JP6104361B2 - 生物変換により長鎖脂肪酸から中鎖ω−ヒドロキシ脂肪酸、α,ω−ジカルボン酸、ω−アミノ脂肪酸を生産する方法 - Google Patents
生物変換により長鎖脂肪酸から中鎖ω−ヒドロキシ脂肪酸、α,ω−ジカルボン酸、ω−アミノ脂肪酸を生産する方法 Download PDFInfo
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Description
1)遺伝子クローニング
オレイン酸加水酵素とアルコール脱水素酵素遺伝子とを含む組換え発現ベクターを作製するために、ステノトロフォモナス・マルトフィリア由来のオレイン酸加水酵素遺伝子とミクロコッカス・ルテウス由来のアルコール脱水素酵素遺伝子のクローニングを行った。
正方向プライマー:5’−gctagcatgtattacagtaatggtaactatgaa−3’(配列番号1)
逆方向プライマー:5’−ggctcgagctatattagtttactttctttca−3’(配列番号2)
正方向プライマー:5’−atcgaattcgtccgagttcacccgtttcga−3’(配列番号3)
逆方向プライマー:5’−atatcaagcttcagccgagcggggtgtcct−3’(配列番号4)
大腸菌E.coli BL21(DE3)は、プラスミド保持のために10g/lグルコース及び適切な抗生剤が含まれるリーゼンバーグ(Riesenberg)培地で培養した。ここで、リーゼンバーグ培地は、4g/lのN(NH4)2HPO4、13.5g/lのKH2PO4、1.7g/lのcitric acid、1.4g/lのMgSO4、及び10ml/lのtrace metal solution(10g/lのFeSO4、2.25g/lのZnSO4、1.0g/lのCuSO4、0.5g/lのMnSO4、0.23g/lのNa2B4O7、2.0g/lのCaCl2、及び0.1g/lの(NH4)6Mo7O24)が含まれており、ミクロコッカス・ルテウスはLB培地で培養した。
まず、実施例1−1)のステノトロフォモナス・マルトフィリア由来のオレイン酸加水酵素遺伝子とミクロコッカス・ルテウス由来のアルコール脱水素酵素遺伝子が挿入されたpACYC/オレイン酸加水酵素/アルコール脱水素酵素発現ベクターを実施例1−2)で培養した大腸菌BL21(DE3)菌株に導入することにより1次形質転換体を作製した。
実施例1で作製した形質転換体を用いて、基質であるリシノール酸からω−ヒドロキシウンデク−9−エン酸を生産した。
実施例1で作製した形質転換体を用いて、基質であるレスクエロール酸からω−ヒドロキシトリデク−11−エン酸を生産した。
実施例1で作製したpACYC/オレイン酸加水酵素/アルコール脱水素酵素発現ベクターとシュードモナス・フルオレッセンス由来のBVMO遺伝子発現ベクター(非特許文献3)を大腸菌BL21(DE3)菌株に導入して形質転換体を作製し、それを用いて基質であるオレイン酸からα,ω−デカンニ酸(セバシン酸)を生産した(図5)。図5aは前記形質転換体を用いて生産された、鎖内にエステル基が導入された脂肪酸の時間の経過に伴う生産量の変化を示すグラフであり、(●)はオレイン酸の濃度を示し、(△)は10−ヒドロキシステアリン酸の濃度を示し、(▽)は10−ケトステアリン酸の濃度を示し、(■)は鎖内にエステル基が導入された脂肪酸の濃度を示す。図5bは反応終了後にエステル加水分解酵素で処理して反応産物をGC/MSで分析した結果を示す図である。オレイン酸から生産された、鎖内にエステル基が導入された脂肪酸は、ほとんどセバシン酸とノルマルオクタノールに変換された。
1)遺伝子クローニング
エステル加水分解酵素とアルコール脱水素酵素遺伝子とを含む組換え発現ベクターを作製するために、シュードモナス・フルオレッセンス由来のエステル加水分解酵素遺伝子とシュードモナス・プチダ由来のアルコール脱水素酵素遺伝子をクローニングした。
正方向プライマー:5’−gcgccatatgatgagcacatttgttgcaaaa−3’(配列番号5)
逆方向プライマー:5’−gcgcctcgagtcagtggtgatggtgatgatgactccgccgccacttt−3’(配列番号6)
正方向プライマー:5’−gcgcggatccgatgtacgactatataatcgtt−3’(配列番号7)
逆方向プライマー:5’−gcgcgcggccgcttagtggtgatggtgatgatgcatgcagacagctat−3’(配列番号8)
まず、実施例5−1)のシュードモナス・フルオレッセンス由来のエステル加水分解酵素とシュードモナス・プチダ由来のアルコール脱水素酵素遺伝子が挿入されたpCOLAduet−1/エステル加水分解酵素/アルコール脱水素酵素発現ベクターを実施例1−2)で培養した大腸菌BL21(DE3)菌株に導入することにより形質転換体を作製した。
実施例1で作製したpACYC/オレイン酸加水酵素/アルコール脱水素酵素発現ベクター、シュードモナス・プチダ由来のBVMO遺伝子発現ベクター、及び実施例5−1)で作製したpCOLAduet−1/エステル加水分解酵素/アルコール脱水素酵素発現ベクターを大腸菌BL21(DE3)菌株に導入して形質転換体を作製した。
1)形質転換体の作製
まず、実施例5−1)のシュードモナス・フルオレッセンス由来のエステル加水分解酵素とシュードモナス・プチダ由来のアルコール脱水素酵素遺伝子が挿入されたpCOLAduet−1/エステル加水分解酵素/アルコール脱水素酵素発現ベクターを大腸菌に導入して1次形質転換体を作製した。
実施例1で作製した形質転換体と実施例6−1)で作製した2次形質転換体を用いて、基質であるオレイン酸からα−アミノエンニ酸を生産した。実施例1で作製した形質転換体を用いて、オレイン酸から鎖内にエステル基が導入された脂肪酸を生産し、実施例6−1)で作製した形質転換体を用いて、鎖内にエステル基が導入された脂肪酸からα−アミノエンニ酸を生産した(図8)。図8は生産されたα−アミノエンニ酸の時間の経過に伴う生産量の変化を示すグラフであり、(■)は鎖内にエステル基が導入された脂肪酸の濃度を示し、(▲)はω−ヒドロキシノナン酸の濃度を示し、(◇)は9−オキソノナン酸の濃度を示し、(◆)はα−アミノエンニ酸の濃度を示す。
Claims (14)
- 加水酵素をコードする遺伝子、アルコール脱水素酵素をコードする遺伝子、BVMOをコードする遺伝子、及びエステル加水分解酵素をコードする遺伝子が導入された形質転換体であって、長鎖脂肪酸から中鎖ω−ヒドロキシ脂肪酸(ω−hydroxy fatty acid )、又はα,ω−ジカルボン酸(α,ω−dicarboxylic acid)を生成する形質転換体。
- 前記長鎖脂肪酸としてオレイン酸(oleic acid(C18))を用いた場合、中鎖ω−ヒドロキシ脂肪酸としてω−ヒドロキシノナン酸(ω−hydroxynonanoic acid(C9))が生成される請求項1に記載の形質転換体。
- 前記長鎖脂肪酸としてオレイン酸を用いた場合、α,ω−ジカルボン酸としてα,ω−デカン二酸(α,ω−decanedioic acid(C10))が生成され、アルコールとしてオクタノール(C8)が生成される請求項1に記載の形質転換体。
- アルコール脱水素酵素をコードする遺伝子、BVMOをコードする遺伝子、及びエステル加水分解酵素をコードする遺伝子が導入された形質転換体であって、ヒドロキシ長鎖脂肪酸から中鎖ω−ヒドロキシ脂肪酸を生成する形質転換体。
- 前記ヒドロキシ長鎖脂肪酸としてリシノール酸(C18)を用いた場合、中鎖ω−ヒドロキシ脂肪酸としてω−ヒドロキシウンデク−9−エン酸(ω−hydroxyundec−9−enoic acid(C11))が生成される請求項4に記載の形質転換体。
- 前記ヒドロキシ長鎖脂肪酸としてレスクエロール酸(lesquerolic acid(C20))を用いた場合、中鎖ω−ヒドロキシ脂肪酸としてω−ヒドロキシトリデク−11−エン酸(ω−hydroxytridec−11−enoic acid(C13))が生成される請求項4に記載の形質転換体。
- アルコール脱水素酵素をコードする遺伝子がさらに導入され、前記長鎖脂肪酸としてオレイン酸(C18)を用いた場合、α,ω−ジカルボン酸としてα,ω−ノナンニ酸(α,ω−nonanedioic acid(C9))が生成される請求項1に記載の形質転換体。
- 加水酵素をコードする遺伝子、アルコール脱水素酵素をコードする遺伝子、BVMOをコードする遺伝子、エステル加水分解酵素をコードする遺伝子、及びアミノ基転移酵素をコードする遺伝子が導入され、長鎖脂肪酸からω−アミノ脂肪酸を生成する形質転換体。
- 前記長鎖脂肪酸としてオレイン酸(C18)を用いた場合、ω−アミノ脂肪酸として ω−アミノノンナン酸(ω−aminononanoic acid(C9))が生成される請求項8に記載の形質転換体。
- (a)請求項1〜9のいずれかの形質転換体と長鎖脂肪酸を反応させて反応物を得るステップと、
(b)前記反応物から分解産物を回収するステップとを含む、長鎖脂肪酸から分解産物を生産する方法。 - 前記長鎖脂肪酸は、オレイン酸、リシノール酸(ricinoleic acid)、12−ヒドロキシステアリン酸(12−hydroxystearic acid)、リノレン酸(linoleic acid)、パルミトレイン酸(palmitoleic acid)、レスクエロール酸又はそれらの組み合わせである請求項10に記載の方法。
- 前記分解産物は、中鎖ω−ヒドロキシ脂肪酸、α,ω−ジカルボン酸、ω−アミノ脂肪酸、アルコール又はそれらの組み合わせである請求項10に記載の方法。
- 前記分解産物は、炭素数5〜14の中鎖ω−ヒドロキシ脂肪酸、α,ω−ジカルボン酸、ω−アミノ脂肪酸、炭素数2〜14のノルマルアルコール又はそれらの組み合わせである請求項10に記載の方法。
- 前記分解産物は、 ω−ヒドロキシノナン酸、ω−ヒドロキシウンデク−9−エン酸、 ω−ヒドロキシトリデク−11−エン酸、α,ω−ノナンニ酸(azelaic acid)、α,ω−デカン二酸(sebacic acid)、 ω−アミノノンナン酸、ヘプタン酸(heptanoic acid)、ノナン酸(nonanoic acid)、ω−ヒドロキシウンデカン酸(ω−hydroxyundecanoic acid)、ω−ヒドロキシトリデカン酸(ω−hydroxytridecanoic acid)、α,ω−ウンデク−2−エンニ酸(α,ω−undec−2−enedioic acid(cis−2−undecene−1,11−dioic acid))、ノルマルオクタノール又はそれらの組み合わせである請求項12に記載の方法。
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PCT/KR2013/002885 WO2013151393A1 (ko) | 2012-04-06 | 2013-04-05 | 생물전환을 통한 장쇄 지방산으로부터 중쇄 오메가-하이드록시지방산, 알파,오메가-디카르복실산, 오메가-아미노지방산을 생산하는 방법 |
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