JP2019528075A - 新規プロモーター及びその用途 - Google Patents
新規プロモーター及びその用途 Download PDFInfo
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- JP2019528075A JP2019528075A JP2019510874A JP2019510874A JP2019528075A JP 2019528075 A JP2019528075 A JP 2019528075A JP 2019510874 A JP2019510874 A JP 2019510874A JP 2019510874 A JP2019510874 A JP 2019510874A JP 2019528075 A JP2019528075 A JP 2019528075A
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Abstract
Description
1−1.新規プロモーター配列を含有する組換えベクターの作製
目的遺伝子の発現を誘導する新規プロモーターを合成するために、コリネバクテリウム属微生物とエシェリキア属微生物由来の様々なプロモーター配列を分析し、それらから配列番号1、2及び3のヌクレオチド配列を有するプロモーターを合成し、それらをそれぞれSPL1、SPL7、SPL13と命名した。
ベクターpECCG117、並びに前述したように作製した組換えベクターpSPL1−GFP、pSPL7−GFP及びpSPL13−GFPと、従来の公知のプロモーターpcj4(特許文献2)を含むp117−CJ4−GFPを、コリネバクテリウム・グルタミカムATCC13032とATCC13869に電気パルス法(非特許文献9)でそれぞれ形質転換し、その後カナマイシン(kanamycin)25mg/Lを含有するルリア−ベルターニ(LB)寒天培地から形質転換菌株を得た。ATCC13032ベースで得られた菌株をそれぞれコリネバクテリウム・グルタミカムATCC13032/pECCG117、コリネバクテリウム・グルタミカムATCC13032/SPL1−GFP、コリネバクテリウム・グルタミカムATCC13032/SPL7−GFP、コリネバクテリウム・グルタミカムATCC13032/SPL13−GFP、コリネバクテリウム・グルタミカムATCC13032/CJ4−GFPと命名した。また、ATCC13869ベースで得られた菌株をそれぞれコリネバクテリウム・グルタミカムATCC13869/pECCG117、コリネバクテリウム・グルタミカムATCC13869/SPL1−GFP、コリネバクテリウム・グルタミカムATCC13869/SPL7−GFP、コリネバクテリウム・グルタミカムATCC13869/SPL13−GFP、コリネバクテリウム・グルタミカムATCC13869/CJ4−GFPと命名した。
SPL1、SPL7及びSPL13プロモーターの活性を確認するために、実施例1−2で得られた形質転換菌株であるコリネバクテリウム・グルタミカムATCC13032/pECCG117、コリネバクテリウム・グルタミカムATCC13032/CJ4−GFP、コリネバクテリウム・グルタミカムATCC13032/SPL1−GFP、コリネバクテリウム・グルタミカムATCC13032/SPL7−GFP、コリネバクテリウム・グルタミカムATCC13032/SPL13−GFP、コリネバクテリウム・グルタミカムATCC13869/pECCG117、コリネバクテリウム・グルタミカムATCC13869/CJ4−GFP、コリネバクテリウム・グルタミカムATCC13869/SPL1−GFP、コリネバクテリウム・グルタミカムATCC13869/SPL7−GFP及びコリネバクテリウム・グルタミカムATCC13869/SPL13−GFPを次の方法で培養し、GFP活性を測定した。
2−1.サイコース生産能の評価
(1)SPL1及びSPL7プロモーター配列を含むATPE発現用ベクター及び形質転換菌株の作製
SPL1及びSPL7を用いて、ATPE(アグロバクテリウム・ツメファシエンスATCC33970由来のサイコースエピメラーゼ)の発現が増加したコリネバクテリウム菌株用ベクターを作製した。pET24−ATPE−2ベクター(配列番号8)を鋳型とし、配列番号9及び10のプライマーでPCR(94℃で30秒間、55℃で30秒間、72℃で1分間の反応を30回)を行い、ATPE遺伝子のORF(Open Reading Frame)を増幅した。増幅したATPE遺伝子と実施例1で作製したコリネバクテリウム菌株用ベクターpSPL1−GFP及びpSPL7−GFPを制限酵素EcoRV及びPstIで処理し、その後前記PCRにより得られたATPE−2をBD In−Fusion kitで作動可能に連結することにより、最終的にコリネバクテリウム菌株用ベクターpSPL1−ATPE−2及びpSPL7−ATPE−2を作製した。
前記過程を経て作製した菌株を実施例1と同じ組成の培地で培養し、その後ATPEの活性を測定した。ATCC13032/pECCG117菌株及びATCC13032/CJ4−ATPE−2菌株は対照群として用いた。
(1)SPL13プロモーター配列を含むUxaE発現用ベクター及び形質転換菌株の作製
GFPが挿入されたCJ4−GFPと実施例1で作製したSPL13−GFPを用いて、サーモトガ・ネアポリタナ由来のタガトースエピメラーゼ遺伝子(UxaE)をクローニングしてコリネバクテリウム菌株用ベクターを作製した。pET28a−TN(m)ベクター(配列番号11)を鋳型とし、配列番号12及び13のプライマーでPCR(94℃で30秒間、55℃で30秒間、72℃で1分間の反応を30回)を行い、TN(m)遺伝子のORF(Open Reading Frame)を増幅した。増幅した遺伝子TN(m)とコリネバクテリウム菌株用ベクターCJ4−GFP及びSPL13−GFPを制限酵素EcoRV及びPstIで処理し、その後ライゲーションを行って最終的にコリネバクテリウム菌株用ベクターpCJ4−TN(m)及びpSPL13−TN(m)を作製した。
前記過程を経て作製した菌株を実施例1の培地及び培養条件と同様に培養及び前処理し、その後UxaEの活性用菌株を確保した。活性評価は実施例2−1と同様の方法で、基質量、反応温度及び時間のみ変更して行った(100g/Lフルクトース溶液を添加して60℃で2時間反応)。その後、遠心分離により上清を回収し、HPLC分析によりタガトース生成量を測定した(図2の(A)及び(B))。反応後のタガトース生成量を表3に示す。
(1)SPL7プロモーター配列を含むpECCG117−SPL7−ilvEベクター及び形質転換菌株の作製
L−アミノ酸の代表的な一例であるL−バリンの生産能を確認するために、バリン生合成主要遺伝子である分枝鎖アミノ酸アミノトランスフェラーゼ(branched-chain amino-acid aminotransferase)をコードするilvE(Ncgl2123)の酵素活性が強化されるように、次の通りpECCG117−CJ7−ilvE及びpECCG117−SPL7−ilvEベクターを作製した。具体的には、ATCC14067染色体を鋳型(template)とし、次の配列番号14及び配列番号15をプライマーとして用いてPCR(94℃で30秒間、55℃で30秒間、72℃で1分間の反応を30回)を行い、NCgl2123遺伝子5’末端にEcoRV、3’末端にPstI制限酵素部位を有する約1104bpのPCR断片を増幅した。前述したように得られたPCR断片を精製し、EcoRV及びPstI制限酵素で処理したpECCG117−CJ7−GFPとpECCG117−SPL7−GFPとそれぞれ混合し、インフュージョンクローニングキット(In-fusion cloning Kit)を用いて連結してベクターを作製し、それらをそれぞれpECCG117−CJ7−ilvE、pECCG117−SPL7−ilvEと命名した。
配列番号14 5’ GAGATCAAAACAGATATCATGACGTCATTAGAGTTC 3’
配列番号15 5’ ATCCCCCGGGCTGCAGTTAGCCAACCAGTGGGTA 3’
前述したように形質転換した3種の菌株のL−バリン生産能を次の方法で培養して分析した。
<生産培地(pH7.2)>
グルコース50g,(NH4)2SO4 20g,コーンスティープソリッド(Corn Steep Solids)20g,KH2PO4 1g,MgSO4・7H2O 0.5g,ビオチン200μg(蒸留水1リットル中)
(1)SPL13プロモーター配列を含むpDZTn−SPL13−gapN1ベクター及び形質転換菌株の作製
L−アミノ酸の代表的な一例であるL−リシンの生産能を確認するために、公知のストレプトコッカス・ミュータンス(Streptococcus mutants)に由来するNADP依存的グリセルアルデヒド−3−リン酸デヒドロゲナーゼ(GapN)の酵素活性が強化されるように、次の通りベクターを作製した。
配列番号16 5’ ATCCTCTAGAGTCGACCAAATGCTCCAACCGTCCGT 3’
配列番号17 5’ CTCGAGGAACTCATTCCTTCTGCTCG 3’
配列番号18 5’ TCTAGAACTAGTGGGCCCGACATCTAATAACCGGGCAG 3’
配列番号19 5’ ATGCCTGCAGGTCGACGCAGACGCACTCGACTACAC 3’
配列番号20 5’ GAATGAGTTCCTCGAGAGAAACATCCCAGCGCTACT 3’
配列番号21 5’ GCCCACTAGTTCTAGATTATTTGATATCAAATACGA 3’
配列番号22 5’ GAATGAGTTCCTCGAGGGCGCTTCATGTCAACAATC 3’
配列番号23 5’ ATTGTTTTGTCATATGTGTTTTGATCTCCTCCAATA 3’
配列番号24 5’ CATATGACAAAACAATATAAAAA 3’
前述したように形質転換した3種の菌株のL−リシン生産能を次の方法で培養して分析した。
<種培地(pH7.0)>
グルコース20g,ペプトン10g,酵母抽出物5g,尿素1.5g,KH2PO4 4g,K2HPO48g,MgSO4・7H2O 0.5g,ビオチン100μg,チアミンHCl 1000μg,パントテン酸カルシウム2000μg,ニコチンアミド2000μg(蒸留水1リットル中)
<生産培地(pH7.0)>
グルコース100g,(NH4)2SO4 40g,大豆タンパク質2.5g,コーンスティープソリッド(Corn Steep Solids)5g,尿素3g,KH2PO4 1g,MgSO4・7H2O 0.5g,ビオチン100μg,チアミン塩酸塩1000μg,パントテン酸カルシウム2000μg,ニコチンアミド3000μg,CaCO3 30g(蒸留水1リットル中)
Claims (7)
- 配列番号1〜3からなる群から選択されるいずれか1つのヌクレオチド配列からなる、プロモーター活性を有する核酸分子。
- 請求項1に記載の核酸分子及び目的遺伝子を含む、遺伝子発現カセット。
- 請求項1に記載の核酸分子又は請求項2に記載の遺伝子発現カセットを含む、組換えベクター。
- 請求項1に記載の核酸分子又は請求項3に記載のベクターを含む、コリネバクテリウム属組換え微生物。
- 前記コリネバクテリウム属微生物は、コリネバクテリウム・グルタミカム又はコリネバクテリウム・アンモニアゲネスである、請求項4に記載の組換え微生物。
- (a)請求項4に記載の組換え微生物を培地で培養するステップと、
(b)前記微生物又は前記微生物を培養した培地から目的物質を回収するステップとを含む、目的物質を生産する方法。 - 前記目的物質は、サイコース、タガトース又はアミノ酸である、請求項6に記載の方法。
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BR112019004161B1 (pt) | 2021-07-27 |
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HUE057965T2 (hu) | 2022-06-28 |
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CN110283823B (zh) | 2023-05-12 |
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MY186296A (en) | 2021-07-06 |
PL3508580T3 (pl) | 2022-04-25 |
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CN108026539B (zh) | 2019-06-07 |
KR101783170B1 (ko) | 2017-09-29 |
JP6679803B2 (ja) | 2020-04-15 |
EP3508580A1 (en) | 2019-07-10 |
WO2018043856A1 (ko) | 2018-03-08 |
BR112019004161A2 (pt) | 2019-09-03 |
CN108559747B (zh) | 2021-08-31 |
DK3508580T3 (da) | 2022-03-07 |
CN108026539A (zh) | 2018-05-11 |
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