Saccharomyces cerevisiae ⳦ࡢゎᯒ

C5C6 ⢾㧗ຠ⋡Ⓨ㓝࡟㈨ࡍࡿ

Saccharomyces cerevisiae ⳦ࡢゎᯒ

2017 ᖺ 3 ᭶

ᓫᇛ኱Ꮫ኱Ꮫ㝔ᕤᏛ◊✲⛉

ᛂ⏝ᚤ⏕≀ᕤᏛᑓᨷ ᚤ⏕≀㑇ఏᕤᏛㅮᗙ

㇂ 㱟 ඾

i

┠ḟ

㡫

⥴ㄽ 1

➨ 1 ❶ ࢟ࢩࣜࢺ࣮ࣝ㈨໬ᛶࡢ Saccharomyces cerevisiae ✺↛ኚ␗యࡢศ㞳࡜≉ᚩ

࡙ࡅ

9

➨ 1 ⠇ ⥴ゝ 9

➨ 2 ⠇ ᐇ㦂ᮦᩱ࡜ᐇ㦂᪉ἲ 11

1.2.1 ⳦ᰴ㸪ࣉࣛࢫ࣑ࢻ㸪࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࣉࣛ࢖࣐࣮ 11

1.2.2 ᇵᆅ 16

1.2.3 㓝ẕࡢᙧ㉁㌿᥮ 18

1.2.4 㓝ẕ㑇ఏᏛ࡛⏝࠸ࡿᶆ‽᪉ἲ 19

1.2.5 㓝ẕᰁⰍయ DNA ࡢㄪ〇 20

1.2.6 High Pure Plasmid Isolation Kit ࢆ⏝࠸ࡓ኱⭠⳦࠿ࡽࡢࣉࣛࢫ࣑ࢻ DNA ᢳ ฟ

21

1.2.7 ࢔࣮࢞ࣟࢫࢤࣝ㟁ẼὋື 22

1.2.8 PCR ἲ࡟ࡼࡿ DNA ᩿∦ࡢቑᖜ 22

1.2.9 ሷᇶ㓄ิỴᐃ 23

1.2.10 㓝ẕࡢ඲ࢤࣀ࣒ሷᇶ㓄ิࡢ෌ゎᯒ 23

1.2.11 cre Ⓨ⌧࡟ࡼࡿ kanMX ࣐࣮࣮࢝ࡢ㝖ཤ 24

1.2.12 ࢟ࢩࣜࢺ࣮ࣝ㈨໬ᛶ✺↛ኚ␗యࡢศ㞳 24

➨ 3 ⠇ ⤖ᯝ 25

1.3.1 Saccharomyces cerevisiae ࡢ࢟ࢩࣜࢺ࣮ࣝ㈨໬ᛶ✺↛ኚ␗యࡢศ㞳 25

1.3.2 Xyt

ኚ␗య SCC7, SCC8, SCC15 ࡢኚ␗⟠ᡤࡢゎᯒ 28

1.3.3 xytѸ7, xytѸ8, xytѸ15 ࡢ≉ᐃ 29

1.3.4 ࢟ࢩࣜࢺ࣮ࣝ㈨໬࡟࠾ࡅࡿ Gal2 ㍺㏦యࡢᙺ๭ 32

ii

➨ 4 ⠇ ⪃ᐹ 35

➨ 5 ⠇ せ⣙ 37

➨ 2 ❶ ࢟ࢩࣜࢺ࣮ࣝ࡜࢟ࢩ࣮ࣟࢫ㈨໬ᛶ Saccharomyces cerevisiae ࡢ࢟ࢩࣜࢺ

࣮ࣝ௦ㅰゎᯒ

38

➨ 1 ⠇ ⥴ゝ 38

➨ 2 ⠇ ᐇ㦂ᮦᩱ࡜ᐇ㦂᪉ἲ 39

2.2.1 ⳦ᰴ࡜ࣉࣛࢫ࣑ࢻ㸪࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࣉࣛ࢖࣐࣮ 39

2.2.2 ᇵᆅ 43

2.2.3 ᙧ㉁㌿᥮㸪㓝ẕᰁⰍయ DNA ࡢㄪ〇࠾ࡼࡧ㓝ẕ㑇ఏᏛ࡛⏝࠸ࡿᶆ‽᪉ἲ 44

2.2.4 High Pure Plasmid Isolation Kit ࢆ⏝࠸ࡓ኱⭠⳦࠿ࡽࡢࣉࣛࢫ࣑ࢻ DNA ᢳ ฟ

44

2.2.5 ࢔࣮࢞ࣟࢫࢤࣝ㟁ẼὋື 44

2.2.6 ሷᇶ㓄ิỴᐃ 44

2.2.7 ᅇᩥᇵ㣴 44

2.2.8 HPLC ศᯒ 44

➨ 3 ⠇ ⤖ᯝ 46

2.3.1 xyt–15 ✺↛ኚ␗య࡜ぶᰴࡢ᎘Ẽⓗ᮲௳ୗ࡛ࡢⓎ㓝ヨ㦂 46

2.3.2 gal80Δ ᰴ࡜ TDH3p–GAL2 ᰴࡢዲẼⓗ࠾ࡼࡧ᎘ẼⓗⓎ㓝ゎᯒ 48

2.3.3 ࢟ࢩࣜࢺ࣮ࣝ௦ㅰ࡟ཬࡰࡍ࢔ࢭࢺ࢔ࣝࢹࣄࢻ (NADH ࡢຠᯝⓗ࡞㓟໬๣ )

ࡢຠᯝ

52

➨ 4 ⠇ ⪃ᐹ 54

➨ 5 ⠇ せ⣙ 56

iii

➨ 3 ❶ ࢟ࢩࣜࢺ࣮ࣝ㈨໬ᛶࡢ Saccharomyces cerevisiae ࢆ⏝࠸ࡓ࢟ࢩࣜࢺ࣮ࣝ

㍺㏦ࢱࣥࣃࢡࡢゎᯒ 57

➨ 1 ⠇ ⥴ゝ 57

➨ 2 ⠇ ᐇ㦂ᮦᩱ࡜ᐇ㦂᪉ἲ 59

3.2.1 ⳦ᰴ࡜ࣉࣛࢫ࣑ࢻ㸪࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࣉࣛ࢖࣐࣮ 59

3.2.2 ᇵᆅ 64

3.2.3 ᙧ㉁㌿᥮㸪㓝ẕᰁⰍయ DNA ࡢㄪ〇࠾ࡼࡧ㓝ẕ㑇ఏᏛ࡛⏝࠸ࡿᶆ‽᪉ἲ 64

3.2.4 High Pure Plasmid Isolation Kit ࢆ⏝࠸ࡓ኱⭠⳦࠿ࡽࡢࣉࣛࢫ࣑ࢻ DNA ᢳฟ

64

3.2.5 ࢔࣮࢞ࣟࢫࢤࣝ㟁ẼὋື㸪 PCR ἲ࡟ࡼࡿ DNA ᩿∦ࡢቑᖜ㸪ሷᇶ㓄ิ

Ỵᐃ

64

3.2.6 ᅇᩥᇵ㣴 65

3.2.7 HPLC ศᯒ 65

3.2.8 ࢟ࢩࣜࢺ࣮ࣝࡢ⣽⬊ෆ࡬ࡢྲྀࡾ㎸ࡳゎᯒ 65

3.2.9 ࢟ࢩࣜࢺ࣮ࣝࡢ⣽⬊እ࡬ࡢ᤼ฟゎᯒ 66

➨ 3 ⠇ ᐇ㦂ࢹࢨ࢖ࣥ 66

➨ 4 ⠇ ⤖ᯝ 69

3.4.1 ࢟ࢩࣜࢺ࣮ࣝࡢྲྀࡾ㎸ࡳࢆ⾜࠺ Gal2, Fps1, Hxt15 69

3.4.2 ࢟ࢩࣜࢺ࣮ࣝࡢ⣽⬊እ㍺㏦ࢆ⾜࠺ Gal2 ࡜ Fps1 71

3.4.3 ࢟ࢩࣜࢺ࣮ࣝࡢ⣽⬊እ㍺㏦࡟ᑐࡍࡿ Hxt15 ࡜ Hxt11 ࡢᙺ๭ 71

➨ 5 ⠇ ⪃ᐹ 74

➨ 6 ⠇ せ⣙ 77

⥲ᣓ 78

ᘬ⏝ᩥ⊩ 81

iv

ᮏㄽᩥ࡟㛵ࡍࡿ୺࡞ሗ࿌ 91

ㅰ㎡ 92

1

⥴ ⥴ㄽ

ෆ㛶ᗓ࣮࣒࣍㸭ෆ㛶ᗓࡢ᪋⟇㸭⥲ྜ⛉Ꮫᢏ⾡࣭࢖ࣀ࣮࣋ࢩࣙࣥ఍㆟㸭ᑓ㛛ㄪᰝ

఍࣭᠓ㄯ఍➼㸭ᡓ␎༠㆟఍࣭࣮࣡࢟ࣥࢢࢢ࣮ࣝࣉ㸭࢚ࢿࣝࢠ࣮ᡓ␎༠㆟఍ ( ➨㸱ᅇ ) ࡀ㸪2014 ᖺ 1 ᭶ 29 ᪥࡟㛤ദࡉࢀࡓ

(http://www8.cao.go.jp/cstp/tyousakai/juyoukadai/energy/ 3kai/ haifu _energy_03.html) ࠋࡑ ࡢ୰ࡢ ㆟㢟 (3) ᖹᡂ 26 ᖺᗘ࢔ࢡࢩࣙࣥࣉࣛࣥ≉ᐃ᪋⟇ࣞࣅ࣮࡛ࣗ㸪 ࠕࣂ࢖࢜⇞ᩱࠖ

࡞࡝ࡢ᪋⟇ࣉࣞࢮࣥࢸ࣮ࢩࣙࣥࡀⓎ⾲ࡉࢀࡓ ( ㈨ᩱ 3-4 㸪ࣂ࢖࢜⇞ᩱ᪋⟇ㄝ᫂㈨ᩱ ) ࠋ

⤒῭⏘ᴗ┬࡛ࡣ㸪኱つᶍ࠿ࡘᏳᐃⓗ࡟ࣂ࢖࢜⇞ᩱࢆ⏕⏘ࡍࡿࡓࡵࡢᢏ⾡㛤Ⓨ࡟㞟୰

ⓗ࡟ྲྀࡾ⤌ࡳ㸪ᅜෆࡢ⏕⏘࡟࡜࡝ࡲࡽࡎ㸪㛤Ⓨ㍺ධࢆᛕ㢌࡟ධࢀࡓࣂ࢖࢜⇞ᩱ⏕⏘

ᢏ⾡ࡢ㛤Ⓨࢆ⾜ࡗ࡚࠸ࡿࠋ㎰ᯘỈ⏘┬࡛ࡣ㸪ᅜෆ࡟㈿Ꮡࡍࡿࣂ࢖࣐࢜ࢫࢆά⏝ࡋ㸪

㎰ᒣ⁺ᮧᆅᇦ࡟࠾ࡅࡿ࢚ࢿࣝࢠ࣮ࡢᆅ⏘ᆅᾘࢆ㐍ࡵࡿࡓࡵ࡟㸪ࡑࢀࡒࢀࡢᆅᇦࢆά

࠿ࡋࡓ◊✲ࢆᐇ᪋ࡍࡿࠋᩥ㒊⛉Ꮫ┬࡛ࡣ㸪㛗ᮇⓗ࡟ࣂ࢖࣐࢜ࢫࡢ฼⏝࣭Ᏻᐃ౪⤥ࡀ

ྍ⬟࡞ᢏ⾡ࡢ๰ฟ࡟ྥࡅ࡚㸪ḟୡ௦ࡢࣂ࢖࣐࢜ࢫᢏ⾡࡟㛵ࡍࡿᇶ♏ⓗ◊✲ࢆ᥎㐍ࡍ

ࡿࠋࡲࡓ㸪ᢏ⾡ⓗㄢ㢟ࢆゎỴࡍࡿࡓࡵࡢ◊✲ࢆ㛵ಀ 3 ┬ᗇ࡛ᐇ᪋ࡍࡿࠋ࢚ࢿࣝࢠ࣮

౪⤥ᵓ㐀㧗ᗘ໬ἲ࡟ᇶ࡙ࡃࣂ࢖࢚࢜ࢱࣀ࣮ࣝ฼⏝┠ᶆࡣ㸪 2017 ᖺࡲ࡛࡟ 50 ୓ kL ࡜ タᐃࡉࢀ㸪࢞ࢯࣜࣥࡢ 3㸣┦ᙜ௨ୖࢆ 2020 ᖺࡲ࡛࡟ᑟධࡍࡿࡇ࡜ࡀ࢚ࢿࣝࢠ࣮ᇶᮏ ィ⏬࡛࠶ࡿࠋ㎰ᒣ⁺ᮧ࡟㇏ᐩ࡟Ꮡᅾࡍࡿ෌⏕ྍ⬟㈨※ࠕࣂ࢖࣐࢜ࢫࠖࡣ㸪ࢢ࣮ࣜ

࣭ࣥ࢖ࣀ࣮࣋ࢩࣙࣥࡀᐇ⌧ࡍࡿ࡜᪂ࡓ࡞㞠⏝࡜⤒῭ᡂ㛗ࢆ⏕ࡳฟࡍ㔜せ࡞㈨※࡜࡞

ࡿࠋࡇࡢࡼ࠺࡞ࣂ࢖࣐࢜ࢫࡢά⏝᥎㐍ࡀᚲせ࡛࠶ࡿࠋࡓࡔࡋ㸪ࣂ࢖࣐࢜ࢫࢆ฼⏝ࡋ ࡓ⏘ᴗࢆ☜❧ࡋ࡚࠸ࡃࡓࡵ࡟ࡣ㸪ᵝࠎ࡞ᢏ⾡ⓗㄢ㢟ࡀᏑᅾࡍࡿࠋ㎰ᒣ⁺ᮧ࡟⏤᮶ࡍ

ࡿࣂ࢖࣐࢜ࢫࡢά⏝ࢆ᥎㐍ࡋ㸪ᆅ⏘ᆅᾘ࡜࢚ࢿࣝࢠ࣮※ࡢከᵝ໬ࢆᅗࡿࡓࡵ࡟㸪㣗

⣊࡜➇ྜࡋ࡞࠸ཎᩱࢆ⏝࠸ࡓ⇞ᩱ໬ࢆ┠ᣦࡋ㸪࢞ࢯ࣭ࣜࣥ㍍Ἔ➼࡟ᑐᢠࡋ࠺ࡿࣂ࢖

࢜⇞ᩱ〇㐀ࢥࢫࢺࡀồࡵࡽࢀࡿࠋࡑࡢ┠ᶆ㐩ᡂࡸ࢚ࢿࣝࢠ࣮ᇶᮏィ⏬࡟ᥖࡆࡽࢀࡓ

ᑟධ㔞ࡢ┠ᶆ㐩ᡂ࡟ຍ࠼㸪㧗௜ຍ౯್≀㉁ࢆ๰ฟࡋ㸪ᐇ㊶ⓗ࡞⏕⏘ᢏ⾡ࡢ☜❧ࢆ┠

2

ᣦࡍࡇ࡜ࡀ㸪ࣂ࢖࣐࢜ࢫ◊✲ࡢ┠ᶆ࡛࠶ࡿࠋ

ࠕࣂ࢖࢜⇞ᩱᢏ⾡◊✲㛤Ⓨࠖ࡟㛵ࡍࡿ⤒῭⏘ᴗ┬ࡢᴫせࡣ㸪௨ୗࡢ㏻ࡾ࡛࠶ࡿࠋࢭ

࣮ࣝࣟࢫ⣔ࣂ࢖࣐࢜ࢫ࠿ࡽ࢚ࢱࣀ࣮ࣝࢆ⏕⏘ࡍࡿࢩࢫࢸ࣒⥲ྜ㛤Ⓨᐇド஦ᴗࢆ⾜࠺ࠋ

஦ᴗࡢᴫせ࡜┠ⓗࡣ㸪㣗⣊࡜➇ྜࡋ࡞࠸ࢭ࣮ࣝࣟࢫ⣔㈨※స≀⏤᮶ࡢ኱つᶍ࡞࢚ࢱࣀ

࣮ࣝ⏕⏘ࡢ◊✲࡟ࡼࡾ㸪஦ᴗ໬࡟ྥࡅࡓࢥࢫࢺపῶࢆ┠ᣦࡍࡇ࡜࡛࠶ࡿࠋࡑࡢࡓࡵ࡟㸪 1 㸧ᐇ⏝໬ࣞ࣋ࣝ࡟࠶ࡿ࢚ࢱࣀ࣮ࣝ⏕⏘ࡢせ⣲ᢏ⾡࡟ࡘ࠸࡚㸪ᢏ⾡ࡢ⥲Ჴ༺ࡋࢆ⾜࠸㸪 ලయⓗ࡟᭱㐺࡞ᢏ⾡ࡢ⤌ྜࡏࢆ᳨ウࡋ㸪 2㸧᳨ウ⤖ᯝ࠿ࡽ㸪ࣉࣞၟ⏝ẁ㝵࡛ࡢ኱つᶍ୍

㈏⏕⏘ࢩࢫࢸ࣒࡟࠾ࡅࡿ㐺⏝ࢆ᝿ᐃࡋ㸪ࢭ࣮ࣝࣟࢫ⣔ࣂ࢖࣐࢜ࢫཎᩱ࡜㐺ྜᛶࡢ㧗࠸

๓ฎ⌮ᢏ⾡㸪㓝⣲ࢥࢫࢺపῶࡢࡓࡵࡢ⢾໬ᢏ⾡࠾ࡼࡧⓎ㓝ຠ⋡ྥୖᢏ⾡ࢆ㑅ᢥࡋ㸪ᢏ

⾡ࡢ⤌ྜࡏホ౯ࢆ⾜࠺➼㸪஦ᴗ໬ࢆぢᤣ࠼ࡓ᳨ド࣭㛤Ⓨࢆᐇ᪋ࡋ㸪 3 㸧⡿ᅜ➼ࡢ᭱᪂ࡢ 㛤Ⓨືྥ㸪ᣢ⥆ྍ⬟ᛶࡢホ౯᪉ἲ➼࡟ࡘ࠸࡚ࡢㄪᰝ◊✲ࢆᐇ᪋ࡍࡿࡇ࡜࡛࠶ࡿࠋ ࢔ࣝ࢝ࣜࡸ㓟⁐ᾮ࡛ࢭ࣮ࣝࣟࢫ⣔ࣂ࢖࣐࢜ࢫࢆᾐₕࡋࡓᚋ࡟⵨Ẽฎ⌮ࡸ⵨Ẽ⇿○

ฎ⌮ࡋ (1, 2)㸪⥆࠸࡚ࢭ࣮ࣝࣛࢮࡸ࣑࣊ࢭ࣮ࣝࣛࢮ࡞࡝ࡢ㓝⣲ฎ⌮࡛ࢢࣝࢥ࣮ࢫ࡜࢟

ࢩ࣮ࣟࢫ࡟ຍỈศゎࡋ࡞ࡀࡽྠ᫬࡟Ⓨ㓝ࡶ⾜࠺ྠ᫬⢾໬୪⾜」Ⓨ㓝ἲࡣ (3, 4) 㸪⃰◲

㓟࡞࡝ࡢ໬Ꮫⓗฎ⌮࡛ࣂ࢖࣐࢜ࢫཎᩱࢆ⢾࡟ຍỈศゎࡍࡿ᪉ἲ (5)࡜ࡣ␗࡞ࡾ㸪◲㓟 ฎ⌮ࡸࡑࡢ◲㓟ᅇ཰ᕤ⛬ࡀᚲせ࡞࠸ࡢ࡛⎔ቃ࡟ඃࡋࡃ㸪⇞ᩱ⏝࢚ࢱࣀ࣮ࣝ⏕⏘ࡢ୺せ

࡞ࣉࣟࢭࢫࡢࡦ࡜ࡘ࡜࡞ࡗ࡚࠸ࡿࠋ㔝⏕ᆺࡢ㓝ẕ Saccharomyces cerevisiae ࡣ㸪ࢢࣝࢥ

࣮ࢫࢆ࢚ࢱࣀ࣮ࣝ࡟ኚ᥮࡛ࡁࡿࡀ㸪࢟ࢩ࣮ࣟࢫࢆ࢚ࢱࣀ࣮ࣝ࡟ࡣ࡛ࡁ࡞࠸ (6) ࠋࡑࡇ

࡛㸪࢟ࢩ࣮ࣟࢫ࠿ࡽ࢟ࢩ࣮ࣝࣟࢫ࡬௦ㅰ࡛ࡁࡿ⤌ࡳ᥮࠼㓝ẕࡀసᡂࡉࢀ࡚࠸ࡿ (6)ࠋ ࡍ࡞ࢃࡕ㸪 Scheffersomyces (Pichia) stipitis ࡢ XYL1 ( ࢟ࢩ࣮ࣟࢫࣜࢲࢡࢱ࣮ࢮ XR ࢆࢥ

࣮ࢻࡍࡿ)࡜ XYL2 (࢟ࢩࣜࢺ࣮ࣝ⬺Ỉ⣲㓝⣲ XDH ࢆࢥ࣮ࢻࡍࡿ)ࢆ S. cerevisiae ࡛Ⓨ⌧

ࡉࡏ࡚࢟ࢩ࣮ࣟࢫࢆ࢟ࢩ࣮ࣝࣟࢫ࡟௦ㅰࡍࡿ࠿㸪 Piromyces sp. (7, 8), Orpinomyces sp.

(9), ࡸ Clostridium ᒓ⣽⳦ (10)࡞࡝ࡢ࢟ࢩ࣮ࣟࢫ࢖ࢯ࣓࣮ࣛࢮ xylA ࢆ S. cerevisiae ࡛

Ⓨ⌧ࡍࡿ⤌ࡳ᥮࠼㓝ẕ⳦ࡀసᡂࡉࢀ࡚࠸ࡿ (11) ࠋ࢟ࢩ࣮ࣝࣟࢫࡣ S. cerevisiae ࡢ࢟ࢩ

ࣝࣟ࢟ࢼ࣮ࢮ࡟ࡼࡗ࡚࢟ࢩ࣮ࣝࣟࢫ 5−ࣜࣥ㓟࡟ኚ᥮ࡉࢀ㸪ḟ࡟࣌ࣥࢺ࣮ࢫࣜࣥ㓟ᅇ

3

㊰ࡢ㓝⣲⩌࡟ࡼࡗ࡚ࣇࣛࢡࢺ࣮ࢫ 6-ࣜࣥ㓟࡜ࢢࣜࢭࣝ࢔ࣝࢹࣄࢻ 3-ࣜࣥ㓟࡟ኚ᥮ࡉ

ࢀ㸪࢚ࣥࣈࢹ࣭࣐ࣥ࢖࣮ࣖ࣍ࣇ࣭ࣃࣝࢼࢫᅇ㊰࡛࢚ࢱࣀ࣮ࣝ࡬࡜ኚ᥮ࡉࢀࡿ (6) ࠋ ⣽⬊ෆ࡬ࡢ࢟ࢩ࣮ࣟࢫ㍺㏦ࡣ㸪࣊࢟ࢯ࣮ࢫ㍺㏦ࢱࣥࣃࢡ㉁࡛࠶ࡿ Hxt7 ࡸ Hxt5 ࠶ࡿ

࠸ࡣ࢞ࣛࢡࢺ࣮ࢫྲྀࡾ㎸ࡳࢆ⾜࠺ Gal2 ࡟ࡼࡗ࡚⣽⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡿ (11, 12) ࠋࡇࢀ

ࡽ㍺㏦ࢱࣥࣃࢡࡢ࢟ࢩ࣮ࣟࢫ࡟ᑐࡍࡿぶ࿴ᛶࡣ㸪Km ್࡛ 100 mM ࠿ࡽᩘ 100 mM ࡜ ᴟࡵ࡚పࡃ㸪ࡲࡓࢢࣝࢥ࣮ࢫ࡟ᑐࡍࡿぶ࿴ᛶࡼࡾࡶప࠸ (13, 14) ࠋࡑࡇ࡛㸪᭱ึ࡟ࢢ

ࣝࢥ࣮ࢫࡀྲྀࡾ㎸ࡲࢀ࡚௦ㅰࡉࢀ㸪⥆࠸࡚࢟ࢩ࣮ࣟࢫࡀྲྀࡾ㎸ࡲࢀ࡚௦ㅰࡉࢀࡿࡓࡵ㸪 ࢢࣝࢥ࣮ࢫ࡜࢟ࢩ࣮ࣟࢫࡢྠ᫬Ⓨ㓝ࡣ㞴ࡋ࠸࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋ Trichoderma reesei (Hypocrea jecorina)ࡢ Trxlt1 ࡣ࢟ࢩ࣮ࣟࢫࡢࡳࢆྲྀࡾ㎸ࡴࡢ࡛ (14)㸪S. cerevisiae ࡢ࢟

ࢩ࣮ࣟࢫྲྀࡾ㎸ࡳ࡟㐺ࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡿࡀ㸪࢟ࢩ࣮ࣟࢫ࡟ᑐࡍࡿぶ࿴ᛶࡣ㸪 Km

್࡛ 130 mM ࠿ࡽ 900 mM ࡜పࡃ㸪෇⁥࡞ྠ᫬Ⓨ㓝࡟ࡣྥ࠸࡚࠸࡞࠸ࠋ

4

Candida intermedia ࡢ࢟ࢩ࣮ࣟࢫ / ࢢࣝࢥ࣮ࢫ −H

ඹ㍺㏦ࢱࣥࣃࢡ Gxs1 ࡢ࢟ࢩ࣮ࣟࢫ࡟

ᑐࡍࡿぶ࿴ᛶࡣ㸪Km = 0.2 mM (30 mg/L)࡜㠀ᖖ࡟㧗࠸ࡀ㸪ࢢࣝࢥ࣮ࢫ࡟ᑐࡍࡿぶ࿴ᛶ ࡢ᪉ࡀࡼࡾ㧗ࡃ㸪ྲྀࡾ㎸ࡳ㔞ࡶ㠀ᖖ࡟ప࠸ (15, 16) ࠋ Gxs1 ࢆࢢࣝࢥ࣮ࢫᏑᅾୗ࡛㧗Ⓨ

⌧ࡉࡏࡿࡓࡵ࡟㸪TDH3 (ࢢࣜࢭࣝ࢔ࣝࢹࣄࢻ-3-ࣜࣥ㓟⬺Ỉ⣲㓝⣲ࢆࢥ࣮ࢻࡍࡿ)ࣉࣟ

࣮ࣔࢱ࣮ࡢᨭ㓄ୗ࡟ GXS1 ࢆ⨨࠸࡚ࡶ࢚ࢱࣀ࣮ࣝ⏕⏘࡟㢧ⴭ࡞ຠᯝࡣཬࡰࡋ࡚࠸࡞࠸

(17)ࠋ࢟ࢩ࣮ࣟࢫ௦ㅰࡀᙅࡅࢀࡤ㸪ྲྀࡾ㎸ࡲࢀࡓ࢟ࢩ࣮ࣟࢫ࠶ࡿ࠸ࡣ୰㛫௦ㅰ≀㉁ࡀ

⵳✚ࡋ㸪⤖ᯝ࡜ࡋ࡚㸪ྲྀࡾ㎸ࡳຠ⋡ࡀྥୖࡋ࡞࠸ࡢ࠿ࡶࡋࢀ࡞࠸ࠋ

࢟ࢩ࣮ࣟࢫ௦ㅰࢆ෇⁥࡟ࡍࡿࡓࡵ࡟㸪࢟ࢩ࣮ࣟࢫ࠿ࡽゎ⢾⣔࡟⮳ࡿᚊ㏿ẁ㝵ࡢ཯ᛂ

ࢆᙉࡵࡿࡼ࠺࡞㑇ఏᏊᵓ㐀ࡢᨵኚࡀヨࡉࢀ࡚࠸ࡿࠋ࢟ࢩ࣮ࣝࣟࢫ࠿ࡽ࢟ࢩ࣮ࣝࣟࢫ 5-

ࣜࣥ㓟࡬ࡢ཯ᛂࡣᚊ㏿཯ᛂࡢࡦ࡜ࡘ࡛࠶ࡾ㸪ࡑࡢࡓࡵ࡟࢟ࢩࣝࣟ࢟ࢼ࣮ࢮࢆ㧗Ⓨ⌧ࡍ

ࡿ࠿㸪࠶ࡿ࠸ࡣ㑇ఏᏊᩘࢆቑࡸࡍࡼ࠺࡟ࡍࡿ࡜௦ㅰᨵၿࡀㄆࡵࡽࢀ࡚࠸ࡿ (18) ࠋࢺࣛ

ࣥࢫ࣏ࢯࣥᤄධࢆ⾜࠸㸪࢟ࢩ࣮ࣟࢫ௦ㅰࡀᨵၿࡍࡿ✺↛ኚ␗యࢆศ㞳ࡋ㸪ཎᅉ㑇ఏᏊ

ࢆ≉ᐃࡋࡓ౛ࡶ࠶ࡿ (19) ࠋࡍ࡞ࢃࡕ㸪࣌ࣥࢺ࣮ࢫࣜࣥ㓟ᅇ㊰ࡢ㓝⣲ࢺࣛࣥࢫ࢔ࣝࢻࣛ

࣮ࢮ (Tal1)ࡢⓎ⌧ࡀᚊ㏿࡛࠶ࡾ㸪㧗Ⓨ⌧࡟ࡼࡾ௦ㅰᨵၿ࡛ࡁࡿࡇ࡜㸪ᶵᵓࡣࡼࡃศ࠿

ࡽ࡞࠸ࡀ Pho13 (p- ࢽࢺࣟࣜࣥ㓟≉␗ⓗ࢔ࣝ࢝ࣜ࣍ࢫࣇ࢓ࢱ࣮ࢮ࡜ࣉࣟࢸ࢕ࣥ࣍ࢫࣇ

࢓ࢱ࣮ࢮάᛶࢆ♧ࡍ)ࢆḞኻࡉࡏࡿ࡜௦ㅰᨵၿࡀ㉳ࡁࡿࠋࡲࡓ㸪࣌ࣥࢺ࣮ࢫࣜࣥ㓟ᅇ㊰

ࡢ㓝⣲Ⓨ⌧ࡣప࠸ࡓࡵ㸪࢟ࢩ࣮ࣝࣟࢫ 5- ࣜࣥ㓟࠿ࡽࣜࣈ࣮ࣟࢫ 5- ࣜࣥ㓟࡬ࡢኚ᥮ࢆ⾜

࠺ L -ࣜࣈ࣮ࣟࢫ 5-ࣜࣥ㓟࢚ࣆ࣓࣮ࣛࢮ (Rpe)㸪ࣜࣈ࣮ࣟࢫ 5-ࣜࣥ㓟࠿ࡽ࣮ࣜ࣎ࢫ 5-ࣜ

ࣥ㓟࡬ࡢኚ᥮ࢆ⾜࠺࣮ࣜ࣎ࢫ 5- ࣜࣥ㓟࢚ࣆ࣓࣮ࣛࢮ (Rki) 㸪࢟ࢩ࣮ࣝࣟࢫ 5- ࣜࣥ㓟࡜

࣮ࣜ࣎ࢫ 5-ࣜࣥ㓟࠿ࡽࢭࢻ࣊ࣉࢶ࣮ࣟࢫ 7-ࣜࣥ㓟࡜ࢢࣜࢭࣝ࢔ࣝࢹࣄࢻ 3-ࣜࣥ㓟࡟㸪

࠶ࡿ࠸ࡣ࢟ࢩ࣮ࣝࣟࢫ 5- ࣜࣥ㓟࡜࢚ࣜࢫ࣮ࣟࢫ 4- ࣜࣥ㓟࠿ࡽࣇࣛࢡࢺ࣮ࢫ 6- ࣜࣥ㓟

࡜ࢢࣜࢭࣝ࢔ࣝࢹࣄࢻ 3-ࣜࣥ㓟࡟ኚ᥮ࡍࡿࢺࣛࣥࢫࢣࢺ࣮ࣛࢮ (Tkl)㸪ࢭࢻ࣊ࣉࢶࣟ

࣮ࢫ 7- ࣜࣥ㓟࡜ࢢࣜࢭࣝ࢔ࣝࢹࣄࢻ 3- ࣜࣥ㓟࠿ࡽࣇࣛࢡࢺ࣮ࢫ 6- ࣜࣥ㓟࡜࢚ࣜࢫࣟ

࣮ࢫ 4-ࣜࣥ㓟࡟ኚ᥮ࡍࡿࢺࣛࣥࢫ࢔ࣝࢻ࣮ࣛࢮ (Tal1)ࡢࡍ࡭࡚ࢆ㧗Ⓨ⌧ࡍࡿ㓝ẕ⳦

5

ࡶసᡂࡉࢀ㸪௦ㅰᨵၿࡀㄆࡵࡽࢀ࡚࠸ࡿ (20−22)ࠋ

⇞ᩱ⏝࢚ࢱࣀ࣮ࣝ⏕⏘㓝ẕ S. cerevisiae KF7 ᰴࡣ㸪จ㞟ᛶ㓝ẕ࡛⪏⇕ᛶࡸ⪏㓟ᛶࡸ

㧗⃰ᗘࡢᗫ⢾⻤ࡶ㛗ᮇ㐃⥆Ⓨ㓝࡛ࡁࡿᐇ⏝㓝ẕ࡛࠶ࡿ (23)ࠋࡲࡓ㸪ங㓟ࢆ㈨໬ࡍࡿᛶ

㉁ࡶేࡏᣢࡗ࡚࠸ࡿ (24) ࠋᐇ⏝㓝ẕ≉᭷ࡢ࣍ࣔࢱࣜࢬ࣒ࡢᛶ㉁ࢆᣢࡘࡀ㸪⬊Ꮚᙧᡂ⬟

ࡸ᥃ࡅྜࢃࡏ⬟ࡣᴟ➃࡟ᝏ࠸ࠋࡑࡇ࡛㸪ࡑࢀࡽࢆᨵၿࡋ㸪࠿ࡘᙧ㉁㌿᥮⬟ࡶᐇ㦂ᐊ㓝 ẕ࡜ྠ➼࠿㸪࠶ࡿ࠸ࡣࡑࢀ௨ୖ࡟ඃࢀࡓ࣊ࢸࣟࢱࣜࢬ୍࣒ಸయ㓝ẕ NAM34−4C ᰴࡀ ᵓ⠏ࡉࢀࡓࠋࡑࡢᰴ࠿ࡽྠ㉁⣔⤫ᰴࡀᵓ⠏ࡉࢀ㸪ࡑࡢᰴ࡟࢟ࢩ࣮ࣟࢫ࠿ࡽ࢟ࢩ࣮ࣝࣟ

ࢫ 5- ࣜࣥ㓟ࡢኚ᥮࡟ᚲせ࡞ TDH3p (TDH3 ࣉ࣮ࣟࣔࢱ࣮)−XYL1−TDH3t (TDH3 ࢱ࣮࣑

ࢿ ࣮ ࢱ ࣮ )−TDH3p−XYL2−TDH3t−TDH3p−XKS1 ( ࢟ ࢩ ࣝ ࣟ ࢟ ࢼ ࣮ ࢮ ࢆ ࢥ ࣮ ࢻ ࡍ

ࡿ )−TDH3t 㑇ఏᏊᵓ㐀ࢆ㓝ẕࡢᰁⰍయ࡟⤌ࡳ㎸ࢇࡔᰴ SCB7 ࡀᵓ⠏ࡉࢀࡓ (25) ࠋࡇࡢ

ᰴࡣ༢୍Ⅳ⣲※࡜ࡋ࡚࢟ࢩ࣮ࣟࢫࢆྵࡴ᭱ᑡᇵᆅ࡛ቑṪ࡛ࡁ㸪᭱ࡶ᪩࠸ୡ௦᫬㛫 G

ࡣ 5 ᫬㛫ࢆ♧ࡋ㸪୍⯡ⓗ࡟ᐇ㦂࡟⏝࠸ࡽࢀ࡚࠸ࡿ㓝ẕࡼࡾࡶቑṪ⬟ࡀ㧗࠸ࠋࡑࡢᰴ࠿

ࡽ G

ࡀ 2.5 ᫬㛫࡜࢟ࢩ࣮ࣟࢫ㈨໬ᛶࡀ㧗ࡃ࡞ࡗࡓ HEX (High efficiency xylose

assimilation) ✺↛ኚ␗యࡀศ㞳ࡉࢀࡓ (25) ࠋࡇࡢ㓝ẕࡢ㑇ఏ⫼ᬒ࡛࢟ࢩ࣮ࣟࢫࡢ㈨໬

ᛶ࡟ཬࡰࡍ㑇ఏᏊࡢゎᯒࡀ⾜ࢃࢀ࡚࠸ࡿࠋࡲࡓ㸪࢟ࢩ࣮ࣟࢫࡣ 10 g/L ௨ୖ࡟࡞ࡿ࡜ቑ Ṫ㜼ᐖࡀ㉳ࡇࡾጞࡵ㸪 180 g/L ⃰ᗘࡢ࢟ࢩ࣮ࣟࢫࢆྵࡴᇵᆅ࡛ࡣቑṪ࡛ࡁ࡞࠸ࡀ㸪ࡑ ࡢࡼ࠺࡞㧗⃰ᗘࡢ࢟ࢩ࣮ࣟࢫᇵᆅ࡛ࡶቑṪ࡛ࡁࡿ SXM (super xylose consumption

metabolism) ኚ␗యࡶศ㞳ࡉࢀ (26) 㸪㧗⃰ᗘ࢟ࢩ࣮ࣟࢫ௦ㅰ࡟ཬࡰࡍ㑇ఏᏊࡢゎᯒࡶ⾜

ࢃࢀ࡚࠸ࡿ (26, 27)ࠋ

⮬↛✺↛ኚ␗యࢆศ㞳ࡋ㸪ࡑࡢ✺↛ኚ␗యࡀᣢࡘኚ␗ࢆḟୡ௦ࢩ࣮ࢣࣥࢧ࡛ゎㄞࡍ

ࡿࠋ⥆࠸࡚㸪⣔ᅗᏛⓗᡭἲࢆ⏝࠸࡚᭷ຊ࡞ཎᅉೃ⿵㑇ఏᏊኚ␗ࢆ⤠ࡾ㎸ࡳ㸪㑇ఏᏛⓗ

࡟≉ᐃࡍࡿ࡜࠸࠺୍㐃ࡢ᪉ἲࡀ㛤Ⓨࡉࢀ㸪᪥ᮏࡔࡅ࡛࡞ࡃᅜ㝿≉チࡢ⏦ㄳࡶ࡞ࡉࢀ࡚

࠸ࡿ (27, 28)ࠋࡇࡢ᪉ἲ࡛ 4 ಶࡢ HEX ኚ␗࡜ 2 ಶࡢ SXM ኚ␗ࡢ඲࡚ࡀゎㄞ࣭≉ᐃࡉ

ࢀ࡚࠸ࡿࠋࡲࡓ㸪 6 ಶࡢኚ␗ࡢ࠺ࡕ 4 ಶࡀඃᛶኚ␗࡛࠶ࡾ㸪㑇ఏᏊ◚ቯ࡛ࡣᚓࡽࢀ࡞

࠸᝟ሗࡀከࠎᚓࡽࢀࡿࡼ࠺࡟࡞ࡗࡓࠋ⮬↛✺↛ኚ␗యࢆศ㞳ࡋ㸪≉ᐃࡍࡿࡲ࡛࡟ 1 ࣨ

6

᭶ࡶ࠿࠿ࡽ࡞࠸࡯࡝ࡢ㎿㏿ࡉ࡛◊✲ࡀ㐍ࡴ≧ἣ࡟࡞ࡗ࡚࠾ࡾ㸪ࡇࡢᡭἲࡣᵝࠎ࡞㡿ᇦ ࡢ◊✲࡟ᙺ❧ࡘ࡜⪃࠼ࡽࢀࡿࠋ

㓝ẕ⳦ࡣ᎘Ẽ≧ែ࡛࢚ࢱࣀ࣮ࣝⓎ㓝ࢆຠ⋡ࡼࡃ⾜࠺ࠋࡇࡢ≧ែ࡛㓟⣲ࢆ౪⤥ࡍࡿ࡜

Ⓨ㓝ࡀᢚไࡉࢀ㸪⢾ࡢᾘ㈝㏿ᗘࡀῶᑡࡍࡿ࡜࡜ࡶ࡟㓟⣲࿧྾ࡀಁ㐍ࡉࢀ㸪㓝ẕ⳦ࡢẚ ቑṪ㏿ᗘࡀቑຍࡋ㸪⳦య཰⋡ࡀ㧗ࡃ࡞ࡿࠋࡇࡢ⌧㇟ࡣࣃࢫࢶ࣮ࣝຠᯝ࡜࿧ࡤࢀ࡚࠸ࡿ

(29) ࠋ୍᪉㸪୍ᐃ௨ୖࡢࢢࣝࢥ࣮ࢫࡀᏑᅾࡍࡿ࡜ዲẼ᮲௳ୗ࡛ࡶ㓟⣲࿧྾ࡀᢚไࡉࢀ

࢚ࢱࣀ࣮ࣝⓎ㓝ࡀ⏕ࡌ㸪㓝ẕ⳦ࡢẚቑṪ㏿ᗘ࡜⳦య཰⋡ࡀῶᑡࡍࡿࢡࣛࣈࢺ࣮ࣜຠᯝ

ࡀ⏕ࡌࡿ (29, 30) ࠋ㓝ẕ⳦ࢆࢢࣝࢥ࣮ࢫᇵᆅ࡛ᇵ㣴ࡋ㸪⏕ࡌࡿ஧㓟໬Ⅳ⣲ࢆ⿵㊊ࡍࡿ

㓟⣲ไ㝈ἲ࡛⮬↛Ⓨ㓝ࡉࡏࡿ࡜㸪ࢢࣝࢥ࣮ࢫ⢾ࡢ 97㸣ࡀ 91㸣ࡢ࢚ࢱࣀ࣮ࣝ࡜ 5.6㸣ࡢ ࢢࣜࢭ࣮ࣟࣝ࡟ኚ᥮ࡉࢀࡿ ( ㉥ᯇ㸪⏣ཱྀ ⚾ಙ ) ࠋࡇࡢ㧗ຠ⋡࢚ࢱࣀ࣮ࣝⓎ㓝ἲ࡛㸪ࢢ

ࣝࢥ࣮ࢫ࡜࢟ࢩ࣮ࣟࢫࢆ⮬↛Ⓨ㓝ࡉࡏࡿ࡜㸪HEX ኚ␗యࡢࡦ࡜ࡘ SCB38 ᰴࡣ㸪⢾ࡢ 89 㸣ࢆ 74 㸣ࡢ࢚ࢱࣀ࣮ࣝ࡜ࡑࢀ௨እࡢ୺せ࡞๪⏘≀࢟ࢩࣜࢺ࣮ࣝ࡜ࢢࣜࢭ࣮ࣟࣝ࡟

ኚ᥮ࡍࡿ (㉥ᯇ㸪⏣ཱྀ ⚾ಙ)ࠋࡇࡢࡼ࠺࡟㸪㧗ຠ⋡࢚ࢱࣀ࣮ࣝⓎ㓝ἲ࡛ࡶࢢࣜࢭ࣮ࣟ

ࣝ࡜࢟ࢩࣜࢺ࣮ࣝࡢ๪⏘≀ࡀ㸪࢚ࢱࣀ࣮ࣝ཰⋡ࡢጉࡆ࡜࡞ࡗ࡚࠸ࡿࠋ

ࢢࣜࢭ࣮ࣟࣝࡣゎ⢾⣔ࡢ୰㛫௦ㅰ≀㉁࡛࠶ࡿࢪࣄࢻࣟ࢟ࢩ࢔ࢭࢺࣥࣜࣥ㓟࠿ࡽࢢ

ࣜࢭ࣮ࣟࣝ -3- ࣜࣥ㓟ࢹࣄࢻࣟࢤࢼ࣮ࢮ (GPD1 ࠾ࡼࡧ GPD2 ࡀࢥ࣮ࢻࡍࡿ ) ࡛ࢢࣜࢭ

࣮ࣟࣝ 3-ࣜࣥ㓟࡟࡞ࡾ㸪⥆࠸࡚ࢢࣜࢭ࣮ࣟࣝ-3-࣍ࢫࣇ࢓ࢱ࣮ࢮ࡛ࢢࣜࢭ࣮ࣟࣝ࡟࡞

ࡿࠋࡇࡢ GPD1 ࡜ GPD2 㑇ఏᏊࢆ◚ቯࡋࡓ SCB38 ᰴࡢㄏᑟయࢆ⮬↛Ⓨ㓝ࡉࡏࡿ࡜㸪 ࢢࣝࢥ࣮ࢫ࡜࢟ࢩ࣮ࣟࢫ⢾ࡢ 96%࠿ࡽ 84㸣ࡢ࢚ࢱࣀ࣮ࣝ࡜ࡑࢀ௨እࡣ࢟ࢩࣜࢺ࣮ࣝ

ࡀ⵳✚ࡋ㸪ࢢࣜࢭ࣮ࣟࣝࡢ⵳✚ࡣᴟഹ࠿࡜࡞ࡿ (31) ࠋࡇࡢࡼ࠺࡟㸪㧗ຠ⋡࢚ࢱࣀ࣮ࣝ

Ⓨ㓝࡛ၥ㢟Ⅼ࡞ࡿࡢࡣ㸪๪⏘≀࢟ࢩࣜࢺ࣮ࣝࡢ⵳✚࡜⪃࠼ࡽࢀࡿࠋ

࢟ࢩ࣮ࣟࢫࢆ࢟ࢩࣜࢺ࣮ࣝ࡬ኚ᥮ࡍࡿ XR 㓝⣲ࡣ㸪཯ᛂ࡟⿵㓝⣲ NADPH ࠶ࡿ࠸ࡣ NADH ࢆせồࡍࡿ (6)ࠋ୍᪉㸪࢟ࢩࣜࢺ࣮ࣝ࠿ࡽ࢟ࢩ࣮ࣝࣟࢫ࡬ኚ᥮ࡍࡿ XDH 㓝⣲

ࡣ㸪཯ᛂ࡟⿵㓝⣲ NAD

ࢆせồࡍࡿ (6) ࠋࡇࡢ⿵㓝⣲せồᛶࡢ୙ᆒ⾮࡟ࡼࡾ࢟ࢩࣜࢺ

࣮ࣝࡀ⵳✚ࡋ㸪⣽⬊እ࡟᤼ฟࡉࢀࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿࡀヲ⣽࡞᤼ฟᶵᵓࡣゎ࠿ࢀ࡚࠸

7

࡞࠸ࠋࡲࡓ㸪⣽⬊እࡢ࢟ࢩࣜࢺ࣮ࣝࡀ⣽⬊ෆ࡟㍺㏦ࡉࢀ࡞࠸࡜ࡶ⪃࠼ࡽࢀࡿࡀ㸪ࡸࡣ

ࡾヲ⣽ࡣศ࠿ࡗ࡚࠸࡞࠸ࠋ⿵㓝⣲ࡢ୙ᆒ⾮ࢆゎỴࡍࡿࡓࡵ࡟㸪⿵㓝⣲せồᛶࢆኚ࠼ࡓ XYL1 ࡸ XYL2 㑇ఏᏊࡢኚ␗యࡀศ㞳ࡉࢀ࡚࠸ࡿࠋࡑࡢࡦ࡜ࡘࡣ㸪XR 㓝⣲ࡢ 270 ␒┠

ࡢ࢔࣑ࣀ㓟 ࣜࢪࣥࢆ࢔ࣝࢠࢽࣥ࡟㸪 272 ␒┠ࡢ࢔ࢫࣃࣛࢠࣥࢆ࢔ࢫࣃࣛࢠࣥ㓟࡟⨨

᥮ࡍࡿኚ␗య࡛࠶ࡿࠋࡇࡢኚ␗࡟ࡼࡾ㸪XR 㓝⣲ࡢ⿵㓝⣲ࡢせồᛶࡀ㸪NADPH ࠿ࡽ

NADH ࡟ ኚ ࢃ ࡿ (32) ࠋ ➨ ஧ ␒ ┠ ࡣ 㸪 XDH 㓝 ⣲ ࢆ 4 㔜 ࢔ ࣑ ࣀ 㓟 ⨨ ᥮ ARSdR

(D207A/I208R/ F209S/N211R)ࡉࡏࡿኚ␗࡛࠶ࡾ㸪XDH 㓝⣲ࡢ⿵㓝⣲せồᛶࡣ㸪NAD

࠿ࡽ NADP

࡟ኚࢃࡿ (33) ࠋࡲࡓ㸪 XR 㓝⣲ࡢ࢔࣑ࣀ㓟⨨᥮ AA (E223A/S271) ࡉࡏࡿࡇ

࡜࡟ࡼࡾ㸪XDH 㓝⣲ࡢ⿵㓝⣲せồᛶࢆ NADPH ౫Ꮡ࡟ࡋࡓኚ␗యࡶᵓ⠏ࡉࢀ࡚࠸ࡿ

(34) ࠋࡇࢀࡽࢆ⤌ࡳྜࡏ࡚࢟ࢩࣜࢺ࣮ࣝࡢ⵳✚ࡀ㍍ῶࡉࢀࡿ࠿ࡀㄪ࡭ࡽࢀ࡚࠸ࡿࡀ㸪

࢟ࢩࣜࢺ࣮ࣝ⵳✚ࡣ㢧ⴭ࡟ࡣῶᑡࡏࡎ㸪ࡑࡢᙳ㡪ࡣഹ࠿࡛࠶ࡿ (6)ࠋኚ␗య࡜㔝⏕ᆺ ࡢ㓝⣲άᛶࡀ␗࡞ࡿࡓࡵ㸪 XR ࡜ XD 㓝⣲཯ᛂ㏿ᗘࡢ୙ᆒ⾮ࡀ⏕ࡌ࡚࠸ࡿྍ⬟ᛶࡀ࠶

ࡿࠋࡲࡓ㸪᎘Ẽⓗ᮲௳ୗ࡛㸪⿵㓝⣲ NADPH/NADP

ࡸ NADH/NAD

ࡢᅇ㌿ࡀ࡝ࡢ⛬ᗘ

࡟㐍ࡴࡢ࠿ࡶ⌧᫬Ⅼ࡛ࡣศ࠿ࡗ࡚࠸࡞࠸ࠋࡑࡇ࡛㸪࢟ࢩ࣮ࣟࢫ࠿ࡽࡢ௦ㅰ࡜࢚ࢱࣀ࣮

ࣝⓎ㓝ࢆゎᯒࡍࡿࡔࡅ࡛࡞ࡃ㸪࢟ࢩࣜࢺ࣮ࣝ࠿ࡽࡢ௦ㅰ࡜Ⓨ㓝ࢆゎᯒࡋ࡚㸪⿵㓝⣲ࡢ ᅇ㌿ࡀ㎿㏿࡟⾜ࢃࢀࡎ࡟௦ㅰᚊ㏿ࡀ⏕ࡌ࡚࠸ࡿྍ⬟ᛶ➼ࢆㄪ࡭ࡿᚲせࡀ࠶ࡿ࡜⪃࠼

ࡽࢀࡿࠋࡑࡢࡓࡵ࡟㸪࢟ࢩࣜࢺ࣮ࣝ㈨໬ᛶࡢ⮬↛✺↛ኚ␗యࢆศ㞳ࡋ㸪ࡑࡢゎᯒࢆ㏻

ࡋ࡚๪⏘≀࢟ࢩࣜࢺ࣮ࣝࡢ⵳✚ࡢせᅉࢆゎ᫂ࡋ㸪㧗ຠ⋡Ⓨ㓝࡟㈨ࡍࡿ㓝ẕ⳦ࡢ㑇ఏᏊ ᵓ㐀ࢆ⪃ᐹࡋࡼ࠺࡜⪃࠼ࡓࠋ

ᮏ◊✲ࡢ➨ 1 ❶࡛ࡣ㸪࢟ࢩࣜࢺ࣮ࣝ㈨໬ᛶࡢ⮬↛✺↛ኚ␗యࢆศ㞳ࡋ㸪ḟୡ௦ࢩ

࣮ࢣࣥࢧゎᯒ࡟ࡼࡗ࡚ೃ⿵ኚ␗㑇ఏᏊࢆ⤠ࡾ㎸ࡳ㸪ࡑࡢ୰࠿ࡽཎᅉ㑇ఏᏊࢆ≉ᐃࡍ

ࡿࡇ࡜ࢆ┠ⓗ࡜ࡋࡓࠋࡲࡓ㸪࢟ࢩ࣮ࣟࢫ㈨໬ᛶࡢ⤌ࡳ᥮࠼㓝ẕ⳦ࡀ㸪࢟ࢩࣜࢺ࣮ࣝ

ࢆ㈨໬ࡋ࡞࠸ᶵᵓࢆ᫂ࡽ࠿࡟ࡍࡿࡇ࡜ࢆ┠ⓗ࡜ࡋࡓࠋ➨ 2 ❶࡛ࡣ㸪࢟ࢩࣜࢺ࣮ࣝ㈨

໬ᛶࡢ⮬↛✺↛ኚ␗యࢆ⏝࠸࡚㸪࢟ࢩ࣮ࣟࢫࡸ࢟ࢩࣜࢺ࣮ࣝࢆⅣ⣲※࡜ࡋࡓዲẼⓗ

࠾ࡼࡧ᎘ẼⓗⓎ㓝ᇵ㣴࡛Ⅳ⣲※ࡢ௦ㅰࢆゎᯒࡋ㸪௦ㅰᚊ㏿ẁ㝵ࢆ≉ᐃࡋ㸪࢟ࢩࣜࢺ

8

࣮ࣝࡢ⵳✚ᶵᵓࢆ᫂ࡽ࠿࡟ࡍࡿࡇ࡜ࢆ┠ⓗ࡜ࡋࡓࠋ➨ 3 ❶࡛ࡣ㸪࢟ࢩࣜࢺ࣮ࣝ㍺㏦

࡟㛵ࢃࡿࢱࣥࣃࢡ㉁ࢆ≉ᐃࡋ㸪ࡇࡢኚ␗య࡜࢚ࢱࣀ࣮ࣝ⏕⏘཰⋡࡟ཬࡰࡍᙳ㡪ࢆ᫂

ࡽ࠿࡟ࡍࡿࡇ࡜ࢆ┠ⓗ࡜ࡋࡓࠋࡇࢀࡽࡢ⤖ᯝࢆࡶ࡜࡟㸪ࢢࣝࢥ࣮ࢫ࡜࢟ࢩ࣮ࣟࢫ࠿

ࡽ㧗ຠ⋡Ⓨ㓝࡟㈨ࡍࡿ㓝ẕ⳦ࡢ㑇ఏᏊᵓ㐀࡟㛵ࡋ࡚⪃ᐹࡍࡿࠋ

9

➨ ➨ 1 ❶ ࢟ࢩࣜࢺ࣮ࣝ㈨໬ᛶࡢ Saccharomyces cerevisiae

✺↛ኚ␗యࡢศ㞳࡜≉ᚩ࡙ࡅ

➨ 1 ⠇ ⥴ゝ

S. cerevisiae ࡟࠾ࡅࡿࢢࣝࢥ࣮ࢫ࠿ࡽ࢚ࢱࣀ࣮ࣝ࡬ࡢ௦ㅰࡣ᎘Ẽⓗ (fermentative)᮲

௳ୗ࡛㉳ࡇࡿࠋゎ⢾⣔ࡢࢢࣜࢭࣝ࢔ࣝࢹࣄࢻ 3- ࣜࣥ㓟⬺Ỉ⣲㓝⣲཯ᛂ࡛ࢢࣜࢭࣝ࢔ࣝ

ࢹࣄࢺ 3-ࣜࣥ㓟࠿ࡽ 1, 3-ࣅࢫ࣍ࢫ࣍ࢢࣜࢭࣜࣥ㓟ࡀ࡛ࡁࡿ㝿࡟㸪⿵㓝⣲ NAD

ࡣ

NADH ࡟ኚࢃࡿࠋ࢔ࣝࢥ࣮ࣝ⬺Ỉ⣲㓝⣲཯ᛂ࡛࢔ࢭࢺ࢔ࣝࢹࣄࢻ࠿ࡽ࢚ࢱࣀ࣮ࣝࡀ࡛

ࡁࡿ㝿࡟㸪 NADH ࡣ᎘Ẽ᮲௳ୗ࡛ࡶ NAD

࡟㑏ඖࡉࢀࡿࠋࡇࡢࡼ࠺࡟㸪⿵㓝⣲

NADH/NAD

ࡢ๭ྜࡣಖࡓࢀ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡿࠋ୍᪉㸪࢟ࢩ࣮ࣟࢫ࠿ࡽ࢚ࢱࣀ࣮ࣝ

࡬ࡢ௦ㅰࡣ㸪⣽⬊⃰ᗘࡀ㧗ࡅࢀࡤ᎘Ẽⓗ᮲௳࡛ࡶ༑ศ࡟㉳ࡇࡿࠋ⤌ࡳ᥮࠼ᆺ㓝ẕ S.

cerevisiae ࡟࠾࠸࡚㸪 Scheffersomyces stipitis ࡢ࢟ࢩ࣮ࣟࢫࣜࢲࢡࢱ࣮ࢮ XR ཯ᛂ࡛࢟ࢩ

࣮ࣟࢫࡣ࢟ࢩࣜࢺ࣮ࣝ࡬௦ㅰࡉࢀࡿࡀ㸪ࡇࡢ࡜ࡁ⿵㓝⣲ NADH ࡀ NAD

࡟ኚࢃࡿ࠿㸪

࠶ࡿ࠸ࡣ NADPH ࡀ NADP

࡟ኚࢃࡿࠋ࢟ࢩࣜࢺ࣮ࣝࡣ㸪 S. stipitis ࡢ࢟ࢩࣜࢺ࣮ࣝ⬺Ỉ

⣲㓝⣲ XDH ཯ᛂ࡛࢟ࢩ࣮ࣝࣟࢫ࡟௦ㅰࡉࢀࡿ᫬࡟㸪NAD

ࡣ NADH ࡟࠿ࢃࡿࠋ᎘Ẽ

ⓗ࡞Ⓨ㓝᮲௳࡛㸪㔝⏕ᆺ㓝ẕ S. cerevisiae ࡀ NADH ࠿ࡽ NAD

࡬ࡢ㓟໬཯ᛂࢆ༑ศ࡟

㉳ࡇࡉ࡞ࡅࢀࡤ㸪 NADH/NAD

ࡢ๭ྜࢆಖࡘ࡟ࡣ XDH ཯ᛂ࡟ᚲせ࡞ NAD

ࡣ XR ཯ᛂ

࡟ࡼࡗ࡚సࡽࢀࡿࡇ࡜࡟࡞ࡿࠋࡇࡢࡇ࡜ࡀṇࡋࡅࢀࡤ㸪 XR 㓝⣲཯ᛂࡀ⿵㓝⣲ NADPH

ࢆ౑⏝ࡍࡿ᫬㸪NAD

୙㊊࡜࡞ࡿࠋ᎘Ẽⓗ࡞Ⓨ㓝᮲௳ୗ࡛ࡶ࣌ࣥࢺ࣮ࢫࣜࣥ㓟ᅇ㊰ࡢ

ഃ㙐཯ᛂ࡛࠶ࡿࢢࣝࢥ࣮ࢫ 6- ࣜࣥ㓟⬺Ỉ⣲㓝⣲ Zwf1 ཯ᛂࡸ 6- ࣍ࢫ࣍ࢢࣝࢥࣥ㓟⬺

Ỉ⣲㓝⣲ (Gnd1 ࡜ Gnd2) ཯ᛂ࡛ NADPH ࡣ⏕ࡌࡿࡢ࡛ (35)㸪XR ཯ᛂ࡟ᚲせ࡞

NADPH ࡣ౪⤥ࡉࢀࡿࠋࡇࡢࡼ࠺࡟㸪 XR ཯ᛂࡣ㐍ࡳ㸪 XDH ཯ᛂࡣ⿵㓝⣲ NAD

ࡢ୙

㊊࡟ࡼࡗ࡚㐜ᘏࡍࡿ࡜⪃࠼ࡽࢀࡿࠋࡑࡋ࡚㸪ࡇࡢࡇ࡜ࡀ࢟ࢩࣜࢺ࣮ࣝࡢ⵳✚ࡢせᅉ࡜

10

ண᝿ࡉࢀࡿࠋࡇࡢࡇ࡜ࢆ☜࠿ࡵࡿ࡟ࡣ㸪࢟ࢩ࣮ࣟࢫ࠿ࡽࡢ௦ㅰ࡜࢟ࢩࣜࢺ࣮ࣝ࠿ࡽࡢ ௦ㅰࢆẚ㍑ࡍࡿᚲせࡀ࠶ࡿࠋ

࢟ࢩ࣮ࣟࢫ࠿ࡽࡢ௦ㅰ࡜࢟ࢩ࣮ࣝࣟࢫ࠿ࡽࡢ௦ㅰࡢሗ࿌౛ࡣ࠶ࡿࡀ(36)㸪࢟ࢩࣜࢺ

࣮ࣝ࠿ࡽࡢ௦ㅰࡸ࢟ࢩࣜࢺ࣮ࣝࡢ⣽⬊ෆ㍺㏦ࡢሗ࿌౛ࡣ࡞࠸ࠋࡑࡇ࡛㸪࢟ࢩࣜࢺ࣮ࣝ

ࢆ㈨໬ࡍࡿ⮬↛✺↛ኚ␗యࢆศ㞳ࡋ㸪ࡑࡢ㑇ఏᏊࢆ≉ᐃࡍࡿࡇ࡜࠿ࡽゎᯒࢆጞࡵࡿࡇ

࡜ࢆ⪃࠼ࡓࠋ⮬↛✺↛ኚ␗యࡢཎᅉ㑇ఏᏊ≉ᐃࡣ㸪᫬㛫࡜ປຊࡀ࠿࠿ࡿ᪉ἲ࡛࠶ࡗࡓࠋ ࡋ࠿ࡋ࡞ࡀࡽ㸪ࢻࣛࣇࢺࢤࣀ࣒ሷᇶ㓄ิࡀゎㄞࡉࢀ (37)㸪ྠ㉁⣔⤫ᰴࢆ౑⏝⳦ᰴ࡟⏝

࠸ࢀࡤ (24, 25) 㸪ኚ␗య DNA ࢆḟୡ௦ࢩ࣮ࢣࣥࢧ࡛ゎᯒࡋ㸪ぶᰴࡸኚ␗య㛫ࡢ㑇ఏᏛ

ⓗ⣔⤫ゎᯒࢆ⾜࠺࡜(27, 28)㸪ೃ⿵㑇ఏᏊኚ␗ࢆ㎿㏿࡟⤠ࡾ㎸ࡵࡿࡇ࡜ࡀ࡛ࡁࡿࡼ࠺࡟

࡞ࡗࡓ (26−28) ࠋࡑࡢᚋ㸪ೃ⿵㑇ఏᏊࡢ㞄᥋ࡍࡿ㒊఩࡟ kanMX ࣐࣮࣮࢝ࢆ⤌ࡳ᥮࠼㸪

ኚ␗ᆺࡢ⾲⌧ᆺࡀኚࢃࡽ࡞࠸ࡇ࡜ࢆ☜ㄆࡋࡓᚋ㸪kanMX ࡜ኚ␗㒊఩ࢆྵࡴ DNA ࡟ࡼ

ࡿぶᰴࡢᙧ㉁㌿᥮యࢆศ㞳ࡋ㸪࢟ࢩࣜࢺ࣮ࣝ㈨໬ᛶࡢ⾲⌧ᆺࡢฟ⌧ࡢ᭷↓࡛≉ᐃࡍࡿ

᪉ἲ࠶ࡿ࠸ࡣ஧㔜ᙧ㉁㌿᥮ἲ࡛㸪ཎᅉ㑇ఏᏊኚ␗ࢆ≉ᐃ࡛ࡁࡿ (26)ࠋࡇࡢࡼ࠺࡟㸪࢟

ࢩࣜࢺ࣮ࣝ㈨໬ᛶࡢ⮬↛✺↛ኚ␗యࢆศ㞳ࡋ࡚ゎᯒࡍࡿ᪉ἲ࡛㸪㈨໬ࡢせᅉࡸᶵᵓࢆ

⡆౽࡛㎿㏿࡟ゎ࡛᫂ࡁࡿ⫼ᬒ࡟࠶ࡿࠋ

⣽⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡓ࢟ࢩࣜࢺ࣮ࣝࡀ㸪㎿㏿࡟௦ㅰࡉࢀࡿ࡜㈨໬ᛶࡢほᐹࢆᐜ᫆

࡟⾜࠺ࡇ࡜ࡀ࡛ࡁࡿࠋ⥴ㄽ࡛㏙࡭ࡓࡀ S. stipitis ࡢ XYL1 ࡜ XYL2 ࡜ෆᅾᛶࡢ XKS1 ࢆ 㓝ẕᰁⰍయୖ࡟⤌ࡳ᥮࠼㸪㧗Ⓨ⌧ࣉ࣮ࣟࣔࢱ࣮࡛࠶ࡿ TDH3 ࣉ࣮ࣟࣔࢱ࣮ (TDH3p)

࡛࢟ࢩ࣮ࣟࢫ௦ㅰ㑇ఏᏊࢆⓎ⌧ࡉࡏࡓ SCB7 ᰴࡣ㸪࢟ࢩ࣮ࣟࢫᇵᆅ࡛ࡶ᭱ࡶ▷࠸ୡ ௦᫬㛫ࡀ 5 ᫬㛫࡛࠶ࡿࠋᚑࡗ࡚㸪࢟ࢩ࣮ࣟࢫ࠿ࡽ࢟ࢩࣜࢺ࣮ࣝ࡬ࡢ Xyl1 㓝⣲཯ᛂ࡛

ࡢ௦ㅰ㸪࢟ࢩࣜࢺ࣮ࣝ࠿ࡽ࢟ࢩ࣮ࣝࣟࢫ࡬ࡢ Xyl2 㓝⣲཯ᛂ࡛ࡢ௦ㅰ㸪࢟ࢩ࣮ࣝࣟࢫ

࠿ࡽ࢟ࢩ࣮ࣝࣟࢫ 5- ࣜࣥ㓟࡬ࡢ Xks1 㓝⣲཯ᛂ࡛ࡢ௦ㅰࡶ෇⁥࡟㐍ࡴ (26) ࠋࡉࡽ࡟㸪 PYK1 㑇ఏᏊ (ࣆࣝࣅࣥ㓟࢟ࢼ࣮ࢮࢆࢥ࣮ࢻࡍࡿ) ෆ࡟⏕ࡌࡓඃᛶኚ␗࡛࠶ࡿ

HEX1−22 ࡟ࡼࡾ᭱ࡶ▷࠸ୡ௦᫬㛫ࡣ 2.5 ᫬㛫࡟࡞ࡿࠋ Pyk1 㓝⣲ࡢάᛶಁ㐍࡟ࡣ㸪ࣇ

ࣛࢡࢺ࣮ࢫ 1㸪6-ࣅࢫࣜࣥ㓟ࡢ⤖ྜࡀᚲせ࡛࠶ࡿࡀ㸪ࢢࣝࢥ࣮ࢫ௦ㅰ࡜ࡣ␗࡞ࡾ࢟ࢩ

11

࣮ࣟࢫ௦ㅰ࡛ࡣ㸪ࡑࡢ౪⤥ࡀᑡ࡞࠸࡜⪃࠼ࡽࢀࡿࠋ࢟ࢩ࣮ࣟࢫ௦ㅰ᫬ࡢ Pyk1 㓝⣲ά ᛶపୗࡢ㍍ῶ࡟ാ࠸࡚࠸ࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿ (26) ࠋ࢟ࢩࣜࢺ࣮ࣝࡢྲྀࡾ㎸ࡳ⬟ࡀప࠸

ሙྜ࡟ࡣ㸪㧗⃰ᗘࡢ࢟ࢩࣜࢺ࣮ࣝࢆ⏝࠸ࡿᚲせࡀ࠶ࡿ࠿ࡶࡋࢀ࡞࠸ࠋ㧗⃰ᗘࡢ࢟ࢩ

࣮ࣟࢫࢆ௦ㅰࡍࡿࡇ࡜ࡀ࡛ࡁ㸪㧗ຠ⋡࢟ࢩ࣮ࣟࢫ㈨໬ࢆᑟࡃᛶ㉁ࡀ࠶ࡿኚ␗࡜ࡋ

࡚㸪SXM1−32 ኚ␗࡜ sxm2−33 ኚ␗ࡀศ㞳ࡉࢀ࡚࠸ࡿ (26)ࠋSXM1−32 ࡜ sxm2−33 ኚ

␗ࡣ㸪 MTH1 㑇ఏᏊ ( ࢢࣝࢥ࣮ࢫࢭࣥࢩࣥࢢఏ㐩⤒㊰ࡢ㈇ࡢไᚚᅉᏊࢆࢥ࣮ࢻࡍࡿ ) ࡢඃᛶኚ␗࡜ GRR1 㑇ఏᏊ (SCF ࣘࣅ࢟ࢳ࣮ࣥࣜ࢞ࢮ」ྜయࡢ F ࣎ࢵࢡࢫࢱࣥࣃࢡ

㉁ᡂศࢆࢥ࣮ࢻࡋ㸪࢝ࢱ࣎ࣛ࢖ࢺᢚไࢆᑟࡃ ) ࡢຎᛶኚ␗࡛࠶ࡾ㸪࠸ࡎࢀࡶ࣊࢟ࢯ

࣮ࢫ㍺㏦ࢱࣥࣃࢡ㉁ࡢ⣽⬊ෆ࡬ࡢ㍺㏦ไ㝈ࢆ㏻ࡋ࡚࢝ࢱ࣎ࣛ࢖ࢺᢚไࡢゎ㝖ࢆ⾜

࠺ࠋࡇࡢࡼ࠺࡟㸪࢟ࢩࣜࢺ࣮ࣝ㈨໬ᛶࡢ✺↛ኚ␗యࢆศ㞳ࡍࡿࡓࡵࡢぶᰴタィࡶ࡛

ࡁࡿࡼ࠺࡟࡞ࡗࡓࠋ

ࡑࡇ࡛㸪➨ 1 ❶࡛ࡣ㸪࢟ࢩࣜࢺ࣮ࣝ㈨໬ᛶࡢ⮬↛✺↛ኚ␗యࢆศ㞳ࡍࡿࡇ࡜㸪ḟୡ ௦ࢩ࣮ࢣࣥࢧゎᯒ࡜㑇ఏᏛⓗ⣔⤫ゎᯒ࡛ཎᅉೃ⿵㑇ఏᏊኚ␗ࢆ⤠ࡾ㎸ࡴࡇ࡜㸪ཎᅉ㑇 ఏᏊኚ␗ࢆ≉ᐃࡍࡿࡇ࡜㸪ేࡏ࡚㸪࢟ࢩࣜࢺ࣮ࣝ㈨໬ᛶ࡜࡞ࡿᶵᵓࢆゎᯒࡍࡿࡇ࡜ࢆ

┠ⓗ࡜ࡋࡓࠋ

➨

➨ 2 ⠇ ᐇ㦂ᮦᩱ࡜ᐇ㦂᪉ἲ

1.2.1 ⳦ᰴ㸪ࣉࣛࢫ࣑ࢻ㸪࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࣉࣛ࢖࣐࣮

ᮏ◊✲ࡢ➨ 1 ❶࡛౑⏝ࡋࡓ⳦ᰴ࡜ࣉࣛࢫ࣑ࢻࢆ⾲ 1-1 ࡟㸪࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࣉࣛ

࢖࣐࣮ࢆ⾲ 1-2 ࡟♧ࡋࡓࠋ㓝ẕ SCB39 ᰴ࡜ SCB40 ᰴࡣ SCB14 ᰴ࠿ࡽᵓ⠏ࡋࡓኚ␗య

࡛࠶ࡾ㸪㧗⃰ᗘ࢟ࢩ࣮ࣟࢫ (180 g/L) ࢆྵࢇࡔᇵᆅ࡟࠾࠸࡚㏿࠸ቑṪࢆ♧ࡍ (26) ࠋ pZeo ࣉࣛࢫ࣑ࢻࡣ㸪kanMX ࣐࣮࣮࢝ࢆ Cre ࢱࣥࣃࢡ㉁ࡢⓎ⌧࡟ࡼࡗ࡚㝖ཤࡍࡿࡓࡵ

࡟⏝࠸ࡓࠋ S. cerevisiae NAM34−4C ࡣ⪏⇕ᛶ࣭⪏㓟ᛶ࣭ங㓟㈨໬ᛶ࣭ᙧ㉁㌿᥮⬟ࡢ㧗

࠸࢚ࢱࣀ࣮ࣝ⏕⏘࡟⏝࠸ࡽࢀ࡚࠸ࡿᐇ⏝㓝ẕࡢㄏᑟయ࡛ᮏ◊✲ࡢぶᰴ࡜ࡋ࡚⏝࠸ࡓ

12

(24)ࠋ኱⭠⳦ DH10B ࡣࣉࣛࢫ࣑ࢻ DNA ࡟ࡼࡿᙧ㉁㌿᥮ࡢཷᐜ⳦࡜ࡋ࡚⏝࠸ࡓ (25)ࠋ

ᮏ◊✲࡛⏝࠸ࡓࣉࣛ࢖࣐࣮㸦 Genenet, Fukuoka, Japan 㸧ࡣ㸪 GENETYX

−MAC 㑇ఏ᝟

ሗฎ⌮ࢯࣇࢺ࢙࢘࢔㸭Macintosh ∧ Primer3㸦ࢮࢿࢸ࢕ࢵࢡࢫ㸪ᮾி㸪᪥ᮏ㸧࠶ࡿ࠸ࡣ Primer3 㸦 http://frodo.wi.mit.edu/primer3/ 㸧࡟ࡼࡗ࡚ࢹࢨ࢖ࣥࡋ㸪⾲ 1-2 ࡟グࡋࡓࠋ S.

cerevisiae 㑇 ఏ Ꮚ ࡢ ሷ ᇶ 㓄 ิ ࡣ 㸪 Saccharomyces Genome Database (http:// www.

yeastgenome.org/) ࡢ᝟ሗ࡟ᇶ࡙࠸ࡓࠋ S. cerevisiae NAM34-4C ᰴࡢࢻࣛࣇࢺࢤࣀ࣒ࡢሷ ᇶ 㓄 ิ ࡣ 㸪 DDBJ/EMBL/GenBank ࢔ ࢡ ࢭ ࢵ ࢩ ࣙ ࣥ ␒ ྕ BAUH01000001 ࠿ ࡽ

BAUH01000154 ࡟Ⓩ㘓ࡉࢀ࡚࠸ࡿࠋ

⾲ 1-1 ᮏ◊✲࡛౑⏝ࡋࡓ⳦ᰴཬࡧࣉࣛࢫ࣑ࢻ

⳦ᰴ 㑇ఏᏊᆺࡲࡓࡣ⾲⌧ᆺ ㉳※㸪⏤᮶㸪ᘬ⏝ᩥ⊩

Saccharomyces cerevisiae

SCB14 MATa pho87Δ ura3Δ::XM8

HEX1−22

25, HEX1–22 mutant showed highly efficient growth in a medium containing low concentration of xylose (20 g/L).

SCB32 MATa pho87Δ ura3Δ::XM8 HEX1−22 Sxm

(SXM1−32 sxmc1−32)

Spontaneously isolated Sxm

mutant obtained from SCB14. Sxm

showed efficient growth in a medium containing high concentration of xylose.

SCB33 MATa pho87Δ ura3Δ::XM8 HEX1−22 Sxm

(sxm2−33)

Spontaneously isolated Sxm

mutant obtained from SCB14

SCB38 MATa pho87Δ URA3::XM1

HEX1−22

25

SCB39 MATa pho87Δ URA3::XM1 HEX1- 22 Sxm

(SXM1−32 sxmc1−32)

Tfm (SCB32: URA3 DNA, URA

, G418−s)

. The URA3 DNA region of pURA3 was amplified using primers F- URA3-STR and R-URA3-

DWN300(GAPDH).

SCB40 MATa pho87Δ URA3::XM1 HEX1- 22 Sxm

(sxm2−33)

Tfm (SCB33: URA3 DNA, URA

,

G418−s). The URA3 DNA region of

pURA3 was amplified using primers F-

13

URA3-STR and R-URA3- DWN300(GAPDH).

NAM34−4C MATα 24

SCC7 MATa pho87Δ URA3::XM1 HEX1- 22 Sxm

(SXM1−32 sxmc1−32) Xyt

(xyt1−7)

Spontaneously isolated Xyt

mutant obtained from SCB39

SCC8 MATa pho87Δ URA3::XM1 HEX1- 22 Sxm

(sxm2−33) Xyt

(xyt1−8)

Spontaneously isolated Xyt

mutant obtained from SCB40

SCC15 MATa pho87Δ URA3::XM1 HEX1- 22 Sxm

(sxm2−33) Xyt

(xyt1−15)

Spontaneously isolated Xyt

mutant obtained from SCB40

SCD200 MATa pho87Δ URA3::XM1 HEX1−22 Sxm

gal80Δ::kanMX

Tfm (SCB39: kanMX DNA, G418−r). The kanMX DNA region of pBlu-LTKTL- TDH3 was amplified using primers F- GAL80-LTKTL and R-GAL80-LTKTL.

SCD201 MATa pho87Δ URA3::XM1 HEX1−22 Sxm

gal80Δ::loxP

The kanMX in SCD200 strain was removed.

SCD202 MATa pho87Δ URA3::XM1 HEX1−22 Sxm

gal80Δ::loxP gal2Δ::kanMX

Tfm (SCD201: kanMX DNA, G418−r). The kanMX DNA region of pBlu-LTKTL- TDH3 was amplified using primers F- GAL2Δ-LTKTL and R-GAL2Δ-LTKTL.

SCD203 MATa pho87Δ URA3::XM1 HEX1−22 Sxm

gal2pΔ::kanMX−TDH3p−GAL2

Tfm (SCB39: kanMX DNA, G418−r). The kanMX DNA region of pBlu-LTKTL- TDH3 was amplified using primers F- GAL2c-LTKTL and R-GAL2c-TDH3p.

SCB42−4A MATα pho87Δ URA3::XM1 HEX1−22 Sxm

Haploid (SCB39 × SCB105−3A)

SCD204 MATa pho87Δ URA3::XM1 HEX1- 22 Sxm

xyt1−7 / MATα pho87Δ URA3::XM1 HEX1−22 Sxm

XYT1

Diploid (SCC7 × SCB42−4A)

SCD205 MATa pho87Δ URA3::XM1 HEX1- 22 Sxm

xyt1−8 / MATα pho87Δ URA3::XM1 HEX1−22 Sxm

XYT1

Diploid (SCC8 × SCB42−4A)

SCD206 MATa pho87Δ URA3::XM1 HEX1- 22 Sxm

xyt1−15 / MATα pho87Δ URA3::XM1 HEX1−22 Sxm

XYT1

Diploid (SCC15 × SCB42−4A)

SCD207 MATa pho87Δ URA3::XM1 HEX1- 22 Sxm

gal80Δ::kanMX / MATα

Diploid (SCD200 × SCB42−4A)

14 pho87Δ URA3::XM1 HEX1−22 Sxm

GAL80

SCD208 MATa pho87Δ URA3::XM1 HEX1- 22 Sxm

/ MATα pho87Δ

URA3::XM1 HEX1−22 Sxm

Diploid (SCB39 × SCB42−4A)

SCD216 MATα pho87Δ URA3::XM1 HEX1−22 Sxm

gal80Δ::kanMX

Tfm (SCB42−4A: kanMX DNA, G418-r).

The kanMX DNA region of pBlu-LTKTL- TDH3 was amplified using primers F- GAL80-LTKTL and R-GAL80-LTKTL.

SCD218 MATa pho87Δ URA3::XM1 HEX1−22 Sxm

xyt1−7 / MATα pho87Δ URA3::XM1 HEX1−22 Sxm

gal80Δ::kanMX

Diploid (SCC7 × SCD216)

SCD220 MATa pho87Δ URA3::XM1 HEX1−22 Sxm

xyt1−8 / MATα pho87Δ URA3::XM1 HEX1−22 Sxm

gal80Δ::kanMX

Diploid (SCC8 × SCD216)

SCD222 MATa pho87Δ URA3::XM1 HEX1−22 Sxm

xyt1−15 / MATα pho87Δ URA3::XM1 HEX1−22 Sxm

gal80Δ::kanMX

Diploid (SCC15 × SCD216)

SCD224 MATa pho87Δ URA3::XM1 HEX1−22 Sxm

gal80Δ::kanMX / MATα pho87Δ URA3::XM1 HEX1−22 Sxm

gal80Δ::kanMX

Diploid (SCD200 × SCD216)

SCC2−11B MATa pho87Δ URA3::XM1 25 SCD302 MATa pho87Δ URA3::XM1

gal80Δ::kanMX

Tfm (SCC2−11B: kanMX DNA, G418-r).

The kanMX DNA region of pBlu-LTKTL- TDH3 was amplified using primers F- GAL80-LTKTL and R-GAL80-LTKTL.

SCD303 MATa pho87Δ URA3::XM1 gal2pΔ::kanMX−TDH3p−GAL2

Tfm (SCC2−11B: kanMX DNA, G418−r).

The kanMX DNA region of pBlu-LTKTL- TDH3 was amplified using primers F- GAL2c-LTKTL and R-GAL2c-TDH3p.

Escherichia coli

DH10B F

mcrA Δ(mrr

hsdRMS mcrBC) Φ80 lacZΔM15 ΔlacX74 recA1

Life technologies, 25

15 endA1 araD139 Δ(ara leu)7697 galU galK λ

rpsL nupG) Plasmid and

amplicon DNA pBluescript II KS+

bla 25

pBlu-LTKTL- TDH3

loxP−TEFp−kanMX−TEFt−loxP−

TDH3p

25

pZeo CEN6 / ARSH4 URA3 GAL1p CRE CYC1t bla ori

TEF1p EM7 Sh ble CYC1t

25

pURA3 URA3p-URA3-URA3t The URA3p-URA3-URA3t DNA region of strain NAM34−4C was amplified using primers F-URA3-UP2.1k(EcoI) and R- URA3-DWN2.1k(EcoRI). The amplicons were treated with restriction enzyme EcoRI and cloned into the EcoRI site of

pBluescript II KS+ to generate pURA3.

a

XM8 ࡣ loxP–TEFp–kanMX–TEFt–loxP–TDH3p–XYL1–TDH3t–TDH3p–XYL2–TDH3t–TDH3p–

XKS1– TDH3t ࡢ㑇ఏᏊᵓ㐀ࢆᣢࡕ㸪loxP, TEF ࣉ࣮ࣟࣔࢱ࣮, kanMX, TEF ࢱ࣮࣑ࢿ࣮ࢱ࣮㸪 TDH3 ࣉ࣮ࣟࣔࢱ࣮㸪XYL1 {Scheffersomyces stipitis ࡢ XYL1 (࢟ࢩ࣮ࣟࢫࣜࢲࢡࢱ࣮ࢮ XR ࢆ ࢥ࣮ࢻࡍࡿ)}㸪TDH3 ࢱ࣮࣑ࢿ࣮ࢱ࣮㸪XYL2 {S. stipitis ࡢ XYL2 (࢟ࢩࣜࢺ࣮ࣝ⬺Ỉ⣲㓝⣲ XD

ࢆࢥ࣮ࢻࡍࡿ)}, XKS1 {S. cerevisiae ࡢ Xks1 (࢟ࢩࣝࣟ࢟ࢼ࣮ࢮࢆࢥ࣮ࢻࡍࡿ)}ࠋ

b

XM1 ࡣ TDH3p–XYL1–TDH3t–TDH3p–XYL2–TDH3t–TDH3p–XKS1– TDH3t ࡢ㑇ఏᏊᵓ㐀ࢆᣢ ࡘࠋ

c

Tfm, ᙧ㉁㌿᥮; Tfm (SCB32: URA3 DNA, URA

, G418−s) ࡣ㸪SCB32 ᰴࡢ URA

DNA ࡟ࡼࡿ

Ura

G418−s ᙧ㉁㌿᥮యࢆ♧ࡍࠋ

d

Haploid (SCB39 × SCB105−3A) ࡣ㸪SCB39 ᰴ࡜ SCB105-3A ᰴࢆ᥃ࡅྜࢃࡏࡿࡇ࡜࡟ࡼࡾᚓ ࡓ஧ಸయࢆ 4 ⬊Ꮚゎ๗ࡋ࡚ᚓࡓ୍ಸయࢆᣦࡋ♧ࡍࠋ

e

Diploid (SCC7 × SCB42−4A) ࡣ㸪SCC7 ᰴ࡜ SCB42−4A ᰴࢆ᥃ࡅྜࢃࡏ࡚ᚓࡓ஧ಸయࢆᣦࡋ

♧ࡍࠋ

16

⾲ 1-2 PCR ቑᖜ࡟⏝࠸ࡓ࢜ࣜࢦࢾࢡࣞ࢜ࢳࢻࣉࣛ࢖࣐࣮

␒ྕ ࣉࣛ࢖࣐࣮ྡ ࣉࣛ࢖࣐࣮ሷᇶ㓄ิ (5’࠿ࡽ 3’)

1 F-URA3-STR atgtcgaaagctacatataagg

2 R-URA3-

DWN300(GAPDH)

gaatttgtgtccattagtggtgttccgtgtgagaactggttccttcatttgcttttgttcca

3 F-GAL80-LTKTL caatctcgatagttggtttcccgttctttccactcccgtcggccgccagctgaag

4 R-GAL80-LTKTL cgttcgctgcactgggggccaagcacagggcaagatgctaggccactagtggatct

5 F-Gal2Δ-LTKTL atggcagttgaggagaacaatatgcctgttgtttcacagcggccgccagctgaa

6 R-Gal2Δ-LTKTL ttattctagcatggccttgtaccacggtttgtcgtcatgtaggccactagtggatct

7 F-Gal2c-LTKTL gcccttcccatctcaagatggggagcaaatggcattatacggccgccagctgaag

8 R-Gal2c-TDH3p gctgtgaaacaacaggcatattgttctcctcaactgccattttgtttgtttatgtgtgtttattcgaa 9 F-URA3-UP2.1k(EcoI) tttgaattcttaagggtcaactaaatacagttg

10 R-URA3-

DWN2.1k(EcoRI)

tttgaattcccggaactcgatgtggt

ࣉࣛ࢖࣐࣮ࡢ࢔ࣥࢲ࣮ࣛ࢖࡛ࣥᣦࡋ♧ࡋࡓ㒊ศࡣ㸪ᰁⰍయ DNA ࡜ࡢ┦ྠ㡿ᇦ࡛࠶ࡿࠋࣉࣛ࢖

࣐࣮9 ࡜ࣉࣛ࢖࣐࣮10 ࡢ࢖ࢱࣜࢵࢡయ࡛ᣦࡋ♧ࡋࡓ㒊ศࡣ㸪ไ㝈㓝⣲ࡢ 6 ሷᇶࡢㄆ㆑㒊఩࡛

࠶ࡾ㸪ኴᏐ࡛♧ࡋࡓ 3 ሷᇶࡣ㸪ไ㝈㓝⣲ษ᩿ࢆᨵၿࡍࡿࡓࡵ࡟ຍ࠼ࡽࢀࡓሷᇶ㒊ศ࡛࠶ࡿࠋ

1.2.2 ᇵ ᇵᆅ

㓝ẕ࢚࢟ࢫ㸪࣌ࣉࢺࣥ㸪ࢢࣝࢥ࣮ࢫ (YPD) ᇵᆅࡣ㸪ࢢࣝࢥ࣮ࢫ 20 g 㸪ࣂࢡࢺ㓝ẕ࢚

࢟ࢫ 10 g㸪ࣂࢡࢺ࣌ࣉࢺࣥ 20 g ࢆ⵨␃Ỉ 1 L ᙜࡓࡾ࡟ྵࡳ pH 5.5 ࡟ㄪᩚࡋ㸪

S. cerevisiae ᰴࡢቑṪ࡟⏝࠸ࡓࠋ࢟ࢩࣜࢺ࣮ࣝ㈨໬ᛶ✺↛ኚ␗ᰴࡢศ㞳࡟౑⏝ࡋࡓᨵ

17

ኚ࢟ࢩࣜࢺ࣮ࣝ᭱ᑠᇵᆅ (LXYP)ࡣ, ࢟ࢩࣜࢺ࣮ࣝ 20 g/L ࡜ప⃰ᗘᰤ㣴ᡂศ (LYP) 3.75 g/L ( ࣂࢡࢺ࣌ࣉࢺࣥ 2.5 g/L, ࣂࢡࢺ㓝ẕ࢚࢟ࢫ 1.25 g/L) ࢆ⵨␃Ỉ 1 L ᙜࡓࡾ࡟ྵ

ࡳ㸪pH4.5 ࡟ㄪᩚࡋࡓࠋ

࢟ࢩ࣮ࣟࢫ㈨໬⬟ࡢゎᯒ࡟౑⏝ࡋࡓᨵኚ࢟ࢩ࣮ࣟࢫᇵᆅ (LYPX) ࡣ㸪࢟ࢩ࣮ࣟࢫ 20

g/L, LYP 3.75 g/L ࢆ⵨␃Ỉ 1 L ᙜࡓࡾ࡟ྵࡳ㸪pH4.5 ࡟ㄪᩚࡋࡓࠋ㓝ẕ࢚࢟ࢫ࣭ࣂࢡࢺ

࣌ࣉࢺ࣭ࣥ࢟ࢩ࣮ࣟࢫ (YPX) ᇵᆅ㸪࠾ࡼࡧ Cre ⤌ࡳ᥮࠼㓝⣲ࡢㄏᑟ࡟౑⏝ࡋࡓ㓝ẕ࢚

࢟ࢫ࣭ࣂࢡࢺ࣌ࣉࢺ࣭ࣥ࢞ࣛࢡࢺ࣮ࢫ (YPG)ᇵᆅࡣ㸪 YPD ᇵᆅ࡟ྵࡲࢀࡿࢢࣝࢥ࣮ࢫ

20 g/L ࢆ㸪࢟ࢩ࣮ࣟࢫ 20 g/L, ࢞ࣛࢡࢺ࣮ࢫ 20 g/L ࡟ࡑࢀࡒࢀ⨨ࡁ᥮࠼ࡓ௨እࡣ YPD

ᇵᆅ࡜ྠࡌ࡛࠶ࡾ㸪 Cre ࣜࢥࣥࣅࢼ࣮ࢮࡢⓎ⌧ㄏᑟ࡟⏝࠸ࡓࠋࢢࣝࢥ࣮ࢫ᭱ᑡ (MSD) ᇵᆅࡣ㸪ࢢࣝࢥ࣮ࢫ 20 g/L 㸪࢔࣑ࣀ㓟࡜ (NH

)

SO

ࢆྵࡲ࡞࠸㓝ẕ❅⣲※࣮࣋ࢫ (YNB w/o, Becton, Dickinson and company sparks, MD21152) 1.7g㸪(NH

)

SO

5g ࢆ⵨␃

Ỉ 1L ᙜࡾ࡟ྵࡳ pH 5.5 ࡟ㄪᩚࡋࡓࠋ࢟ࢩ࣮ࣟࢫ᭱ᑠᇵᆅ (MSX) ࡣ㸪 MSD ᇵᆅࡢࢢ

ࣝࢥ࣮ࢫ 20 g/L ࢆ࢟ࢩ࣮ࣟࢫ 20 g/L ࡟⨨ࡁ᥮࠼ࡓࠋྜᡂ᏶඲ (SC)ᇵᆅࡣ㸪1 L ᙜࡓ

ࡾ 20 g ࢢࣝࢥ࣮ࢫ㸪 6.7 g YNB w/o 㸪 0.83 g ྜᡂ᏶඲ῧຍΰྜ≀ (2.0 g ࢔ࢹࢽࣥ㸪 2.0 g L-࢔ࣝࢠࢽࣥ㸪2.0 g L-ࣄࢫࢳࢪࣥ㸪2.0 g L-࢖ࢯࣟ࢖ࢩࣥ㸪2.0 g L-ࣟ࢖ࢩࣥ㸪2.0 g L-

ࣜࢪࣥ㸪 2.0 g L- ࣓ࢳ࢜ࢽࣥ㸪 2.0 g L- ࣇ࢙ࢽࣝ࢔ࣛࢽࣥ㸪 2.0 g L- ࢭࣜࣥ㸪 2.0 g L- ࢺࣞ

࢜ࢽࣥ㸪2.0 g L-ࢺࣜࣉࢺࣇ࢓ࣥ㸪2.0 g L-ࢳࣟࢩࣥ㸪2.0 g L-࢘ࣛࢩࣝ㸪2.0 g L-ࣂࣜࣥ)

ࢆྵࡳ㸪 pH 5.5 ࡟ㄪᩚࡋࡓࠋᇵᆅ࡟ࡣᚲせ࡟ᛂࡌ࡚᭱⤊⃰ᗘࡀ࢔ࢹࢽ࡛ࣥ 50 mg/L 㸪

࢘ࣛࢩ࡛ࣝ 50 mg/L㸪࢔࣑ࣀ㓟࡛ 40 mg/L ࡜࡞ࡿࡼ࠺࡟ຍ࠼ࡓࠋ G418 ஧◲㓟ሷ (G418) ࡣ㸪ᚲせ࡟ᛂࡌ࡚᭱⤊⃰ᗘࡀ 360 mg/L ࡜࡞ࡿࡼ࠺࡟ຍ࠼ࡓࠋᅛᙧᇵᆅ࡟ࡣ㸪ᇵᆅ 1 L ᙜࡓࡾ 20 g ࡢᐮኳࢆຍ࠼ࡓࠋ⬊Ꮚᙧᡂ (SpoK2)ᇵᆅࡣ㸪㓑㓟࣒࢝ࣜ࢘ 10 g㸪1 g/L 㓝 ẕ࢚࢟ࢫ㸪 0.5 g/L ࢢࣝࢥ࣮ࢫ㸪 20 g/L ᐮኳἓࢆ⵨␃Ỉ 1 L ᙜࡓࡾ࡟ྵࡳ㸪 pH 5.5 ࡟ㄪ

ᩚࡋࡓࠋ

኱⭠⳦ࡢቑṪᇵᆅ࡜ࡋ࡚⏝࠸ࡓ Luria-Bertani (LB) ᇵᆅࡣ㸪ࣂࢡࢺࢺࣜࣉࢺࣥ 10 g 㸪

ࣂࢡࢺ㓝ẕ࢚࢟ࢫ 5 g㸪 NaCl 10 g ࢆ⵨␃Ỉ 1 L ᙜࡓࡾ࡟ྵࡳ pH 7.2 ࡟ㄪᩚࡋࡓ (38)ࠋ

18

ᅛᙧᇵᆅ࡟ࡣᇵᆅ 1 L ᙜࡓࡾ 15 g ࡢᐮኳࢆຍ࠼ࡓࠋࣅࢱ࣑ࣥࡣᚲせ࡟ᛂࡌ࡚㸪ࢳ࢔࣑

ࣥࢆ᭱⤊⃰ᗘ 5 mg/L ࡜࡞ࡿࡼ࠺࡟ຍ࠼ࡓࠋᢠ⏕≀㉁ࡣᚲせ࡟ᛂࡌ࡚㸪᭱⤊⃰ᗘࡀ࢔

ࣥࣆࢩࣜࣥ (Amp)࡜࢝ࢼ࣐࢖ࢩࣥ (Km)࡛ 50 μg/mL ࡜࡞ࡿࡼ࠺࡟ຍ࠼ࡓࠋࢥࣥࣆࢸ

ࣥࢺ࡞኱⭠⳦ࡢㄪ〇࡟⏝࠸ࡓ M9 ᇵᆅࡣ (38) 㸪 NH

Cl 1.0 g 㸪 Na

HPO

6.0 g 㸪 KH

PO

3.0 g㸪NaCl 0.5 g㸪1 M MgSO

2 mL㸪200 g/L ࢢࣝࢥ࣮ࢫ 10 mL㸪1M CaCl

0.1 mL ࢆ

⵨␃Ỉ 1 L ࠶ࡓࡾ࡟ྵࡳ pH7.5 ࡟ㄪᩚࡋࡓࠋ

1.2.3 㓝 㓝ẕࡢᙧ㉁㌿᥮

S. cerevisiae ࡢᙧ㉁㌿᥮ࡣ㸪Gietz ࡜ Woods ࡢ᪉ἲ࡛⾜ࡗࡓ (39)ࠋཷᐜ⳦ࢆ 5 mL ࡢ 2 × YPDA ᇵᆅ࡟᳜⳦ࡋ㸪 30°C ୍࡛ᬌ᣺┞ᇵ㣴ࡋ (120 ᅇ / ศ  ᚟᣺┞ ) 㸪྾ගᗘ A

ࢆ ᐃࡋࡓ (2.5 × 10

⣽⬊/mL)ࠋணࡵ 30°C ࡟ಖ ࡋࡓ 2 × YPDA ᇵᆅ 50 mL ࡟㸪ึ

Ⓨ⃰ᗘࡀ 5 × 10

⣽⬊ /mL ࡜࡞ࡿࡼ࠺࡟ᇵ㣴ᾮࢆຍ࠼ࡓࠋ 30°C 120 rpm ࡛⣙ 4 ᫬㛫᣺

┞ᇵ㣴ࡋ㸪⣽⬊⃰ᗘࡀ 2.0 × 10

⣽⬊/mL ࡟࡞ࢀࡤ㸪9100 × g㸪2 ศ㛫㐲ᚰศ㞳ࡋࡓࠋ

ୖ⃈ࡳࢆᤞ࡚㸪࣎ࣝࢸࢵࢡࢫ࣑࢟ࢧ࣮࡛ΰࡐࡓࠋ 20 mL ࡢ⁛⳦Ỉࢆຍ࠼ᠱ⃮ࡋ㸪㐲ᚰ ศ㞳ࡋ (9100 × g, 2 ศ)ୖ⃈ࡳࢆᤞ࡚㸪 ࣎ࣝࢸࢵࢡࢫ࣑࢟ࢧ࣮࡛ΰࡐ࡚⣽⬊ࢆὙίࡋࡓࠋ Ὑίࡣ 2 ᅇ⾜ࡗࡓࠋ⣽⬊ᠱ⃮ᾮࢆ 1.5 mL ࡢ࣐࢖ࢡࣟ㐲ᚰ⟶࡟⛣ࡋ㸪 13,000 × g ࡛ 1 ศ 㛫㐲ᚰศ㞳ࡋ㸪ୖ⃈ࡳࢆᤞ࡚㸪࣎ࣝࢸࢵࢡࢫ࣑࢟ࢧ࣮࡛ΰࡐ㸪⁛⳦Ỉࢆຍ࠼࡚ 1.0 mL ᐜ㔞࡜ࡋࡓ (1.0 × 10

/mL) ࠋ㓝ẕ⣽⬊ᠱ⃮ᾮ 100 μL ࢆ 1.5 mL ࡢ࣐࢖ࢡࣟ㐲ᚰ⟶࡟⛣

ࡋ㸪13,000 × g ࡛ 30 ⛊㛫㐲ᚰศ㞳ࡋ㸪ୖ⃈ࡳࢆᤞ࡚ࡓࠋᙧ㉁㌿᥮ΰྜ⁐ᾮࢆ⾲ 1-3 ࡢ ᐜ㔞࡛ㄪ〇ࡋ㸪ᙧ㉁㌿᥮ΰྜ⁐ᾮ 360 μL ࢆ࣐࢖ࢡࣟ㐲ᚰ⟶࡟⛣ࡋ㸪࣎ࣝࢸࢵࢡࢫ࣑

࢟ࢧ࣮࡛Ⰻࡃΰࡐ㸪 42Υ࡛ 40 ศಖ ࡋࡓࠋ 1 ศ 13,000 × g ࡛㐲ᚰศ㞳ࡋࡓᚋ㸪 1 mL ࡢ

⁛⳦Ỉࢆຍ࠼ࡓࠋ࣐࢖ࢡࣟࣆ࣌ࢵࢺࢳࢵࣉ࡛ỿẊ㓝ẕࢆΰࡐ㸪ࡑࡢᚋ࣎ࣝࢸࢵࢡࢫ࣑

࢟ࢧ࣮࡛ᨩᢾࡋࡓࠋ0.5 mL ࡢ YPD ᾮయᇵᆅࢆຍ࠼㸪30Υ࡛ 4 ᫬㛫ಖ ࡋࡓᚋ㸪G418

ࢆྵࡴ YPD 㑅ᢥᇵᆅ࡟ሬ᢯ࡋ㸪 30 Υ࡛ 2 ᪥㟼⨨ᇵ㣴ࡋࡓࠋ

19

⾲ 1-3 ᙧ㉁㌿᥮᫬࡟౑⏝ࡍࡿΰྜ⁐ᾮࡢ๭ྜ

ヨ⸆ ΰྜ⁐ᾮ

PEG3500 50%w/v

240 μL

㓑㓟ࣜࢳ࣒࢘ 1M

36 μL

↻Ἓ ss-࢟ࣕࣜ࢔ DNA

50 μL

ࣉࣛࢫ࣑ࢻ DNA 㸩Ỉ 34 μL

඲㔞 360 μL

50 (w/v)%࣏࢚ࣜࢳࣞࣥࢢࣜࢥ࣮ࣝ⁐ᾮࡣ㸪50 g ࡢ࣏࢚ࣜࢳࣞࣥࢢࣜࢥ࣮ࣝ 3350 ࡟⁛⳦Ỉ

35 mL ࢆຍ࠼࣮࢜ࢺࢡ࣮ࣞࣈࡋࡓࠋ

1 M LiCl ⁐ᾮࢆㄪᩚᚋ࡟࣮࢜ࢺࢡ࣮ࣞࣈ⁛⳦ࡋࡓࠋ

࢟

ࣕࣜ࢔ DNA ࡢㄪ〇ࡣ Gietz ࡜ Woods ࡢ᪉ἲ࡛⾜ࡗࡓ (39)ࠋࡍ࡞ࢃࡕ㸪200 mg ࡢ㧗ศ㔞ࡉࡅ

⢭Ꮚ DNA ࢆ 100 mL ࡢ TE ⦆⾪ᾮ (10 mM Tris-HCl, pH8.0−1.0 mM EDTA)࡟ຍ࠼ࡓࠋ10 mL ࡢ

ࣆ࣌ࢵࢺ࡛⧞ࡾ㏉ࡋୖୗࡋ࡚ DNA ࢆ๧᩿ࡋࡓࠋࡑࡢᚋ㸪࣐ࢢࢿࢸ࢕ࢵࢡࢫࢱ࣮࣮࡛ࣛ 2−3 ᫬ 㛫᏶඲࡟⁐ゎࡍࡿࡲ࡛᧠ᢾࡋࡓࠋᚲせ࡟ᛂࡌ࡚ 4°C ୍࡛ᬌಖ ࡋࡓࠋ㐺㔞࡟ศࡅ㸪−20°C ࡛ ಖᏑࡋࡓࠋ౑⏝๓࡟ 1.0 mL ࡢ࢟ࣕࣜ࢔ DNA ࢆ 5 ศ㛫Ἓ㦐ࡉࡏࡓᚋ㸪ịỈ୰࡛෭ࡸࡋࡓࠋ㞟

⳦୰࡟ࡇࡢ᧯సࢆ⾜ࡗࡓࠋ

1.2.4 㓝 㓝ẕ㑇ఏᏛ࡛⏝࠸ࡿᶆ‽᪉ἲ

(1) ⬊Ꮚᙧᡂ࡜⬊Ꮚᙧᡂࡢほᐹ

⿕ヨ㦂㓝ẕ⳦ࢆ 30°C㸪1 ᪥ YPD ᅛᙧᇵᆅୖ࡛㟼⨨ᇵ㣴ࡋࡓࠋ⁛⳦ࡋࡓ∎᳿ᯞ࡛ቑ Ṫࡋࡓࢥࣟࢽ࣮ࢆ⬊Ꮚᙧᡂᇵᆅ࡟⛣ࡋ㸪 30°C 㸪 2−3 ᪥㟼⨨ᇵ㣴ࡋ࡚⬊Ꮚࢆᙧᡂࡉࡏࡓࠋ ࢧࣥࣉࣝࢆ⁛⳦∎᳿ᯞ࡛ྲྀࡾ㸪 ࢫࣛ࢖ࢻ࢞ࣛࢫୖ࡟⨨࠸ࡓ 5 μL ࡢ⁛⳦Ỉ࡟ᠱ⃮ࡋࡓࠋ ගᏛ㢧ᚤ㙾࡛ (300 ಸ㸪ᑐ≀ࣞࣥࢬ ×20 㸪᥋║ࣞࣥࢬ ×10 㸪୰㛫ኚಸ ×1.5 㸪࢜ࣜࣥࣃ

ࢫගᏛ㢧ᚤ㙾 BH2) ⬊Ꮚᙧᡂࢆほᐹࡋࡓࠋ (2) ᥋ྜᆺุᐃࡢࡓࡵࡢ㞟ᅋ᥋ྜ

⁛⳦ࡋࡓⓑ㔠⥺࡛㓝ẕ⳦⣽⬊ࢆ 2 mL ࡢ YPD ᾮయᇵᆅ࡟᳜⳦ࡋࡓࠋࡉࡽ࡟㸪ゎᯒࡎ

ࡳࡢ MATa ᰴ࠾ࡼࡧ MATα ᰴࢆ⁛⳦ࡋࡓⓑ㔠⥺࡛ྠࡌᇵᆅ࡟᳜⳦ࡋ㸪 30Υ࡛㟼⨨ᇵ㣴

20

ࡋࡓࠋ

(3) ᥋ྜᏊࡢุᐃ

㞟ᅋ᥋ྜࡋࡓᇵ㣴ᾮࢆ 30Υ࡛ 1 ᪥㟼⨨ᇵ㣴ࡋࡓࠋࡑࡢᇵ㣴ᾮ 5 μL ࢆࢫࣛ࢖ࢻ࢞ࣛ

ࢫ࡟ࡢࡏࡓࠋࡑࡢୖ࡟࢝ࣂ࣮࢞ࣛࢫࢆࡢࡏ㸪ගᏛ㢧ᚤ㙾࡛ほᐹࡋࡓࠋ MATa ࡜ MATα

࡜ࡢ᥃ࡅྜࢃࡏࡓ⁐ᾮࢆࢫࣛ࢖ࢻࢢࣛࢫࡢ㞄࡟⨨ࡁ㸪ẚ㍑ࡋ࡞ࡀࡽ㢧ᚤ㙾ほᐹࡋࡓࠋ

⣽⬊ࡀ඾ᆺⓗ࡞୍ಸయ㓝ẕ࡜␗࡞ࡾ㸪⣽⬊ᙧࡀ୙つ๎࡟࡞ࢀࡤ᥋ྜ࡟ྥ࠿ࡗ࡚࠸ࡿ࡜

ุ᩿ࡋࡓࠋ

(4) ࣑ࢡ࣐ࣟࢽࣉ࣮ࣞࢱ࣮࡟ࡼࡿ㓝ẕ⳦ࡢ஧ಸయศ㞳

㞟ᅋ᥋ྜࡋࡓᇵ㣴ᾮࢆ 30Υ࡛㟼⨨ᇵ㣴ࡋ㸪ᩘ᫬㛫ᚋ࡟୍ᗘ ࣎ࣝࢸࢵࢡࢫ࣑࢟ࢧ࣮

࡛ΰྜࡋ㸪 1 ᪥㟼⨨ᇵ㣴ࡋࡓࠋ᪂ࡋ࠸ YPD ᾮయᇵᆅ 2 mL ࡟ 75 μL ࡢᇵ㣴ᾮࢆຍ࠼㸪 30Υ࡛ 1 ᪥㟼⨨ᇵ㣴ࡋࡓࠋᇵ㣴ᾮࢆⅆ⅖⁛⳦ࡋࡓⓑ㔠⪥࡛ 20 mL ࡢ YPD ᅛᙧᇵᆅࡢ

ୖ࡟㍕ࡏࡓࠋ࣑ࢡ࣐ࣟࢽࣉ࣮ࣞࢱ࣮ ( ࢩ࣮ࣥ࢞ MSM ࢩࢫࢸ࣒ 400 㸪 Singer Instruments, Roadwater, Watchet, Somerset TA23 0RE, UK) ࢆ⏝࠸࡚㸪඾ᆺⓗ࡞஧ಸయ㓝ẕ࡛࠶ࡿ༸

ᙧ࡟㏆࠸ᙧࡢ༢⣽⬊ࢆศ㞳ࡋࡓࠋ 30°C ࡛ 2 ᪥㛫 㟼⨨ᇵ㣴ࡋ㸪༢⣽⬊࠿ࡽቑṪࡋࡓ஧

ಸయࢆᚓࡓࠋ

(5) Ꮚᄞ⬊Ꮚࡢゎ๗

⬊Ꮚᙧᡂᇵᆅୖࡢ⣽⬊ࢆ 300 μg/mL ᭱⤊⃰ᗘ࡛ zymolyase20-T ࢆྵࡴ 0.15 M ࣜࣥ

㓟࣒࢝ࣜ࢘⦆⾪ᾮ pH7.5 ࡢ 75 μL ࡟ᠱ⃮ࡋ㸪 30°C ࡛ 20 ศಖ ࡋࡓࠋ⁛⳦ࡋࡓⓑ㔠⪥

࡛⬊Ꮚᠱ⃮ᾮࢆྲྀࡾ㸪 YPD ᅛᙧᇵᆅୖ࡟⛣ࡋࡓࠋ࣑ࢡ࣐ࣟࢽࣉ࣮ࣞࢱ࣮࡛ 4 ⬊Ꮚࢆ༢

⬊Ꮚࡎࡘゎ๗ࡋࡓᚋ㸪 30°C ࡛ 2 ᪥࠿ࡽ 3 ᪥㛫㸪㟼⨨ᇵ㣴ࡋࡓࠋ

1.2.5 㓝 㓝ẕᰁⰍయ DNA ࡢㄪ〇

DNA ᢳฟࡣ Gen ࡜ࡿࡃࢇ TM (㓝ẕ⏝) High Recovery (ࢱ࢝ࣛࣂ࢖࢜ᰴᘧ఍♫㸪ி

㒔㸪᪥ᮏ ) ࢆ⏝࠸㸪࢟ࢵࢺ౪⤥ඖࡢㄝ᫂᭩࡟ᚑࡗ࡚ㄪᩚࡋࡓࠋ⡆༢࡟ࡣ㸪 30 Υ 1 ᪥ YPD

ᐮኳᇵᆅୖ࡛ᇵ㣴ࡋࡓ㓝ẕ⳦ࢆ 2 mL ࡢ YPD ᾮయᇵᆅ࡟ 1 ⓑ㔠⥺᳜⳦ࡋ㸪㐺ᙜ࡟ᕼ㔘

21

ࡋࡓࠋࡑࡢ 0.1 mL ࢆ 20 mL ࡢ YPD ᾮయᇵᆅ࡟᳜⳦ࡋ㸪30°C ୍࡛ᬌ᣺┞ᇵ㣴ࡋࡓࠋ A

=1.0 ࡟㏆࠸ᇵ㣴ᾮࢆ௨ୗࡢ᧯స࡟⏝࠸ࡓࠋᇵ㣴ᾮࢆ 5 mL ᐜ࢚ࢵ࣌ࣥ࡟ 2−3mL ࡎ ࡘศὀࡋ㸪12,000 rpm 5 ศ㐲ᚰࡋࡓࠋୖ⃈ࡳࢆᤞ࡚ 1 mL ࡢ⁛⳦Ỉࢆຍ࠼࡚Ὑίࡋ㸪ࡶ

࠺ 1 ᗘ㐲ᚰࡋࡓࠋ 0.5 mL ࡢ⁐ᾮ A ࢆ⳦యỿẊ࡟ຍ࠼࡚࣎ࣝࢸࢵࢡࢫ࣑࢟ࢧ࣮࡛ΰྜ

ࡋࡓࠋ᣺┞ᇵ㣴ᶵ࡟ධࢀ㸪 37°C ࡛ 1 ᫬㛫ࡺࡿࡸ࠿࡟᣺┞ࡋࡓࠋࡲࡓ㸪᫬ࠎྲྀࡾฟࡋ⦆

ࡸ࠿࡟ΰྜࡋࡓࠋ 0.1 mL ࡢ⁐ᾮ B ࢆຍ࠼㸪✜ࡸ࠿࡟ΰྜᚋ㸪 70 Υ࡛ 10 ศ㛫ຍ⇕ࡋࡓࠋ

0.2 mL ࡢ⁐ᾮ C ࢆຍ࠼㸪✜ࡸ࠿࡟ΰྜᚋ㸪ị୰࡛ 5 ศ㛫෭ࡸࡋࡓࠋ 12,000 rpm ࡛ 5 ศ

㛫㐲ᚰศ㞳ࡋ㸪ỿẊࢆ྾ࢃ࡞࠸ࡼ࠺ὀព῝ࡃୖ⃈ࡳࢆྲྀࡾ㸪᪂ࡋ࠸ 1.5 mL ᐜࢧࣥࣉ

ࣝ⟶࡟⛣ࡋࡓ (ỿẊ≀ࢆྲྀࡾ㝖ࡃࡓࡵ࡟ྠࡌ᧯సࢆ 2 ᅇ⾜ࡗࡓ)ࠋᅇ཰ࡋࡓᾮ㔞ࡢ 1/2 㔞ࡢ࢖ࢯࣉࣟࣃࣀ࣮ࣝࢆຍ࠼㸪⦆ࡸ࠿࡞࢖ࣥࣂ࣮ࢪ࡛ࣙࣥΰྜࡋࡓࠋ 12,000 rpm ࡛ 5 ศ㛫㸪4°C ࡛㐲ᚰᚋ㸪ୖ⃈ࡳࢆᤞ࡚ࡓࠋࡼࡃ෭࠼ࡓ 99.5% ࢚ࢱࣀ࣮ࣝࢆ 1 mL ຍ࠼࡚

㐲ᚰศ㞳ࡋୖ⃈ࡳࢆᤞ࡚ࡓࠋḟ࡟㸪ࡼࡃ෭࠼ࡓ 70% ࢚ࢱࣀ࣮ࣝࢆ 0.5 mL ຍ࠼㸪 12,000 rpm ࡛ 1 ศ㛫㐲ᚰศ㞳ࡋ㸪ୖ⃈ࡳࢆᤞ࡚࡚஝⇱ࡉࡏࡓࠋᖖ ࡢ࣑ࣜ Q Ỉࢆቨࡢ DNA

ࢆ⁐࠿ࡍࡼ࠺࡟ 0.1 mL ຍ࠼ࡓࠋ

1.2.6 High Pure Plasmid Isolation Kit ࢆ ࢆ⏝࠸ࡓ኱⭠⳦࠿ࡽࡢࣉࣛࢫ࣑ࢻ DNA ᢳฟ ࣉࣛࢫ࣑ࢻᢳฟ࡟ࡣ㸪High Pure Plasmid Isolation Kit (ࣟࢵࢩ࣭ࣗࢲ࢖࢔ࢢࣀࢫࢸ࢕

ࢵࢡᰴᘧ఍♫㸪ᮾி㸪᪥ᮏ ) ࢆ⏝࠸㸪౪⤥ඖࡢࣉࣟࢺࢥ࡛ࣝㄪᩚࡋࡓࠋ 5 mL ࡢ LB ᾮ య㑅ᢥᇵᆅ࡟ (ᚲせ࡟ᛂࡌ࡚ᢠ⏕≀㉁㸪ࣅࢱ࣑ࣥࢆῧຍ)㸪ࣉࣛࢫ࣑ࢻࢆಖᣢࡍࡿ኱⭠

⳦ࢆ㸯ⓑ㔠⪥᳜⳦ࡋ㸪 37°C ୍࡛ᬌ᣺┞ᇵ㣴ࡋࡓࠋ㐲ᚰศ㞳 (2300 × g 㸪 3 ศ㛫㸪 4°C) ࡛ 㞟⳦ࡋୖ⃈ࡳࢆᤞ࡚ࡓࠋ ⳦య࡟ High Pure Plasmid Isolation Kit ࡢ RNase/Suspension Buffer (1) ࢆ 0.25 mL ຍ࠼㸪ࡼࡃΰ࿴ᚋ࡟ Lysis Buffer (2) ࢆ 0.25 mL ຍ࠼㸪⦆ࡸ࠿࡟ΰ࿴ࡋ㸪 ị୰࡛ 5 ศ㛫ಖ෭ࡋࡓࠋࡇࢀ࡟ணࡵị୰࡛෭ࡸࡋ࡚࠾࠸ࡓ Binding Buffer (3)ࢆ 0.35 mL ຍ࠼㸪⦆ࡸ࠿࡟ΰ࿴ࡋ㸪ị୰࡛ 5 ศ㛫ಖ෭ࡋࡓࠋ㐲ᚰศ㞳ࡋ (13,000 × g 㸪 10 ศ㛫㸪

4°C)㸪ỿẊ≀ࡸᾋ㐟≀ࢆྲྀࡽ࡞࠸ࡼ࠺࡟ୖ⃈ࡳࢆ Collection Tubes ࡟ࢭࢵࢺࡋࡓ High

22

Pure Filter Tubes ࡟ᅇ཰ࡋࡓࠋ㐲ᚰศ㞳ࡋ (20,400 × g㸪1 ศ㛫㸪4°C)㸪Collection Tubes

࡟⁀ࡲࡗࡓ⁐ᾮࢆᤞ࡚㸪 Wash Buffer II (5) ࢆ 0.7 mL ࡢ High Pure Filter Tubes ࡟ຍ࠼ࡓࠋ 㐲ᚰศ㞳ࡋ (20,400 × g㸪1 ศ㛫㸪4°C)㸪Collection Tubes ࡟⁀ࡲࡗࡓ⁐ᾮࢆᤞ࡚㸪㐲ᚰ ศ㞳ᚋ (20,400 × g 㸪 1 ศ㛫㸪 4°C) 㸪 High Pure Filter Tubes ࢆ⁛⳦ࡋࡓ࢚ࢵ࣌ࣥࢻࣝࣇ࡟

ࢭࢵࢺࡋࡓࠋHigh Pure Filter Tubes ࡟ Elution Buffer (6)ࢆ 0.1 mL ຍ࠼㸪㐲ᚰศ㞳ࡋࡓ (20,400 × g 㸪 1 ศ㛫㸪 4°C) ࠋ㐲ᚰᚋ㸪 High Pure Filter Tubes ࢆᤞ࡚࢚ࢵ࣌ࣥࢻࣝࣇ࡟ᚓ

ࡽࢀࡓᾮࢆࣉࣛࢫ࣑ࢻ DNA ⁐ᾮ࡜ࡋࡓࠋ

1.2.7 ࢔ ࢔࣮࢞ࣟࢫࢤࣝ㟁ẼὋື

1/2× TBE ⦆⾪ᾮ ( ⦆⾪ᾮ 1 L ᙜࡓࡾ 5.4 g Tris-HCl 㸪 2.75 g ࣍࢘㓟㸪 0.47 g EDTA ࣭ 2Na ࢆྵࡴ)࡟ 0.8ࠥ2.0 %࡜࡞ࡿࡼ࠺࡟࢔࣮࢞ࣟࢫ (ᐆ㓇㐀♫〇 L03 ࡶࡋࡃࡣࢼ࢝ࣛ

࢖♫〇 ) ࢆຍ࠼㸪 121°C ࡛ 1 ศ㛫ࡢ⵨Ẽຍ⇕࡛⁐ゎᚋ࡟ࢤࣝࢆసᡂࡋࡓࠋ 1−10 μL ࡢ

DNA ⁐ᾮ࡟ᑐࡋ࡚ 1−2 μL ࡢ࣮ࣟࢹ࢕ࣥࢢ⦆⾪ᾮ (0.25% bromophenol blue㸪0.25%

xylene cyanol 㸪 30% ࢢࣜࢭ࣮ࣟࣝ ) ࢆຍ࠼㸪ࢤࣝࡢ࢙࢘ࣝ࡟ࢳ࣮ࣕࢪࡋࡓࠋ࣑ࢽࢤࣝ㟁

ẼὋືࢩࢫࢸ࣒ (mupid-2 ࢥࢫࣔࣂ࢖࢜ᰴᘧ఍♫〇)ࢆ⏝࠸࡚ 50 V㸪90 ศ㛫㟁ẼὋື

ࡋࡓࠋ 0.5 μg/mL ࡢ࢚ࢳࢪ࣒࢘ࣈ࣐ࣟ࢖ࢻࢆྵࡴ TBE ⦆⾪ᾮ࡟ 15 ศ㛫ᾐࡋ࡚ᰁⰍࡋ ࡓࠋᰁⰍࡋࡓࢤࣝࢆࢺࣛࣥࢫ࢖࣑ࣝࢿ࣮ࢱ࣮ୖ࡛ほᐹࡋࡓࠋ

1.2.8 PCR ἲ࡟ࡼࡿ DNA ᩿∦ࡢቑᖜ

PCR ཯ᛂࡣ㸪 KOD FX (TOYOBO ♫〇 ) ࢆ⏝࠸࡚⾜ࡗࡓࠋ཯ᛂヨ⸆࡟㗪ᆺ DNA ࡜

ࣉࣛ࢖࣐࣮ࢆῧຍࡋࡓ (ྜィ 50 μ0)ࠋ㍍ࡃࢫࣆࣥࢲ࢘ࣥࡋ㸪94°C ࡟ಖᣢࡉࢀࡓࢧ࣮

࣐ࣝࢧ࢖ࢡ࣮ࣛ࡟ࢭࢵࢺࡋࡓࠋ PCR ཯ᛂ᫬㛫ࡣቑᖜ᩿∦ࡢ኱ࡁࡉ࡟ࡼࡗ࡚ኚ࠼ࡓࠋ 1 kb ࡢ᩿∦ࢆቑᖜࡍࡿ࡜ࡁࡣ㸪95°5 ࡛ 15 ⛊㸪50°0 ࡛ 30 ⛊㸪60°C ࡛ 1 ศࡢࢧ࢖ࢡࣝࢆ

25 ᅇ⧞ࡾ㏉ࡋࡓࠋࡑࡢᚋ 68°8 ࡛ 5 ศಖᣢࡍࡿࡇ࡜࡟ࡼࡗ࡚ቑᖜࡋࡓࠋ

23

1.2.9 ሷ ሷᇶ㓄ิỴᐃ

Applied Biosystems 3130 ࢪ࢙ࢿࢸ࢕ࢵࢡ࢔ࢼࣛ࢖ࢨࢆ౑⏝ࡋሷᇶ㓄ิỴᐃࡋࡓࠋ㓄

ิỴᐃ⏝ࡢ࢟ࢵࢺࡣ㸪BigDye® Terminator v1.1 Cycle Sequencing Kit ࡲࡓࡣ BigDye®

Terminator v3.1 Cycle Sequencing Kit ࢆ౑⏝ࡋࡓࠋ㗪ᆺ DNA ࡢᕼ㔘ࡣ㸪ቑᖜࡍࡿ᩿∦

ࡢࢧ࢖ࢬࢆ⪃៖ࡋ㐺ษ࡞⃰ᗘ࡛⾜ࡗࡓࠋࡍ࡞ࢃࡕ㸪ࣉࣛࢫ࣑ࢻ DNA ࡛࠶ࢀࡤ㸪 150㹼

300 ng ࡟࡞ࡿࡼ࠺࡟ᕼ㔘ࡋࡓࠋ⢭〇ࡋࡓ㗪ᆺ DNA ࢆ౑⏝ࡋ㸪 96°C ࡛ 1 ศ㛫ࡢ DNA

ኚᛶᚋ㸪 96°C ࡛ 1 ศ㛫 DNA ኚᛶ㸪 50°C ࡛ 5 ศ㛫 ࢔ࢽ࣮ࣜࣥࢢ㸪 60°C ࡛ 4 ศ㛫 DNA ఙ㛗཯ᛂࢆ 25 ࢧ࢖ࢡࣝ⾜ࡗࡓࠋࡑࡢᚋ㸪 4°C ࡢịỈ୰࡛ᛴ෭ࡋࡓࠋ࢚ࢱࣀ࣮ࣝỿẊ ࡋ㸪DNA ࢆᅇ཰ࡋࡓࠋ࢚ࢱࣀ࣮ࣝỿẊࡣ㸪5 μL ࡢ 125 mM EDTA࣭2Na ࢆຍ࠼ࡓᚋ㸪 60 μL ࡢ 99.5% ࢚ࢱࣀ࣮ࣝࢆຍ࠼㸪Ⰻࡃΰྜࡋ㸪ᐊ ࡛ 15 ศ㛫㟼⨨ࡋࡓࠋ 20,400 × g

࡛ 20 ศ㛫 㐲ᚰศ㞳ࡋୖ⃈ࡳࢆ㝖ཤࡋࡓࠋࡑࡢᚋ㸪60 μL ࡢ 70%࢚ࢱࣀ࣮ࣝࢆຍ࠼㸪

20,400 × g ࡛ 5 ศ㛫 㐲ᚰศ㞳ࡋ㸪ୖ⃈ࡳࢆ㝖ཤࡋࡓࠋ 5 ศ㛫ࡢῶᅽ஝⇱ᚋ㸪࢔࣑ࣝ࣍

࢖࡛ࣝそ࠸㸪4°C ࡛ಖᏑࡋࡓࠋ౑⏝ࡍࡿ㝿ࡣ㸪20 μL ࡢ࣒࣍ࣝ࢔࣑ࢻࢆຍ࠼ 95Υ࡛ 3 ศ㛫 ኚᛶᚋ㸪 4°C ࡢịỈ୰࡛ᛴ෭᧯సࢆ⾜ࡗࡓࠋㄪᩚࡋࡓ DNA ⁐ᾮࡢ඲㔞ࢆ཯ᛂࣉ

࣮ࣞࢺ࡟ධࢀ㸪ࢩ࣮ࢣࣥࢧ࡟ࢭࢵࢺࡋ㸪ሷᇶ㓄ิỴᐃࡋࡓࠋ

1.2.10 㓝ẕࡢ඲ࢤࣀ࣒ሷᇶ㓄ิࡢ෌ゎᯒ

ኚ␗య DNA ࢆ౪⤥ඖࡢࣉࣟࢺࢥࣝ (Life Technologies, Inc., Carlsbad, CA, USA) ࡟ᇶ

࡙ࡁ SOLiD 5500xl ࢩࢫࢸ࣒࡛㸪ᅜ❧◊✲㛤Ⓨἲே ⏘ᴗᢏ⾡⥲ྜ◊✲ᡤ (⏘⥲◊)࡛ゎ

ᯒࡉࢀࡓࠋ⏘⥲◊ࡣ㸪 NEDO ᅜ❧◊✲㛤Ⓨἲே ᪂࢚ࢿࣝࢠ࣮࣭⏘ᴗᢏ⾡⥲ྜ㛤Ⓨᶵ ᵓࡢࠕࣂ࢖࣐࢜ࢫ࢚ࢿࣝࢠ࣮ᢏ⾡◊✲㛤Ⓨ㸭ࣂ࢖࢜⇞ᩱ〇㐀ࡢ᭷⏝せ⣲ᢏ⾡㛤Ⓨ஦ᴗ 㸭᭷⏝ᚤ⏕≀ࢆ⏝࠸ࡓⓎ㓝⏕⏘ᢏ⾡ࡢ◊✲㛤Ⓨࠖࣉࣟࢪ࢙ࢡࢺ␒ྕ P13011 ࡛ጤクඛ

᪥᥹ᰴᘧ఍♫㸪ᏛᰯἲேྩࡀῡᏛᅬ ᓫᇛ኱Ꮫ㸪ᅜ❧◊✲㛤Ⓨἲே ⏘ᴗᢏ⾡⥲ྜ◊✲

ᡤ ( ⏘⥲◊ ) 㸪୍⯡㈈ᅋἲேࣂ࢖࢜࢖ࣥࢲࢫࢺ࣮ࣜ༠఍ࡢ୰ࡢࡦ࡜ࡘࡢᶵ㛵࡛࠶ࡿࠋࡍ

࡞ࢃࡕ㸪ᚓࡽࢀࡓಶࠎࡢሷᇶ㓄ิࢆཧ↷ࡍࡿ NAM34−4C ᰴࡢࢤࣀ࣒ DNA 㓄ิ࡟ᩚ

24

ิ ࡉ ࡏ ࡓ (BAUH01000001 to BAUH01000154) ࠋ ኚ ␗ ࡋ ࡓ 㓄 ิ ࢆ ࢹ ࣮ ࢱ ゎ ᯒ

LifeScope2.5 ࢯࣇࢺࡢᶆ‽ࣃ࣓࣮ࣛࢱ࣮ࡢࢭࢵࢺ࡛ࣂࣜ࢔ࣥࢺࢥ࣮࣮ࣛ (diBayes) ࡟

ࡼࡾ᳨ฟࡋࡓࠋࡉࡽ࡟㸪ᚲせ࡞᝟ሗࡣ㸪ཧ↷ࢤࣀ࣒ࡢࢻࣛࣇࢺ㓄ิᰁⰍయ␒ྕ㸪㑇ఏ Ꮚྡࡶࡋࡃࡣ㞄᥋㑇ఏᏊྡ ( ኚ␗ࡀ㑇ఏᏊ㛫࡟࠶ࡿሙྜ ) 㸪 Ruby ࣉࣟࢢ࣒࡛ࣛグ㍕ࡉ

ࢀ࡚࠸ࡿኚ␗ࡢಙ㢗ᛶࢆ♧ࡍ P ್࡞࡝ࢆᢳฟࡋࡓࠋぶᰴࡸ 3 ᰴࡢ࢟ࢩࣜࢺ࣮ࣝኚ␗య ࡢኚ␗ࢆẚ㍑ࡋ㸪ཎᅉ㑇ఏᏊኚ␗ࡢೃ⿵ࢆ P ್ࡢ㧗࠸㡰࡟ᢳฟࡋࡓࠋ⥆࠸࡚㸪ぶᰴ࡜

3 ᰴࡢ࢟ࢩࣜࢺ࣮ࣝኚ␗య㛫࡛㑇ఏᏛⓗ⣔⤫ゎᯒࢆ⾜࠸㸪ೃ⿵㑇ఏᏊኚ␗ࢆ⤠ࡾ㎸ࢇ

࡛࠸ࡗࡓࠋ

1.2.11 cre Ⓨ Ⓨ⌧࡟ࡼࡿ kanMX ࣐࣮࣮࢝ࡢ㝖ཤ

kanMX 㑅ᢥ࣐࣮࣮࡛࢝㑇ఏᏊ᧯సࡋࡓᰴ࠿ࡽ kanMX ࢆྲྀࡾ㝖ࡃ᪉ἲࡣ㸪Guldener

ࡽࡢ᪉ἲ (40) ࡟ᚑࡗࡓࠋ loxP−kanMX–loxP 㡿ᇦࢆྵࡴᰴ࡟㸪 cre Ⓨ⌧ࣉࣛࢫ࣑ࢻ pZeo

ࢆᙧ㉁㌿᥮ἲ࡛⛣ࡋ㸪ࢮ࢜ࢩࣥ⪏ᛶ㌿᥮యࢆᚓࡓࠋḟ࡟㸪5 mL ࡢࢮ࢜ࢩࣥࢆຍ࠼ࡓ

YPDAU ᇵᆅ࡟⿕ヨ㦂⳦ࢆ 1 ⓑ㔠⥺᳜⳦ࡋ㸪 30°C ࡛⣙ 16 ᫬㛫᣺┞ᇵ㣴ࡋࡓࠋࡇࡢᇵ

㣴ᾮࡢ A

ࢆ ᐃࡋ㸪⃮ᗘࡀ 1 ௨ୖ࡟࡞ࡗ࡚࠸ࢀࡤᇵ㣴ᾮ 4 mL ࢆ 3500 × g, 1 ศ㛫

㐲ᚰศ㞳ࡋࡓࠋୖ⃈ࡳࢆᤞ࡚㸪࣎ࣝࢸࢡࢫ࣑࢟ࢧ࣮࡛ΰࡐ㸪 4 mL ࡢ⁛⳦Ỉ࡟ᠱ⃮ࡋ ࡓࠋࡇࡢ᧯సࢆ 2 ᅇ⧞ࡾ㏉ࡋ㸪⣽⬊ࢆὙίࡋࡓࠋ⳦యࢆ 1 mL ࡢ⁛⳦Ỉ࡟ᠱ⃮ࡋ㸪100 μL ࢆ 5 mL ࡢ YPGalAU ᇵᆅ࡟᳜⳦ࡋ㸪 30°C ࡛ 1 ᫬㛫ᇵ㣴ࡋࡓࠋᇵ㣴ᾮ 4 mL ࢆ 3500

× g, 1 ศ㛫㐲ᚰศ㞳ࡋࡓࠋୖ⃈ࡳࢆᤞ࡚㸪࣎ࣝࢸࢡࢫ࣑࢟ࢧ࣮࡛ΰࡐ㸪4 mL ࡢ⁛⳦Ỉ

࡟ᠱ⃮ࡋࡓࠋࡇࢀࢆ 2 ᅇ⧞ࡾ㏉ࡋ㸪⣽⬊ࢆὙίࡋࡓࠋᠱ⃮ᾮࢆ㐺ᙜ࡟ᕼ㔘ࡋ࡚㸪 YPDAU ᖹᯈᇵᆅ࡟ሬ᢯ࡋ㸪 30Υ࡛ 1−2 ᪥㟼⨨ᇵ㣴ࡋࡓࠋ⏕ࡌࡓࢥࣟࢽ࣮ࢆ YPDAU 㸪

YPDAU+G418 㸪ࢮ࢜ࢩࣥࢆྵࡴ YPDAU ᖹᯈᇵᆅ࡟ࡑࢀࡒࢀ 50 ಶࡎࡘ⛣ࡋ㸪 G418 ឤ

ཷᛶᰴࢆ㑅ᢥࡋࡓࠋ

ࣉࣛࢫ࣑ࢻࢆ㝖ཤࡍࡿࡓࡵ࡟㸪 G418 ឤཷᛶ࡛ࢮ࢜ࢩࣥ⪏ᛶᰴࢆ 5 mL ࡢ YPADAU

ᇵᆅ࡟ 1 ⓑ㔠⥺᳜⳦ࡋ㸪30Υ࡛⣙ 16 ᫬㛫᣺┞ᇵ㣴ࡋࡓࠋᇵ㣴ᾮࢆ㐺ᙜ࡟ᕼ㔘ࡋ࡚㸪

25

YPDAU ᇵᆅ࡟ሬ᢯ࡋ㸪30Υ࡛ 1 ᪥㟼⨨ᇵ㣴ࡋࡓࠋ⏕ࡌࡓࢥࣟࢽ࣮ࢆ YPDAU㸪ࢮ࢜

ࢩࣥࢆྵࡴ YPDAU ᖹᯈᇵᆅ࡟ 50 ಶࡎࡘ⛣ࡋ㸪ࢮ࢜ࢩࣥឤཷᛶᰴࢆ㑅ᢥࡋࡓࠋ

1.2.12 ࢟ ࢟ࢩࣜࢺ࣮ࣝ㈨໬ᛶ⮬↛✺↛ኚ␗యࡢศ㞳

S. cerevisiae SCB39 ᰴ࡜ SCB40 ᰴࡢᰤ㣴⣽⬊ࢆ YPD ᇵᆅ pH 4.5 ࡛ 24 ᫬㛫㸪᣺┞

ᇵ㣴 (30°C, 120 rpm) ࡋࡓࠋ㐲ᚰศ㞳 (4°C , 2400 × g, 1 ศ ) ࡛㞟⳦ࡋ㸪⁛⳦Ỉ࡟ᠱ⃮ࡋ ࡓࠋึⓎ⣽⬊⃮ᗘ A

= 0.014 ࡜࡞ࡿࡼ࠺࡟ 5 mL ᐜ㔞ࡢ L ᆺヨ㦂⟶ෆࡢࠊ࢟ࢩࣜࢺ࣮

ࣝ 100 g/L ࢆྵࢇ࡛࠸ࡿ XLYP ᇵᆅࡶࡋࡃࡣ LYP ᇵᆅ 5 mL ࡬࡜᳜⳦ࡋࡓࠋ

➨ 3 ⠇ ⤖ᯝ

1.3.1 Saccharomyces cerevisiae ࡢ࢟ࢩࣜࢺ࣮ࣝ㈨໬ᛶ✺↛ኚ␗యࡢศ㞳

⣽⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡓ࢟ࢩࣜࢺ࣮ࣝ௦ㅰࡢ᪩࠸ᰴࡀ㸪࢟ࢩࣜࢺ࣮ࣝ㈨໬ᛶ✺↛ኚ

␗యࢆศ㞳ࡍࡿぶᰴ࡟ࡣᮃࡲࢀࡿࠋࡑࡇ࡛㸪HEX1−22 ࡜ SXM1−32 ࠶ࡿ࠸ࡣ

HEX1−22 ࡜ sxm2−33 ኚ␗ࢆᣢࡘ࢟ࢩ࣮ࣟࢫ㈨໬ᛶࡀ㧗࠸⤌ࡳ᥮࠼㓝ẕ⳦ S.

cerevisiae SCB39 ᰴ (MATa URA3::TDH3p-XYL1-TDH3t−TDH3p-XYL2-TDH3t−TDH3p- XKS1-TDH3t HEX1−22 SXM1−32 sxmc1−32) ᰴࡶࡋࡃࡣ SCB40 ᰴ (MATa

URA3::TDH3p-XYL1- TDH3t−TDH3p-XYL2-TDH3t−TDH3p-XKS1-TDH3t HEX1−22 sxm2)

ࢆぶᰴ࡜ࡋ࡚✺↛ኚ␗యࢆศ㞳ࡍࡿࡇ࡜࡟ࡋࡓࠋ

᭱ึ࡟㸪࢟ࢩࣜࢺ࣮ࣝ௨እࡢᰤ㣴≀࡛࠶ࡿ࣌ࣉࢺࣥ࡜㓝ẕ࢚࢟ࢫࡢ, ぶᰴ SCB39

࡟ᑐࡍࡿቑṪࡢᙳ㡪ࢆㄪ࡭ࡓࠋ࣌ࣉࢺࣥ࡜㓝ẕ࢚࢟ࢫࢆ㸪ࡑࢀࡒࢀ 2.5 g/L ࡜ 1.25 g/L, 5.0 g/L ࡜ 2.5 g/L, 10 g/L ࡜ 5.0 g/L㸪࠾ࡼࡧ 20 g/L ࡜ 10 g/L ྵࡴ 5 mL ࡢ 20 g/L ࢟ ࢩࣜࢺ࣮ࣝᇵᆅ pH 4.5 ࢆ⏝࠸㸪 ᗘ 35 Υ࡛ ⮬ググ㘓ィࣂ࢖࢜ࣇ࢛ࢺࣞࢥ࣮ࢲ࣮࡛

ቑṪࢆゎᯒࡋࡓࠋ࣌ࣉࢺࣥ 20 g/L ࡜㓝ẕ࢚࢟ࢫ 10 g/L ࢆྵࡴ࢟ࢩࣜࢺ࣮ࣝᇵᆅ࡛

26

ࡣ㸪10 ᫬㛫௨㝆ࡶ⦆ࡸ࠿࡞ቑṪࢆ♧ࡋࡓࡀ㸪࣌ࣉࢺࣥ 2.5 g/L, 㓝ẕ࢚࢟ࢫ 1.25 g/L

࡛ࡣ㸪ቑṪࡀほᐹࡉࢀ࡞࠿ࡗࡓ ( ᅗ 1-1a) ࠋࡲࡓ㸪ࡑࡢ㛫ࡢ⃰ᗘ࡛ࡣ㸪⃰ᗘ࡟ᛂࡌ࡚

୧⪅ࡢ୰㛫ⓗ࡞ቑṪࢆ♧ࡋࡓࠋᚑࡗ࡚㸪㧗࠸ᰤ㣴≀ࡢ⃰ᗘ࡛ࡣ㸪࢟ࢩࣜࢺ࣮ࣝ㠀౫ ᏑࡢቑṪࢆ♧ࡋ࡚࠾ࡾ㸪࢟ࢩࣜࢺ࣮ࣝ౫ᏑࡢቑṪࢆゎᯒࡍࡿࡓࡵ࡟ࡣ㸪࣌ࣉࢺࣥ 2.5 g/L ࡜㓝ẕ࢚࢟ࢫ 1.25 g/L ࡀⰋ࠸࡜⤖ㄽࡋࡓࠋ

⥆࠸࡚㸪࣌ࣉࢺࣥ 2.5 g/L ࡜㓝ẕ࢚࢟ࢫ 1.25 g/L ࢆྵࡴ࢟ࢩࣜࢺ࣮ࣝᇵᆅࡢ࢟ࢩࣜ

ࢺ࣮ࣝ⃰ᗘࢆ 20 g/L ࠿ࡽ 140 g/L ࡲ࡛⃰ᗘࢆኚ໬ࡉࡏ࡚ቑṪࢆゎᯒࡋࡓࠋ࢟ࢩ࣮ࣟࢫ

㈨໬ᰴ SCB39 ࡜ SCB40 ࡣ㸪࢟ࢩ࣮ࣟࢫ㈨໬࡟ᚲ㡲࡛࠶ࡿ XYL1, XYL2, XKS1 ࡢ 3 㑇 ఏᏊ㸪㧗ຠ⋡࢟ࢩ࣮ࣟࢫ௦ㅰኚ␗ HEX1−22㸪㧗⃰ᗘ࢟ࢩ࣮ࣟࢫ௦ㅰኚ␗ SXM1−32

࠶ࡿࡣ sxm2−33 ࢆᣢࡗ࡚࠸ࡿࡀ㸪࠸ࡎࢀࡢᇵᆅ࡛ࡶ⏕⫱ࡣぢࡽࢀ࡞࠿ࡗࡓ ( ᅗ 1-

1b㸪ᅗ 1-1d)ࠋ⃰ᗘ໙㓄࡟ࡼࡿ࢟ࢩࣜࢺ࣮ࣝࡢ⣽⬊ෆ㍺㏦ࡶ㉳ࡇࡗ࡚࠸࡞࠸࡜⪃࠼ࡽ

ࢀࡿࠋᚑࡗ࡚㸪࣌ࣉࢺࣥ 2.5 g/L, 㓝ẕ࢚࢟ࢫ 1.25 g/L 㸪࢟ࢩࣜࢺ࣮ࣝ 100 g/L ࢆྵ

ࡴ࢟ࢩࣜࢺ࣮ࣝᇵᆅ࡛ቑṪࡍࡿ✺↛ኚ␗యࢆศ㞳ࡍࡿࡇ࡜࡟ࡋࡓࠋ

SCB39 ࡜ SCB40 ᰴࢆ࢟ࢩࣜࢺ࣮ࣝ 100 g/L ࢆྵࡴ XLYP ᇵᆅ࡛ 30 Υ 5−7 ᪥㛫㸪

ࣂ࢖࢜ࣇ࢛ࢺࣞࢥ࣮ࢲ࣮࡛ᇵ㣴ࡋࡓᚋ㸪㏿ࡸ࠿࡟ቑṪࡍࡿ⊂❧ࡋࡓኚ␗యࢆ SCB39

ᰴ࠿ࡽ 1 ಶ (SCC7) 㸪 SCB40 ᰴ࠿ࡽ 2 ಶ (SCC8, SCC15) ྲྀᚓࡋࡓࠋ୍᪉㸪࢟ࢩࣜࢺ࣮

ࣝࢆྵࡲ࡞࠸ LYP ᇵᆅ࡛ࡣ㸪ቑṪࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋᚓࡽࢀࡓ࢟ࢩࣜࢺ࣮ࣝ㈨໬

ᛶࡢ✺↛ኚ␗యࢆ࣑ࢡ࣐ࣟࢽࣉ࣮ࣞࢱ࣮࡛༢⣽⬊ศ㞳ࡋ㸪 3 ಶࡢ࢟ࢩࣜࢺ࣮ࣝ㈨໬ᛶ ࡢ✺↛ኚ␗యࡢቑṪࢆ࢟ࢩࣜࢺ࣮ࣝᇵᆅ࡛ㄪ࡭ࡓࠋぶᰴ SCB39 ᰴ࡜ẚ㍑ࡋ࡚ኚ␗య SCC7 ᰴ࡛ࡣ㸪࢟ࢩࣜࢺ࣮ࣝᇵᆅ࡛㢧ⴭ࡞ቑṪࡀぢࡽࢀ ( ᅗ 1-1c) 㸪ぶᰴ SCB40 ࡜ẚ

㍑ࡋ࡚ 2 ಶࡢኚ␗య SCC8 ࡜ SCC15 ࡣ㸪㢧ⴭ࡞ቑṪࡀぢࡽࢀࡓ (ᅗ 1-1d)ࠋࡑࡇ࡛㸪

࢟ࢩࣜࢺ࣮ࣝ㈨໬ᛶ✺↛ኚ␗యᰴࡢ⾲⌧ᆺࢆ Xyt

࡜グ㍕ࡋ㸪ࡇࡢ⾲⌧ᆺࢆᑟࡃኚ␗

ࢆ SCC7 ᰴ࡛ࡣ xyt−7, SCC8 ᰴ࡛ࡣ xyt−8, SCC15 ᰴ࡛ࡣ xyt−15 ࡜௬࡟グ㍕ࡍࡿࡇ࡜

࡟ࡋࡓࠋ

27

ᅗ 1-1 S. cerevisiae ᰴ SCB39㸪SCB40 ࡜ Xyt

✺↛ኚ␗యࡢቑṪࠋ(a) ࣌ࣉࢺࣥ࡜㓝ẕ࢚࢟ࢫ⃰ᗘࡢ㸪

S. cerevisiae SCB39 ᰴࡢ࢟ࢩࣜࢺ࣮ࣝ㈨໬ᛶ࡟ཬࡰࡍᙳ㡪ࠋSxm

ᰴ SCB39 ᰴࡢ⣽⬊ࢆ 5 mL ࡢ 2.5−20

g/L ࣌ࣉࢺࣥ࡜ 1.25−10 g/L 㓝ẕ࢚࢟ࢫࢆྵࡴ࢟ࢩࣜࢺ࣮ࣝᇵᆅ pH 4.5 ࡟᳜⳦ࡋ㸪35Υ࡛᣺┞ᇵ㣴ࡋ ࡓࠋ⣽⬊⃰ᗘࢆࣂ࢖࢜ࣇ࢛ࢺࣞࢥ࣮ࢲ࣮࡛ ᐃࡋࡓࠋグྕ: ۵, 20 g/L ࣌ࣉࢺࣥ࡜ 10 g/L 㓝ẕ࢚࢟ࢫ;

ᇞ, 10 g/L ࣌ࣉࢺࣥ࡜ 5 g/L 㓝ẕ࢚࢟ࢫ; □, 5 g/L ࣌ࣉࢺࣥ࡜ 2.5 g/L 㓝ẕ࢚࢟ࢫ; ۍ, 2.5 g/L ࣌ࣉࢺࣥ

࡜ 1.25 g/L 㓝ẕ࢚࢟ࢫࠋ(b) SCB39 ᰴࡣ㸪࢟ࢩࣜࢺ࣮ࣝ㠀㈨໬ᛶ࡛࠶ࡿࠋSCB39 ᰴࡢ⣽⬊ࢆ 5 mL ࡢ

20−140 g/L ࡢ࢟ࢩࣜࢺ࣮ࣝࢆྵࡴ LYP ᇵᆅ pH 4.5 ࡟᳜⳦ࡋ㸪35Υ࡛᣺┞ᇵ㣴ࡋࡓࠋ⣽⬊⃰ᗘࢆࣂ࢖

࢜ࣇ࢛ࢺࣞࢥ࣮ࢲ࣮࡛ ᐃࡋࡓࠋグྕ: ۵, 20 g/L; ᇞ, 40 g/L; ۃ, 60 g/L; ڽ, 80 g/L; ۇ, 100 g/L; □, 120 g/L; ۍ, 140 g/Lࠋ(c) Xyt

✺↛ኚ␗యࡢቑṪࠋXyt

ኚ␗య (SCC7) ࡜ぶᰴ (SCB39) ࢆ 5 mL ࡢ XLYP ᇵ ᆅ pH 4.5 ࡟᳜⳦ࡋ㸪35Υ࡛᣺┞ᇵ㣴ࡋࡓࠋグྕ: ۵㸪SCB39; ᇞ, SCC7ࠋ (d) Xyt

✺↛ኚ␗యࡢቑṪࠋ Xyt

ኚ␗య (SCC8 ࡜ SCC15) ࡜ぶᰴ (SCB40) ࢆ 5 mL ࡢ XLYP ᇵᆅ pH 4.5 ࡟᳜⳦ࡋ㸪35Υ࡛᣺┞ᇵ 㣴ࡋࡓࠋグྕ: ۵㸪SCB40; ᇞ, SCC8; □, SCC15ࠋ(c)࡜(d)ࡢࢹ࣮ࢱࡣ㸪3 ᅇࡢ⊂❧ࡋࡓᐇ㦂࡟ࡼࡿᖹᆒ

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