɌȑɚ: 9 (11)óƓě'Āƾ" Ɍ7:9 C2 7 C6 óčȽ(ĀȎɎǝ' naphtha =cmRL~^H†!Úˣ9"!˚̞:

9TD†€IV'¸ʛ̽ƠĶ""3%C24C3óčȽ'naphtha 7'˚̞ȢœcmRL~^H†'ɶÉɃ°Š9ʚǢC4óčȽ'

¸ʛ̽'‹̊9"ƒƇ: 9C4óčȽ(ʼȳɐ%ŀĦ̽œ$

ɅnjȰ!Ķ̽¸ʛċʳ$'(erythritol '0!8erythritol 7ȆʔóąƆ%

6 butene4butadiene'˚̞Ƙ˖ɥɺ͆ɔ: 9$7ͫ

ěöˏě!($ erythritol =óŁĀƾ" Ɍ92%(͸º!86 8͸Ɋɋċʳ$ˇǨ'͆ɔ"ńº$Āƾ7'Ɋɋȉ27: 9

-">#'ʎ@€P†€ûʎ4™ʎGQʎ'@€`kb=óŁɖ%̮

Ë @€P†€%IJ ˚̞: 9!erythritol (ƝĿˇʏ̵Ȃ!

9Moniliella megachiliensis$#=Ɍglucose7'ɔ̵Ɋɋ%6 Ɋɋ

: 9M. megachiliensis=Ɍɔ̵Ȑ%69erythritol'ŢǯɖɊɋ(Ɣ

Ĥ!ɐ%Ì͵ ɨɽ:GUc€Ƙ˖!9 (12)M. megachiliensis( hJi]'Ŭˑ7Ú͛:ˇ²!8ǘɥɺ!ɌM. megachiliensis

SN-124AǨ3͸ʎȮůɈįŠ7Ú͛:ˇǨ!9 (13, 14)Ţǯɖ%Ɍ

7: 9ˇǨ(ǘÚ˾Ǩ= UV ȵŐ4IJɒ˳ɔæ%6 ƀ7:ɻȳIJɒ Ǩ!8çɋȽ" Ɋɋ:9 glycerol 'Ɋɋʳ° erythritol =͸Ɋɋ 9M. megachiliensis(Ș̚ĥVaV%Ɔʃ erythritol=ɊƓ 9"

ʭ7: 8'6$ʖʲÒ%ˌɸ9°ÚĿǓǷóčȽ=̫čȩ̇

(compatible solute)"Ę*'6$Ƚ̇=ʖʲÒ%ˌɸ9ˇ²ƹĴĬĕ

: 9 (15, 16)̫čȩ̇'ɊƓǷDzlj7%$ 9'(

͍7:y`€ɊȽ'0!8Ĵ'ɋǯ‰ǓɌ$ɊȽ!(ˣlj:

$

‡ʿ%ɊȽ(ɈįVaV%Ɔʃ ̫Ɔ9ʳê=Æ 9̫Ɔ ʳê=̶̦̉$VaVɈįǜ¦Š!('Ƿʳ(ˊá͍:ĭč

%6 (Ǽ%ʽ9Ș̚ĥVaV% 3ʖʲ(ijɐ'Ș̚ĥ'IJó%

Ŏ ƋŧƊ=ʞƢ92'ǷDz=Æ 9'ǷDz(759ɊȽ%

Ḁ̌ɖ%˲27:Ș̚ĥƆʃ"ʠɲ:9 (17)ɟǩɊȽ!9̵Ȃ%(

‡ʿ%ĀǩɊȽ4¡'ĴʖʲɊȽ"(Ȅ̎%$7$p€'Ș̚ĥ%̫Ɔ 93'Ĵ͸Ș̚ĥVaV̵Ȃʖʲ%́˄:ĭčȘ̚ĥţ

%6 ʖʲÒ'ȆÚƉ̝%ʖʲʹ=̜̦ ʖʲij%ƧÙ::%¬

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Ŏ (i@" É2Ș̚ĥ'ƉȬ$‰Lj%7:ʖʲ7(

ʵȆ̈9ʖʲ'ɊɇǷʳ'Ĵ(̫ǻ$ʖʲƓÚ'Ȯů4ʖʲʹ=͔

BG„ò̲=äƬ" 92ʵȆ(ʖʲ'Ɋʱ%țã$źͣ=Ą.

(18)'2ʖʲ(Ș̚ĥƆʃ" VaV%68Ŵ̈:\x†

U7'ġƂ'2%ǁ%ʖʲÒ'Ș̚ĥ=‰Lj ʖʲÒij'Ș̚ĥ ţ=ˣș9ƅ˞9

ʖʲ͗ň'ĀĢ'΀(̦ů'Ef€K†ș̄!9"ʭ7: 9 (19)·)pH '°Š%Ŏ ʖʲÒ%ŀĦ9´è'ȆʔBG„(ʖ ʲʹ%ŀĦ9H⁺-ATPase%6 ƧÙ:pH%͈9rxGV[TVʞ Ƣ: 9"ʭ7: 9 (19)H⁺-ATPase %69ȆʔBG„'

ƧÙ%(ATP'ș̄=ƅ˞"92ǰ2 Ĵ'Ef€K†=ș̄9

"%$9ɈįVaV́˄$ʖʲ%Ș̚ĥ$#'VaV́˄:9

"ɈįVaVƆʃąƆ VaVʯƊǷDzȔƊó9"ʭ7:

9ɈįVaVƆʃ!(̜ŧ'ʖʲƆʃ"ďdz%ʖʲ˗Ŝ'X„R†[

„jL̇ƐɢTMc€%6 ʖʲÒƍĬ«̨ʏ=  ̍Ô4ʬˬ Ž¿Ͱ'˶ʆ˕<:9 (20)‡ʿɖ%İǿǔ'ʖʲ(cAMP¹ŀƊ'Protein

kinase AȔƊó: 8ʖʲİǿ%͈Œ9̭«Ŀʩ'̍Ôŷ¼̠

:9 (21)‡ǂProtein kinase AȔƊó: 9ȿƑ!(VaVʯƊ

%͈Œ 9̭«Ŀʩ'ɔɅ(ˊáƁ:9 (22)İǿǔ%9ʖʲ%

ɈįVaVƆʃɊ9"œ$"3cAMP¹ŀƊ'Protein kinase A'‹

ȔƊóCCCCT /( AGGGG 'Įī̲Ü!DzƓ:9VaVƆʃ̲Ü

(STRE: stress response element)4VaVƆʃƊ'TV̲Ü=Ǔ9̭«Ŀ'ɔ

Ʌʖʲ¾˿Ƿʳ=Ǔ9̵ʔ4ʖʲ¾˿Ƚ̇'ɊčƓ%͈Œ9£˺ʏ'¼

̠Ą*ʖʲ¾˿Ƚ̇'˳ő/(ˌɸ˕<:9":9 (22, 23, 24, 25) y`€ɊȽ!9Saccharomyces cerevisiae ͸Ș̚ĥVaVǜ¦Š%7 :9"ʖʲʹ%řĦ9Ș̚ĥX„R†Ș̚ĥVaV=ƐĈ9 (26) ƐĈ:VaV(VaVƆʃƊMAPK (Mitogen-activated protein kinase)H VN†b'1!9 HOGʙ̋;High osmolarity glycerol pathwayͿ=ʙɎ Ș̚ĥVaVƆʃ=˕Ș̚ĥX„R†%(ʹ̜̂X„R†kV]U„J c†Y!9Sln1"ʖʲ̇ƆʃK{†[†!9Sho1=ʙ92'ʙ̋

ŀĦ:Ⱥǂ'0!3͸Ș̚ĥǜ¦Š!'Ș̚ĥƆʃ% 9TM c€«̨ċʳ":9 (26, 27, 28, 29)Ș̚ĥX„R†%68MAPKHVN

†bȔƊó:9"MAPK HVN†b'Ǘɿ%¯ʧ9 Hog1 ' threonine

"tyrosine'ŽǾī„̷ó:Hog1'ȔƊó̈9 (18)ȔƊó Hog1 (ʖʲ̇7̝4%ǩÒ%ɳ˕̍ÔĢĿ!9 Hot1 '„̷ó=

˕ (29, 30)„̷óŽHog1"Hot1ɜšµɌMsn1=  glycerol ɊƓ% 9̵͂ʔ!9Glycerol-3-phosphate dehydrogenase 1 (GPD1)̭«Ŀ'

̍Ôͦĩ%ŀĦ9STRE%ʚč9 (30):%68Hog1RNA polymerase II=(2"9īǘ̍Ô˘ʧ=o‚y†[†%L€†aGPD1̭«Ŀ' ɔɅ˳ő=¼glycerol'ɊƓ¼̠:9/Ș̚ĥVaV=Ĉ9

"ďnj%Ȇ4glycerol'ʹ̙̑[„jL̇!9 Fps1ͅĉ9:%6

8glycerol'ʹijȕÙ=Ļˇ²Ò!'glycerol'ˌɸ̈9Ș̚ĥV

aV˶ƺŽ(Fps1 =͆ĉ9"!ˇ²Ò'̦è$ glycerol 'ƧÙ=˕

"ʭ7: 9 (31)

‡ǂM. megachiliensis% 9erythritol'ɊƓʙ̋(ˣʎʏ"q„a†V

„ ̷ ġ ̋ ' Ñ Ż % 6 Ɋ Ɠ : erythrose-4-phosphate ǹ ! Erythrose-4-phosphate phosphatase (E4PP)'µɌ!ȲǷ„̷ʵ͛ erythrose

"$87%Erythrose reductase (ER)'µɌ%68erythritolɊƓ:9 :%Ŏ ˣʎʏ!ɊƓ:9fructose-6-phosphate Phosphoketolase 'µɌ

!erythrose-4-phophate "$8ǹ!Erythritol-4-phosphate dehydrogenase 'µ Ɍ%6 erythritol-4-phosphate "$8ǑŽ% Phosphatase %6 erythritol ɊƓ:9ʙ̋9äʮ( Candida magnoliaePseudozyma tsukubaensis 4Torula corallina$#Ĵ'̵Ȃ%˟7:Žʮ(“̷ˇ%˟7:9 (32, 33, 34,

35)ˣʎʏ(glucose=pyruvic acid/!£˺ LE„̷ġ̋+¸Œ92

'̻˞$ʙ̋!9q„a†V„̷ʙ̋3ʎ£˺'̻˞$ʙ̋' 1 ! 8ʙ̋‰%(˂ͳǃ@vg̷eLG]b$#ǓɌȽ̇'čƓī̇"

$9erythrose-4-phosphate 4 ribose-5-phosphate ͇Ƚ̇" ¯ʧ 9 ë M. megachiliensis(glycerol683erythritol=Ĵ̽%ɊƓ9"7 y ` € Ɋ Ƚ 6 8 3 ˛ ͚ $ ʎ £ ˺ ʙ ̋ = Ǔ 9 " ʭ 7 : 9 M.

megachiliensis Ɏǝ'ER%( 3ɵͩ'@BZSBwɨ˲: 8:

:ER1ER2ER3 "Đ¢7: (36)/:7' cDNA =ćƀ Ķ ʸˇ!ɔɅʚǢNADPHŀĦŠ!glyceraldehyde"erythrose%Ŏ ͸ ȾɒƊ=ɪ"7C4 Ą* C3 @€`kb=̮Ë9 Yeast aldo-keto

reductase family %ś9̵ʔ!9"ƫŅ: (37)$7C.

magnoliae ' ER ! ŀ Ħ ɨ ˲ : 9 Xylose reductase subfamily ' KXXXGF(Y/G)XG motif M. megachiliensis % (ɨ˲: $ (37, 38)/ǘ̵ʔ(NADPH=˙ĢĿ" ˤľµɌ=ɪNADH!(ˤ ľµɌ=ɪ$ (37)

:/! M. megachiliensis % 9erythritol ɊƓ'̵͂ʔ!9 G6PDH

(Glucose-6-phosphate dehydrogenase)TKL (Transketolase)TAL (Transaldolase) ER'[„jL̇p€!'ˣǟ(̠27: 9 (39)̭«Ŀp€!'Ƿ ʳˣǟ(˕<: $/M. megachiliensis% 3Hog1̭«Ŀ'ŀ Ħɨ˲: 8Ș̚ĥ'TMc€«̨ǷDz% Hog ʙ͈̋Œ 9

"lj7%$89ǘɥɺ!(erythritolɊɋˇ!9M. megachiliensis

% ER̭«Ŀ=Ē1Ė̔Įī̲Ü=ćƀ9"'ɔɅïƑ4Ƿʳˣ ǟ=˕"Ą*VaVƆʃnj% 9ʎ£˺ǷDz=lj7%9"!

VaVƆʃ%¬erythritolɊƓ'Ƿʳˣljɔ̵%69erythritolɊɋ'Ǒ

̫ó=Ǯ˧9""

Fig. 1 Stress response and production of compatible solute.

Fig. 2 Metabolic pathway of polyol fermentation in M. megachiliensis.

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Moniliella megachiliensis %% 99

erythritol ɊɊƓƓ̵̵͈͈̟̟ʔʔ

Erythrose reductase ̭̭««ĿĿ''ǷǷʳʳˣˣǟǟ""

erythritol ɊɊƓƓnjnj''ȾȾŅŅ

ʁ 1 ʆ ʡ˥

ɊȽ(ɈįVaV%Ɔʃ 9ʇģÒ!̫Ɔ9ʳê=Æ 8 '6$ǷDz=VaVƆʃ"ɲ: 9 (17)

S. cerevisiae % (VaVTMc€«̨ʙ̋!9HOG ʙ̋= 

̫čȩ̇" glycerol=ʖʲÒ%ˌɸʖʲÒij'Ș̚ĥţ=ˣș 9glycerolɊƓ%͈Œ9GPD (Glycerol-3-phosphate dehydrogenase)%(2 '@BZSBwŀĦ:: GPD1GPD2 "Đ¢7: 9 (40) GPD1 ̭«Ŀ(Ș̚ĥƆʃ%"3$ɔɅ̽'İëɨ˲: 9‡ǂ GPD2̭«Ŀ(Ņŧɖ%ɔɅ˕<: 8‡ǹ£˺'‡"ʭ7: 9 (41)'GPD1̭«Ŀ'open reading frame (ORF)'ɛä%VaVƆʃ

%͈Œ9": 9STRE'ŀĦ˲27: 9 (30)M. megachiliensis

% 3MAPJc†YHVN†b'MAPK%9Hog1'ŀĦɨ˲

: 8 (42)͸Ș̚ĥǜ¦Š!3Ɋʱċʳ$ɇɎ(VaV=ƐĈ̫č

ȩ̇" glycerol 4 erythritol =ɊƓ92"ʭ7: 9' glycerol ( S. cerevisiae "ďdz'£˺!9"ʭ7: 9‡ǂerythritol ( ˣ ʎ ʏ " q „ a † V  „ ̷ ġ ̋ ' Ñ Ż % 6 Ɋ Ɠ : erythrose-4-phosphateʵ„̷ó:erythrose%£˺:Ž%erythritolɊ Ɠ̵͈̟ʔ'ER (Erythrose reductase)%6 erythritolɊƓ:9:7'

"68ER̭«Ŀ(EVa†€ɊƓ'̵͂ʔ": 9ĶʸˇɔɅ

%69̵ʔȔƊ%6 M megachiliensis'ER(NADPH!(ąƆ=ɪ NADH !(ąƆ=ɪ$ī̇ȾɒƊ=Ǔ 9"ɢ7: 9 (37) /ER̭«Ŀ%( 3 '@BZSBwŀĦER1̭«ĿER2 ̭«Ŀ ER3̭«Ŀ"Đ¢7: 9:7ER̭«Ŀ3ŀĦ 9"68 S. cerevisiae'GPD1̭«Ŀ"ďdz%M. megachiliensis(Ɉį%Ɔ :7=

¶Ú 9"ɪĝ:9/8ER̭«ĿerythritolɊƓ'̻˞$

Ƿʳ=Ɲ 9"ʭ7: 9

̫čȩ̇" erythritol =ɊƓ9ƃɊȽ( ER ̭«Ŀ9('ďdz' Ƿʳ=Ǣ": 9Aldo-keto reductase family%ś9̵ʔ=Ǔ 8 erythrose7erythritol+"£˺ 9 (43)'!3Ķ̽%erythritol=Ɋ Ɠ9ƃɊȽ(M. megachiliensis4C. magnoliae$#‡̱!9 (1432)

ſ erythritol =ɊƓ$ɊȽ(q„a†V„̷ġ̋%6 ɊƓ:

erythrose-4-phosphate¤͌'erythritol=ɊƓ92'̵ʔ=Ǔ $"

ʭ7:9y`€ɊȽ!9S. cerevisiae3erythritol=ɊƓ$ER̭

«Ŀ"ɜďƊ=ɪGCY1 (galactose-inducible, crystailin-like yeast protein) ̭«Ŀ

=Ǔ 9 (44)GCY1̭«Ŀ3Aldo-keto reductase family%ś9̵ʔ!

8ER"'ɜďƊ(40%ɴů!9S. cerevisiaeǓ9̭«Ŀ'!(Ǒ 3͸GCY1(Glycerol dehydrogenase" glycerol7dihydroxyacetone+ '£˺!ˤľȔƊ=ɪ"–Ǝ: 9 (45)/VaVƆʃ%͈Œ

9̭«Ŀ!9"3ɢ7: 9 (46, 47)S. cerevisiae(OgwƍĬ

͆ɪ: 9"38¡'ˇǨɎǝ'̭«Ŀ=S. cerevisiae%őÎ '̭«Ŀ'Ƿʳ=˶,9ǂȐ<59ɒɵɔɅ3ŭ˕<: 9

ǘɾ!(ER̭«Ŀ'Įī̲Ü'ˣǟ 6*ɔɅ̽ˣǟ"S. cerevisiae'ǧ ͱ˞ȉƊǨ!9BY4741Ǩ"'GCY1̭«ĿǸƯǨ=Ɍ ER̭«Ŀ=ŷ áɔɅ9"!ER̭«Ŀ'Ƿʳˣǟ=˕

ʁ 2 ʆ ņͷǚƾ 6*ǂȐ

1 ņͷǚƾ 6*ˮˏ…ǷĠ 1-1 ˇ²

ǘɥɺ% ˇ²( (ɀ) ͫěʠčɥɺƕ ƃɊȽi„L7Ú˾:

M. megachiliensis SN-124A=ɌM. megachiliensis(YMAĪħ [0.5% peptone (ǰǞ˚ˏŢǯǨųªɬ), 0.3% bacto malt extract, 0.3% bacto yeast extract, 2.0%

glucose, 2.0% INA Agar (©̯ͫěŢǯ)] 'ƿ͡Īħ%Va†Lˇ²÷Ú

%Ɋʱ"=ɨ˲Ž4˚C !¾ʅ

Saccharomyces cerevisiae BY4741Ǩ (MATa his3!1 leu2!0 met15!0 ura3!0)"' GCY1̭«ĿǸƯǨ (MATa his3!1 leu2!0 met15!0 ura3!0 gcy1::KanMx4)(:

: YPD Īħ [2.0% peptone (ǰǞ˚ˏŢǯ), 1.0% bacto yeast extract, 2.0%

glucose, 2.0% INA Agar (©̯ͫěŢǯ)]!Īͱ

̭«Ŀ'L‚†d„M%(Escherichia coli DH5!=Ɍ LBĪħ [2.0 % Bacto Triptone, 1.0 % Bacto Yeast Extract, 2.0 % NaCl, 2.0% INA Agar (©̯ͫěŢǯǨų

ªɬ)]! 37˚C!Īͱ

1-2 ǷĠ

PCRąƆĄ*0.2 mL]{†n'B„J{p†a%(TM~UD„aR†yR

BL~† (Biometra) 3( T1 R†yRBL~† (Biometra) =Ɍ1.5 mL]{†n'̪ƄÚ͛%(ù‰̪ƄǷ KUBOTA 1120‚†[†RA-50J (’¾

ɍИ) =Ɍ50 mL]{†n (CORNING®) '̪ƄÚ͛%(Õþ̪ƄǷ

RSL-05AVB„M‚†[†50 mL"4|d^a (±’͇˚µƕ) =Ɍ/

Ⱦ%˗˩$͍8ˮˏ(ĚÍʒˏŢǯ'Ⱦʓˮˏ=ɌȆ(Milli-Q̉

ʒȆ˘ʧ (MILLIPORE) !ƀ̉ʒȆ=G†aL†nȪˇ Ɍ/

Īħ( ES-215 (Ǩųªɬ…av†ʍŢ) =˶˚ˮˏ 6* 0.2 mL

(WATOSON) Ą*1.5 mL]{†n (Axygen, Bio-BIK) (ES-315 (Ǩųªɬ…a v†ʍŢ) =Ɍ G†aL†nȪˇˆˢm~VP( Drying Sterilizer

SG810 (zuaɯŁǨųªɬ) =Ɍ ”ȷȪˇ

΁ Moniliella megachiliensis'ˇ²Īͱ

2-1 ɣǔɖ$Ș̚ĥVaVǜ¦Š% 9Īͱ

M. megachiliensis=2% glucose=Ē1GYĪħ [0.5% bacto yeast extract, ˩̏

:Ȯů'glucose]!200 rpm30˚C'ǜ¦Š'3"!OD6001.0%̨9 /!Ĺȅɖ%ƥ"ĪͱOD600 1.0 %̨ˇ²=3,000 rpm10 Ú 4˚C!̪ƄÚ͛%6 ͙ˇ'‡̱ˇ²=20% glucose=Ē1GYĪħ%

ɳ 30Ú͇Īͱ͙ˇˇ²(TE buffer (pH 8.0)=Ɍ 2ġȓȖ

-80˚C% Õ×¾ŀ

2-2 ̈́ǔɖ$Ș̚ĥVaVǜ¦Š% 9Īͱ

M. megachiliensis=2% glucose/(20% glucose=Ē1GYĪħ!200 rpm 30˚C'ǜ¦Š'3"!12nj͇772nj͇/!Ĺȅɖ%Īͱ12nj͇"%

ˇ²=ġĆƕŅ'nj͇Īͱˇ²(3,000 rpm4˚C10Ú͇̪ƄÚ͛

=˕ ͙ˇ͙ˇˇ²(TE buffer (pH 8.0)=Ɍ 2ġȓȖ-80˚C

% Õ×¾ŀ

2-3 ͸ʎȮůǜ¦Š% 9Īͱ

M. megachiliensis=2%20%40%60% glucose':=Ē1GYĪħ

!200 rpm30˚C'ǜ¦Š'3"Ĺȅɖǜ¦Š!7Dž͇ĪͱĪͱˇ²

(3,000 rpm4˚C10Ú͇̪ƄÚ͛=˕ ͙ˇ͙ˇˇ²(TE buffer

(pH 8.0)=Ɍ 2ġȓȖ-80˚C% Õ×¾ŀ

΂ Moniliella megachiliensis Ogv^Lm?†U~Bn~†7'VL†

d„M"Erythrose reductasḙ«Ŀ'̲Üˣǟ

M. megachiliensis'Ogv^Lm?†U~Bn~†(ŸɥɺŇ!Dzʈ:

3'=Ɍ

3-1 ‡ǹVL†d„M

25,000L‚†„=Ē1 5 'j^N†U„MȩȚ 1 µL =̀Ĩ"Go Taq

DNA Polymerase (Promega)=Ɍ o~†L\BLaPCR=˕Ɍ

primer(ER1.2-for"ER1.2-rev (ER1̭«Ŀ"ER2̭«Ŀ)ER3-for"ER3-rev (ER3̭«Ŀ)!9'i„bǮÙ!R„o€=2ǹVL†d

„M%¸

3-2 ER1̭«Ŀ"ER2̭«Ŀ'ȾŅ

ER1"ER2(cDNA'ɜďƊ98%"͸2Ⱥǂ'0%Ⱦɒɖ$primer

=˪˦9"!$'2ER1̭«Ŀ"ER2̭«Ŀ'˼ß(

::'ǀȺ=PCR!İŨEZ-10 Spin Column PCR Product Extraction Kit

(BIO BASIC INC)=Ɍ DNA'ʍ˚=˕Ž%AluI (TaKaRa)!á̵͍

ʔșó=˕ÛǀnjER1 ̭«Ŀ(Ûǀ:9ER2 ̭«Ŀ(Ûǀ :$

3-3 ɚɖ'̭«Ŀ=Ē1m?†U'͍Ņ

3-3-1 m?†UȩȚ'[B[#]D^L

1 ǹVL†d„M!ɚɖ'̭«Ŀ=Ē1"ɨ˲:m?†UȩȚ=

SM Buffer [50 mM Tris-HCl (pH 7.5), 0.028% NaCl, 0.01% MgSO4Ή7 H2O, 0.01%

gelatin (DIFCO LABORATORIES)] !10Á100ÁĄ*1,000Á%ť̺1.5 mL]{†n%ť̺m?†UȩȚ 1 $LĶʸˇXL-1 Blue MRA 200 $L=ë 37˚C20 Ú͇B„J{p†aƐǤ'Ž”ȷˮͷʅ%ƐǤ m?†UȩȚ%72Ȥ2 LB soft agar 4 mL=Ͷ̕lq^a

!ë̍ÂȝĚLB/ Maltose / MgSO4o†a [LBĪħ%0.16 % w/v MaltoseΉ H2O, 0.2% MgSO4Ή7H2O=ë3']%ƱLB soft ager ”Ž37 ˚C

!ʗĵĪͱ'ŽČo†a'o~†Lƹ=ƹ1 $L8'o~†

Lƹ=ʄÙ

3-3-2 m?†UȩȚ'ƐǤ

ʄÙ1 $L8'o~†Lƹ=ī%m?†UȩȚ=ĶʸˇXL-1 Blue

MRA %ƐǤ1.5 mL ]{†n%ť̺m?†UȩȚĶʸˇ XL-1 Blue

MRA=ë37˚C20Ú͇B„J{p†a'Ž”ȷȪˇˮͷʅ

%ƐǤm?†UȩȚ72Ȥ2 LB soft ager 4 mL=Ͷ̕l q^a!ë̍ÂȝĚo†a'ƹ([B[#]D^L!ƀ7:o~

†Lƹ"o†a'ǡƹ=Éġ96%˪ŅLB soft ager "'

ȝčȚ=LB/ Maltose / MgSO4o†a %/37˚C!ĪĪͱ

3-3-3 PCR%69ɨ˲

ƐǤo†a%Ŏ SM buffer=4 mLȕo~†L=ȩÙ 'ȩȚ1 $L%Ŏ PCR (initial denaturation 95˚C for 2 min; 30 cycles 95˚C for 30 min, 56˚C (ER1,2) or 55˚C (ER3) for 30 min, 72˚C for 30 min; final elongation 72˚C

2 min, 4˚C pause)=˕ɚɖ'̭«Ŀ=Ē1m?†U'ǓȲ=ɨ˲

3-4 ɚɖ'̭«Ŀ=Ē1m?†U'û͛

m?†U'VL†d„M1/101/20%$";![B[†]D^

L=˕o†a7û͛ċʳ$o~†L=1"8SM Buffe 10

$LÎ:1.5 mL]{†n%ƒȭs€_^LV=Ɍ ȬƲƞŽ

1µL=̀Ĩ" o~†L\BLaPCR=˕'µǯ!i„bǮÙ :7ɚɖ'̭«Ŀû͛!3'"

3-5 m?†U7'DNAʍ˚

û͛j^N†U„MȩȚ7100Áť̺ˮƾ4 $L"XL-1 Blue MRA

(STRATAGENE) 400 $L=1.5 mL]{†n%Î:37˚C20Ú͇ƐǤ

ƐǤˮƾ101 $L=LB soft ager 4 mL%ƒȭŽLB/Maltose/MgSO4

Īħ ˦4ǡ%/37˚C!Īͱ 6*ƐǤ

Īͱ 6*ƐǤŽSM buffer 4 mL%4˚C4nj͇Ș8 mL15 mL] {†n2ǘ%ć8chloroform 3 mL=ë 3,000 rpm30Ú͇4˚C!̪ƄÚ

͛=˕15 mL]{†n4ǘ%‰ȟ=ġĆDNase (TaKaRa) "RNase (d

^t„U†„)=ë37˚C30Ú͇ąƆŽPEGȩȚ9 mL=ëȇȆ

!5nj͇ƶʧ3,000 rpm1nj͇4˚C!̪ƄÚ͛Ž‰ȟ=͎Ă

Ȍȁ=SM buffer 1.5 mL!ȩˣ500 $L1.5 mL]{†n%Úȑ

0.5 M EDTA 20 $L"10 % SDS 5 $L=ë 60˚C1Ú͇ëȤPCI=500

$Lë 1Ú͇s€_^LV=Ɍ ȬƲƞ=˕12,000 rpm5Ú͇

ŇȤ!̪ƄÚ͛‰ȟ=ġĆÓůPCI 500 $L=ë ďǜ¦!̪ƄÚ

͛=˕‰ȟ=ġĆ7%chloroform 500 $L=ë ďǜ¦!̪Ƅ Ú͛=˕‰ȟ=ġƉȟ=360 $LġĆ3 M sodium acetate (pH 5.2)

"100% ethanol 900 $Lë-80˚C!30Ú͇ƶʧ14,000 rpm20Ú͇

4˚C!̪ƄÚ͛=˕‰ȟ=͎Ă70 % ethanol=500 $Lë Óƒȭ

Ž14,000 rpm5Ú͇4˚C!̪ƄÚ͛=˕'µǯ=2ġ˕

ͪ”Ž%ȪˇȆ!ȩˣ

3-6 RnL‚†d„M

ʍ˚m?†UDNA = NotI (TaKaRa)=Ɍ á̵͍ʔșó=˕37˚C

!ąƆpBluescript II SK(+) (Stratagene)3ďdz%á̵͍ʔșó=˕

á ͍ ̵ ʔ ș ó = ˕ pBluescript SK (+)( E. coli Alkaline Phosphatase

(TOYOBO)=Ɍ @€HmFVm?[#YØɇ3˕::ąƆʗ

• Ž E [ g † € Ȍ ȁ % 6 ġ Ć DNA = 2"Rapid Ligation Buffer (Promega)"T4 DNA Ligase (Promega) =Ɍ 16˚C!ąƆ

~BO†T}„o~Vvb=E. coli DH5!=Ɍ Ź̇̍ƭ~B O†T}„ȩȚ %P„l_„aX€ 200 $L=ëɹ4%lq^_A„M ȇ‰!30Ú͇B„J{p†T}„Ž42˚C!30ɰ͇ëȷÓ*ȇ‰!

3Ú͇ƶʧ37˚C%–2Ȥ2 SOCĪħ 800 mL=ëlq^_

A „ M ” ȷ Ȫ ˇ ȡ 0 ˮ ͷ ʅ % ɳ 37˚C ! 1 nj ͇ Ș ̚ Ī ͱ

LB/Ampicillin/IPTG/X-gal o†a%ˇȚ 100 $L P„~†Uǫ!ʔdžo

†_A„Mo†a”7ˍ=37˚C !Īͱ:7'Ƴ µ(L†„p„]Ò!˕ĪͱP‚d†'ɕˁP‚d†=̬

ƛGo Taq DNA Polymerase (Promega)%69P‚d†\BLaPCR=˕

ąƆ˟7:P‚d†% (LB/AmpicillinĪħ%37˚C!Īͱˇ

²ġĆ=˕Ž%EZ-10 Spin Columu Plasmid DNA MiniPreps Kit (BIO BASIC INC)=Ɍ o~Vvb'ġĆ=˕

3-7 Įī̲Ü'ˣǟ

1 assay%0.2 mL]{†n%Big Dye® Sequencing Buffer 3 µLReading Reaction Mix 2 µL1 µM Primer 3.2 µLo~Vvb 300 ngɜŸǑŽ% 20 µL

%$96%ȪˇȆ=ë:7'ȝčȚ=95˚C!2 minB„J{p†a 95˚C!10 sec50˚C!5 sec60˚C!4 min=25 cyclesʦ8̗4˚C! PCRąƆ=ʗ•ąƆȚ=1.5 mL]{†n%ɳ125 ·M EDTA=2 µL 3 M sodium acetate (pH 5.2)=2 µL100 % ethanol=50 µLëȇ‰!20Ú͇

ƶʧ4˚C!14,000 rpm20 Ú͇̪ƄŽ‰ȟ=͎Ă70 ͽ ethanol 70 µL

!Óƒȭ'Ž4˚C!14,000 rpm5Ú͇̪ƄŽ‰ȟ=͎ĂŇȤ!ͪ”

ŽABI3100 (Applied Biosystems)/(ABI3100xl (Applied Biosystems)!Įī̲

Ü'ˣǟ=˕

Įī̲Ü'ˣǟ%( DNASIS-Mac v3.5 (HITACTI)Ą* GENETYX-Mac v15 (GENETYX)= Ɍ ˕ Į ī ̲ Ü ƍ Ĭ ( NCBI (National Center for Biotechnology Information:http://www.ncbi.nlm.nih.gov/ )=ʙɎ BLASTǮʕ=

˕

΃ ˇ²ÒtG†€'ƜÙ"ȥŅȐ 4-1 ˇ²ÒtG†€'ƜÙ

Īͱˇ²'‡̱=2 mL'ȪˇȆ!Óƒȭ'1 mL=10 Ú

͇ȶȍŽ%100 % trichloroacetic acid 200 mL=ë s€_^LVvJ R†=Ɍ 20Ú͇ȝĚ12,000 rpm!10Ú͇̪ƄÚ͛ ƀ7:‰

ȟ=ˇ²Òˌɸʎ" Úǟ=˕

4-2 ʎÚǟ

BG„L‚uaM~mA†TV_w (ICS-3000DIONEX)=Ɍ ˇ²Ò 6*ˇ²ij'tG†€Ē̽=ȥŅR„o€( 25 $L őÎ500 mM NaOH%68ȩ͛H~w(Carbo Pac MA-1 (DIONEX)=Ɍȕ̝(

0. 3 mL/min 'ǜ¦!j€Vb@„q‚xaǮÙĠ (ED-50̾͝ǰ)

(DIONEX)!ǮÙǴȨȚ" 50 $M100 $M'ȩȚ=ȥŅ™ȰǮ

̽Ȑ%6 ȮůǖɢȩȚ'ȥŅ=˕ˮƾ(Ǒ3͸ǮÙ:9ʎǮ

̽ʢÒ%ʑ/9ǮÙů%$96%ť̺ Úǟ

4-3 ˇ²”ȹ̻̽'ȥŅ

`TO†[†%Ɍ9THO€"c„i„M@€vH^o= 160 ˚C

! 1 nj͇ø”ȷȪˇTHO€=ƻ`TO†[†Ò!@€vH^o=

ÕþÕþŽʍŋ͝Ŀķɱ (ǨųªɬE†…@„b…`B GH-202)= Ɍ ʍɱˇ²̻̽ȥŅɌ%¾ŀˇȚ=500 $L@€vH^o%ȫŠ

80˚C5nj͇”ȹ”ȹˇ²(@€vH^o"Ñ%ʍɱ@€

vH^o"'ţ=ˇ²̻̽"

΄ Moniliella megachiliensis7'RNAƜÙ"cDNAčƓ 5-1 total RNA'ƜÙ

-80˚C%×ʚˇ²=“̿%Î:Ț²ɼʔ=ë$7“ǫ!ɧɦ

ɧɦˇ²'100 mg ɜŸ=1.5 mL]{†n%Î:%ƜÙ buffer [100 mM Tris-HCl (pH8.0), 10 mM EDTA (pH8.0), 100 mM LiCl, 1% SDS]!ͯĚ mDg†€ȩȚ= 400 $L ë7%ƜÙ buffer 400 $L " CI Ț [chloroform: isoamyl alcohol = 24 : 1]=ës€_^LV=Ɍ ȬƲƞ

12,000 rpm15Ú͇ŧȤ% ̪ƄÚ͛=˕ŠŜ"‰Ŝ%Ú͛

‰Ŝ=ǁ1.5 mL]{†n%ɳPCI [ƜÙbuffer!ͯĚmDg†€

ȩȚ: chloroform: isoamyl alcohol = 25: 24 : 1]=‰Ŝ"ʂ̽ës€_^LV=

Ɍ ȬƲƞÓů12,000 rpm15Ú͇ŧȤ!̪ƄÚ͛=˕‰

Ŝ=ǁ 1.5 mL ]{†n%ɳ7%ɳ]{†n%ʂ̽'

chloroform=ë 12,000 rpm15Ú͇ŧȤ% ̪ƄÚ͛=1.5 mL]{

†n%‰Ŝ=ɳɳ‰Ŝ'1/3̽'10 M LiCl=ë s€_^LV

=Ɍ ȬƲƞ-20˚C!ʗĵÕþÕþ]{†n(Õþ̪ƄǷ

=Ɍ 13,200 rpm20Ú͇4˚C!RNA=Ȍȁ ‰ȟ=͎Ă2 M

LiCl 500 $L=ë 13,200 rpm20Ú͇4˚C!ȓȖq^a=ͪ”

ȪˇȆ 400 $L!Óȩˣ'ŽDNase I (TaKaRa) =Ɍ 4˚Cǜ¦Š!ąƆ ąƆ=ʗ•92%PCI=500 $Lë s€_^LV=Ɍ

ȬƲƞ12,000 rpm15Ú͇ŧȤ% ̪ƄÚ͛=˕ŠŜ"‰Ŝ

%Ú͛‰Ŝ=ǁ1.5 mL]{†n%ɳʂ̽'chloroform=ë

12,000 rpm15Ú͇ŧȤ% ̪ƄÚ͛=˕Óů‰Ŝ=ǁ1.5 mL

]{†n%ɳʂ̽'2-propanol=ë Ʋƞ'Žȇ!10 Ú͇Õþ

ÕþŽ13,200 rpm20Ú͇4˚C=˕RNA=Ȍȁ70% ethanol=

ë 13,200 rpm5Ú͇4˚CÕþ̪ƄÚ͛'Žͪ”:=Ȫˇ

Ȇ50 $L%ȩGene spec=Ɍ ȮůȥŅ=˕

5-2 cDNA'čƓ

PrimeScript^TM Reverse Transcriptase (TaKaRa) =Ɍ cDNAčƓ=˕]

{†n%Ⱥ̓čƓ%Ɍ9primer(dT-15-primer=Ɍ

΅ Real time PCRȐ%69ɔɅ̽ˣǟ

6-1 Real Time PCR%Ɍ9Ǯ̽ʢµ˚'2'pL[†Dzʈ

Real Time PCR!İŨ9̭«ĿǀȺ=Go Taq DNA Polymerase (Promega)

=Ɍ PCR (initial denaturation 95˚C for 2 min; 30 cycles 95˚C for 30 min, 56˚C (ER1,2), 55˚C (ER3) or 59˚C (GAPDH) for 30 min, 72˚C for 30 min; final elongation 72˚C 2 min, 4˚C pause)%6 İŨpGEM^®-T Easy Vector (Promega)=Ɍ

̭«ĿǀȺ=RnL‚†d„Mƀ7:o~Vvb'̭«Ŀ̲Ü=ɨ˲

Ž%Ǯ̽ʢ!Ɍ92'pL[†"

6-2 Real time PCR

LightCycler^® FastStart DNA Master SYBR GreenI (Roche)=Ɍ Real Time PCR

=˕LightCycler^® FastStart Reaction Mix SYBR GreenI, 10"conc. (Roche)'Î ]{†n1ǘ%Ŏ LightCycler^® FastStart Enzyme (Roche)10 µL=ë 1.5 mL ] { † n % Ŏ LightCycler^® FastStart Reaction Mix SYBR GreenI,10"conc. (Roche)"LightCycler^® FastStart Enzyme (Roche)'ȝčȚ=1"%

$96%ëforward primer"reverse primer=::ʗȮů 0.5 µMMgCl2

stock solution25 mM=ʗȮů3 mM%$96%ë 1 assay18 µL%$

96%ȪˇȆ!Ņ̽20Áť̺cDNA 2 µL=LightCycler^® Capillaries (20 µL)%ë 7LightCycler^® FastStart Enzyme (Roche)=Ē1ȝčȚ=18 µL ë

ąƆ(95˚C!10Ú͇'Ž95˚C!10 sec54˚C!10 sec72˚C!8 sec'R BL€=35ġ˕'Ž95˚C/!Ȥů=‰Lj 7@d†„M'

Ȥů%5˚C‰LjȤů/!Š¤͌0.1˚C/sec '̝ů!Ȥů=Š

¶Ɍo~Bu†( ER1 ̭«Ŀ 6* ER2 ̭«Ŀ( ER1.2-RT-for "

ER1.2-RT-revER3 ̭«Ŀ( ER3-RT-for " ER3-RT-revGPD1 ̭«Ŀ%(

GPD1-RT-for " GPD1-RT-rev GAPDH ̭ « Ŀ % ( GAPDH-RT-for "

GAPDH-RT-rev=Ɍ

Real Time PCR'ˣǟ%(LightCycler^® Software Ver.3.5 (Roche)=Ɍ ˣǟ

=˕

Ά Saccharomyces cerevisiae%69Erythrose reductasḙ«Ŀ'ɒɵɔɅ 7-1 ER̭«ĿŷáɔɅpL[†'Dzʈ

M. megachiliensis'cDNA68KOD-Plus (TOYOBO)=Ɍ ER̭«Ŀ=

::İŨEJZeL@†YȔƊ=ĸȔŽ%Go taq DNA

Polymerase (Promega)=Ɍ İṶ̃«Ŀ'3’Ǘɿ%Įī̲Ü'A=¢ë

'̭«ĿǀȺ=pGEM^®-T Easy Vector (Promega)%RnL‚†d„M İŨĮī̲Üǻ­̈́ 9Įī̲Ü=ˣǟǻ

­̈́ ̲Ü=ER1̭«Ŀ 6*ER2̭«Ŀ'ĭčXbaI (TaKaRa)"SacI (TaKaRa)ER3̭«Ŀ'ĭčHindIII (TaKaRa)"XbaI (TaKaRa)!Įī̲Ü=Û ǀďʘ0č<'á̵͍ʔ!Ûǀ @€HmFVm?[#YØɇ=

pDB05pL[†"~BO†T}„=˕E. coli DH5a%̭«ĿőÎ ER

̭«ĿŷáɔɅpL[†=Ĵ̽%ƀ (Fig. 3)cDNA'İŨ%Ɍo~Bu

†(ER1̭«Ŀ 6*ER2̭«ĿER1.2-XbaI-for"ER1.2-SacI-revER3̭«

Ŀ'İŨ%(ER3-HindIII-for"ER3-XbaI-rev=Ɍ 8:7o~Bu†

%(á̵͍ʔ˲˼9̲Ü¢ë: 9

7-2 ̭«ĿőÎ

̭«ĿőÎ9S. cerevisiae BY4741Ǩ"'GCY1̭«ĿǸƯǨ=YPDĪħ 3 mL=ˮͷʅ%ǭˇ30˚C!16nj͇Īͱ'ˇȚ=YPDĪħ100 mL

=500 mLŊˆˢm~VP%ǭˇƥ"ĪͱǷ ([HKÕǷǨųªɬ) =Ɍ

200 rpm ! OD600 0.40.6 %̨9/!ĪͱĪͱˇ²'

50 mL=50 mL]{†n=Ɍ 3,000 rpm10 Ú4˚C!̪ƄÚ͛%6 ͙ ˇĪħ=͎Ă10 mM Tris-HCl (pH7.5)0.1M LiAcȝčȚ!Óƒȭ Ó*ďǜ¦!̪ƄÚ͛'µǯ=2ġʦ8̗500 µL'10 mM Tris-HCl

(pH7.5)0.1M LiAcȝčȚ!ȩˇ 1.5 mL]{†n%ˇȚ=100 µL

ć8őÎ9̭«Ŀ=1 µgɜŸë7%10 mM Tris-HCl (pH7.5)0.1M LiAc45% PEG (%4000)ȝčȚ 300 $L=1 mLë 30˚C!15Ú͇ëȤ

'Ž42˚C!30Ú͇ëȤ10,000 rpm1Ú͇̪ƄÚ͛=˕͙ˇ

TE=1 mLë Óƒȭ'100 µL=SD-LeuĪħ%Ʊ30˚C!Ī ͱɊʱ˟7:ˇ²(P‚d†\BLaPCR (initial denaturation 95˚C for 2 min; 30 cycles 95˚C for 30 min, 56˚C (ER1,2) or 55˚C (ER3) for 30 min, 72˚C for 30 min; final elongation 72˚C 2 min, 4˚C pause)=˕Ź̇̍ƭ'ɨ˲=˕

7-3 ̵ʔȔƊ'ȥŅ

7-3-1 [„jL̇ƜÙ

͙ˇR„o€%Ŏ ˇ²'̻%Ŏ ʂ̽'!-@€vc (12 $m)

"'3Á̽'0.2 M phosphate buffer (pH 7.5)=ë“̿=Ɍ ˇ²ɧɦ=

˕ɧɦˇ²=3,000 rpm!10Ú͇̪ƄÚ͛‰ȟ=ʊ[„jLƜ ÙȚ" BCA^TM Protein Assay kit (PIERCE23227) =Ɍ Ȯů=ȥŅ 7-3-2 ER'̵ʔȔƊ

1.5 mL]{†n%ʗȮů50 mM phosphate buffer (pH 6.5) 3 mM erythrose (ƒP†NvH€) 5 mM NADPH20 $g ɜŸ'ʊ[„jLƜÙȚ=ë30˚C

!30Ú͇ąƆȶȍ ąƆ=ÄǺŽBG„L‚uaM~mA

†TV_w (ICS-3000DIONEX) =Ɍ erythritol'ɊƓ̽=ɨ˲

ʁ 3 ʆ ʚǢ

1 Moniliella megachiliensisOgv^Lm?†U~Bn~†7'VL†d

„M"Erythrose reductasḙ«Ŀ'̲Üˣǟ

ER1̭«ĿER2̭«ĿER3̭«Ŀ=Ē1m?†U=Ogv^Lm?†U

~Bn~†7::û͛9"!/'m?†UDNA

=ʍ˚NotI%69á̵͍ʔșó'ŽpBluescript II SK(+)=Ɍ RnL‚†

d„M=Ė̔Įī̲Ü'ˣǟ=˕

cDNA% 9::'ɜďƊ(ER1̭«Ŀ"ER2̭«Ŀ!97%ER1̭

«Ŀ"ER3̭«Ŀ!76%ER2̭«Ŀ"ER3̭«Ŀ!76%=ɪER1̭«Ŀ

"ER2̭«Ŀ(ͩ®Ɗ͸"ɨ˲: m?†U=VL†d

„M"%68Ė̔Įī̲Ü3ͩ® 9"ɨ˲!ER1̭

«Ŀ'‰ȕ2,174 bp 6*Šȕ1,102 bpER2̭«Ŀ'‰ȕ2,041 bp 6*Šȕ 1,097 bpER3̭«Ŀ'‰ȕ3,392 bp 6*838 bp=ˣǟ9"!ER1

̭«Ŀ"ER2̭«Ŀ(cDNA% (ɜďƊ͸"(ɨ˲!

'ˣǟ%68‰Šȕ% 3ɜďƊ͸"ɨ˲:/ER3

̭«Ŀ'‰ȕ-148 bp"-80 bp'¯ʧ%STRE˟:ER1̭«ĿER2

̭«Ŀ%3STREŀĦ::‰ȕ7-1,072 bp-1,071 bp"ER3

̭«Ŀ683͆ļx]Gd„7͛:¯ʧ%ɨ˲: (Fig. 4):'̭

«Ŀ% 3Ė̔Įī̲Ü%tG†€'ɊƓ%͈Œ 9̭«Ŀ(˟7

:L~V[†ŹƓ( $

2 VaVƆʃ%69tG†€ˌɸ̽"Erythrose reductasḙ«Ŀ'ɔɅ̽

ǟ

2-1 VaVÝǔƆʃ%69ˇ²'tG†€ɊƓ̽'IJó

20% glucose!60Ú͇Ș̚ĥVaV=́˄ˇ²"́˄ $ˇ²

% erythritol"glycerol'ɊƓ̽% Ǯ˧'ʚǢǑĶ30

Ú͇Øɇˇ²% Øɇ $ˇ²683glycerol!ʐ2Á

erythritol!ʐ3Áİë ɊƓ̽" (glycerolʐ75 $mol/mg cell%Ŏerythritol(ʐ35 $mol/mg cell!8glycerol'ǂĴɊƓ:

(Fig. 5a)

2-2 VaVÝǔƆʃ%69ˇ²'ɔɅ̽Ņ̽ˣǟ

20% glucose!30Ú͇Ș̚ĥVaV=́˄ˇ²"́˄ $ˇ²

% ER1̭«Ŀ 6*ER2̭«ĿER3̭«ĿGPD1̭«Ŀ'ɔɅ̽

=Real Time PCR%6 Ņ̽ˣǟhCVJ†l„M̭«Ŀ"

GAPDH̭«Ŀ=Ɍ'ʚǢ=Fig. 5b%ɪVaV́˄'$ˇ²

20% glucose%69VaV=́˄ˇ²"3%ER1 6*ER2̭«Ŀ'ɔ

Ʌ̽ER3̭«Ŀ'ɔɅ̽%IJó(˟7:$‡ǂGPD1̭«Ŀ'ɔɅ

̽(ʐ60Á'İë˟7:

3 erythritolɊƓnj'ȾŅ"Erythrose reductasḙ«Ŀ'ɔɅ̽ǟ

3-1 tG†€ɊƓnjǔ'ʙnjIJó

erythritol'ɊƓ'ʙnjIJó=˶,92%2% glucoseǜ¦Š"20% glucose ǜ¦Š!Īͱ=˕'ˇ²Ò%ˌɸ:ʎ"ĪħǾŀglucose̽ 6*

ˇ²̻̽=ȥŅ'ʚǢ=Fig. 6%ɪ2% glucoseǜ¦Š!(erythritol ˌɸ̽24nj͇¤͌‰Lj36nj͇!ǑĶ%̨glycerol'ɊƓ̽(

<!-">#IJó˟7:$/36nj͇'njȰ!Īħ'glucose ǣȠ %3<7erythritol'ɊƓ˕<: 20% glucose ǜ¦Š!(24nj͇/!(glycerol'ɊƓɘ>%˕<: :¤͌(

Ȣœ'Çđ˟7:‡ǂerythritol ( 12 nj͇¤͌ 36 nj͇/!(İë'Ç đ˟7:48nj͇¤͌˃ũȢœÇđ˟7:

3-2 ͟Ǻǔ/!Īͱnj'ER1̭«Ŀ 6*ER2̭«ĿER3̭«ĿGPD1

̭«Ŀ'ɔɅ̽ˣǟ

2%Ą*20% glucoseǜ¦Š!'12nj͇772nj͇/!'ER1̭«Ŀ 6*

ER2̭«ĿER3̭«ĿGPD1̭«Ŀ'ɔɅ̽=Real Time PCR=Ɍ ˶, 2-2"ďdz%hCVJ†l„M̭«Ŀ%(GAPDH̭«Ŀ=ɌʚǢ

=Fig. 7%ɪER1̭«Ŀ 6*ER2̭«Ŀ'ɔɅ̽(24nj͇Ž%ƃİ

'Ž°Š'%ŎER3̭«Ŀ'ɔɅ̽(24nj͇!ǑĶ%̨Ž3-

">#°Š9"$/ER3̭«Ŀ(2%68320%'ǂ͸ɔ

Ʌ 820% glucoseǜ¦Š'12nj͇Īͱnj"24nj͇Īͱnj'Ȅ̎!(Ž

ʮ25Áİë 2% glucoseǜ¦Š!72nj͇Īͱˇ²!3ER3̭

«Ŀ'ɔɅ̽(İë ‡ǂ12 nj͇¤‰ĪͱĭčGPD1 ̭«Ŀ