JP5264778B2 - コリネバクテリアを利用してグリセロールを含む炭素源から発酵産物を生産する方法 - Google Patents
コリネバクテリアを利用してグリセロールを含む炭素源から発酵産物を生産する方法 Download PDFInfo
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- JP5264778B2 JP5264778B2 JP2009547170A JP2009547170A JP5264778B2 JP 5264778 B2 JP5264778 B2 JP 5264778B2 JP 2009547170 A JP2009547170 A JP 2009547170A JP 2009547170 A JP2009547170 A JP 2009547170A JP 5264778 B2 JP5264778 B2 JP 5264778B2
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- C12P13/00—Preparation of nitrogen-containing organic compounds
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- C12N9/10—Transferases (2.)
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Description
コリネバクテリウム ジフテリアのグリセロール利用関連遺伝子の塩基配列は、既に明白に明かされて公開されている。米国国立衛生研究所ジーンバンク(NIH GeneBank)からコリネバクテリウム ジフテリアのグリセロール利用関連タンパク質であるGlpF、GlpK、GlpDをコーディングする遺伝子(glpF, glpK及びglpD)及び周辺塩基配列に対する情報を入手した。コリネバクテリウム ジフテリアのGlpFジーンバンク許可番号は、NC_940539.1、GlpKのジーンバンク許可番号は、NC_940538.1、GlpDのジーンバンク許可番号は、NC_940540.1であった。それぞれの遺伝子は、ゲノム上で連続的に存在して、これを利用し、一回のPCR法を通じて、3種の遺伝子の全部を単一ポリヌクレオチドに増幅した。コリネバクテリウム ジフテリアのグリセロール利用関連遺伝子のPCR法による増幅には、配列番号1と2のプライマーが使用された。
配列番号1 : 5' GATGCGGCCGCGCTGTGTGGCGTATGTCG3'
配列番号2 : 5'GATGCGGCCGCAATCATCAAACCCAACCCCA 3'
得られたプラスミドを制限酵素のNotIで切って、グリセロール利用関連遺伝子が含まれているDNA切片を得た後、これを大腸菌−コリネバクテリウムシャトルベクターのpECCG117でクローニングして、大腸菌TOP10に形質転換した。製作された菌株を大腸菌CO02-0014と命名し、これをKCCMに受託番号KCCM 10834Pとして寄託した(KCCM (Korean Culture Center of Microorganisms) of KFCC (Korean Federation of Culture Collection), the International Depository Authority located at 361-221, Hongje-1-Dong, Seodaemungu-Gu,Seoul, Korea, on January 8, 2007)。
配列番号7: 5' GGAAATCTGGGCCAACACGCGCCAAGCC 3'’
配列番号8: 5' GGCTTGGCGCGTGTTGGCCCAGATTTCC 3'
pECCG117-cdi glpDFK-1とpECCG117-cdi glpDFK-2をそれぞれコリネバクテリウム グルタミカムATCC13032に電気パルス法を利用して導入した後、バクトペプトン10g/L、イースト抽出液10g/L、ビーフ抽出液5g/L、NaCl 2.5g/L、カナマイシン25μg/mLの含まれたプレートで培養した。得られたコロニーに対し、PCRクローニング法を通じて、グリセロール利用関連遺伝子が含まれるプラスミドを有しているコロニーを獲得した。これをそれぞれコリネバクテリウム グルタミカムATCC13032/pECCG117-cdi glpDFK-1、コリネバクテリウム グルタミカムATCC13032/pECCG117-cdi glpDFK-2と命名した。
コリネバクテリウム グルタミカム/pECCG117-cdi glpDFK-1及びコリネバクテリウム グルタミカム/pECCG117-cdi glpDFK-2のグリセロール利用性を確認するために、まず固体状の最小培地でそれぞれの前記プラスミドを含むコリネバクテリウム グルタミカムATCC13032と、含まないコリネバクテリウム グルタミカムATCC13032を培養した。そのための最小培地の組成は、下記のようである。
コリネバクテリウム グルタミカムの最小培地組成(pH 7.2):グリセロール10g/L、KH2PO4 1g/L、K2HPO4 2g/L、MgSO4・H2O 0.4g/L、尿素 2g/L、(NH4)2SO4 5g/L、NaCl 0.5g/L、ニコチン酸アミド5mg/L、パントテン酸カルシウム1mg/L、チアミン3mg/L、ビオチン200μg/L、微量元素1mL、寒天(Agar)20g/L
グリセロールを利用し、コリネバクテリアの成長だけではなく、実際有用物質の生産も可能であるかを調べるために、発現ベクターのpECCG117-cdi glpDFK-1をグルタミン酸生産菌株であるコリネバクテリウム グルタミカムSM5(KFCC-11112)に、実施例3と同様な方法により導入した。コリネバクテリウム グルタミカム SM5及びコリネバクテリウム SM5/pECCG117-cdi glpDFK-1に対し、ブドウ糖のみを炭素源として使用する場合、グリセロールのみを炭素源として使用する場合、それぞれを必要とされる比率で混ぜて使用した場合について、グルタミン酸の生産性を比較した。上記のSM5及び発現ベクターを含むSM5を一白金耳量種培地に接種した後、30℃で18時間培養した。種培地の構成成分は、ペプトン1g/L、酵母エキス0.5g/L、肉汁0.5g/L、グルコース1g/L、塩化ナトリウム0.25g/L、尿素0.13g/Lであり、液相のpHは、7.2である。発酵を行うために、種培養液1mLを培養培地に接種して、30℃で24時間培養した。培養培地の成分は、HSM 3mL、ビオチン1μg/L、廃糖蜜0.05g/L、硫酸アンモニウム0.1g/L、硫酸鉄0.002g/L、硫酸マンガン0.002g/L、硫酸マグネシウム0.05g/L、チアミン塩酸500μg/L、リン酸第一カリウム0.2g/L、尿素0.95g/Lであり、炭素源を培養条件にしたがって添加した。培地のpHは、7.2であった。その結果、L-グルタミン酸の生成を確認することができ、これを比較して表1に示した。
グリセロールを利用したコリネバクテリアの成長だけではなく、実際有用物質の生産も可能であるかを調べるために、発現ベクターのpECCG117-cdi glpDFK-1をリシン生産菌株であるコリネバクテリウム グルタミカム CF 905(KFCC-10881)に、実施例3と同様な方法により導入した。
原糖20g、ペプトン10g、酵母抽出物5g、尿素1.5g、K2HPO4 8g、MgSO4・7H2O 0.5g、ビオチン100μg、チアミン塩酸塩 1000μg、カルシウム-パントテン酸2000μg、ニコチンアミド2000μg(工程水1リットル基準)。
生産培地(pH 7.0):
ブドウ糖100g、(NH4)2SO4 40g、大豆タンパク質(Soy protein) 2.5g、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、カルシウム-パントテン酸 2000μg、ニコチンアミド3000μg、CaCO3 30g(工程水1リットル基準)
Claims (11)
- 配列番号9の塩基配列を有する、グリセロールを利用可能にする遺伝子組み合わせのglpDFKで形質転換されたことを特徴とするコリネバクテリウム グルタミカム。
- 配列番号3の塩基配列を有する、グリセロールを利用可能にする遺伝子組み合わせのglpDFKで形質転換されたことを特徴とするコリネバクテリウム グルタミカム。
- 前記コリネバクテリウム グルタミカム (Corynebacterium glutamicum)は、コリネバクテリウム グルタミカムSM5(KFCC-11112)及びコリネバクテリウム グルタミカムCF905(KFCC-10881)からなる群から選択されることを特徴とする、請求項1または2に記載のコリネバクテリウム グルタミカム。
- 配列番号3または配列番号9の塩基配列を有する、グリセロールを利用可能にする遺伝子組み合わせのglpDFKで形質転換されたコリネバクテリウム グルタミカムを、炭素源としてグリセロールが一部あるいは全部含まれた培養培地に接種して培養する段階と、前記培養物から発酵産物を分離する段階とを含むことを特徴とする、グリセロールを利用して発酵産物を生産する方法。
- 前記glpDFKがコリネバクテリウム ジフテリア(Corynebacterium diphtheriae)NCTC13129由来の配列番号9の塩基配列を有することを特徴とする、請求項4に記載の方法。
- 前記glpDFKがコリネバクテリウム ジフテリア(Corynebacterium diphtheriae)NCTC13129由来の配列番号3の塩基配列を有することを特徴とする、請求項4に記載の方法。
- 前記形質転換が、前記glpDFK遺伝子を含むベクターにより行われることを特徴とする、請求項4に記載の方法。
- 前記形質転換が、前記glpDFK遺伝子を含むベクターで宿主細胞を形質転換した後、当該宿主細胞から得られるプラスミドを電気パルス法でコリネバクテリアに導入して行われることを特徴とする、請求項4に記載の方法。
- 前記glpDFK遺伝子を含むベクターで形質転換された宿主細胞が大腸菌CO02−0014(受託番号:KCCM 10834P)であることを特徴とする、請求項8に記載の方法。
- 前記コリネバクテリウム グルタミカム (Corynebacterium glutamicum)は、コリネバクテリウム グルタミカム SM5(KFCC-11112)及びコリネバクテリウム グルタミカム CF 905(KFCC-10881)からなる群から選択されることを特徴とする、請求項4に記載の方法。
- 前記発酵産物がグルタミン酸またはリシンであることを特徴とする、請求項4に記載の方法。
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