JP7452900B2 - 乳酸耐性の向上を有する酵母およびその使用 - Google Patents
乳酸耐性の向上を有する酵母およびその使用 Download PDFInfo
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- JP7452900B2 JP7452900B2 JP2022508758A JP2022508758A JP7452900B2 JP 7452900 B2 JP7452900 B2 JP 7452900B2 JP 2022508758 A JP2022508758 A JP 2022508758A JP 2022508758 A JP2022508758 A JP 2022508758A JP 7452900 B2 JP7452900 B2 JP 7452900B2
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- lactic acid
- haa1
- myr2787
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- yeast
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N1/16—Yeasts; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Description
本明細書で使用される技術的用語または科学的用語は、別段の定めがない限り、当業者によって理解されるような定義を有する。本発明の理解を容易にするために、専門語の説明を以下に定める。
この方法を使用して、合成線状DNA断片、PCR断片、または制限酵素によって生成された断片から、プラスミドを構築することができる。
キット、たとえばNEBuilder HiFi DNA Assembly Cloning Kit(New England BioLabs、Ipswitch、米国、マサチューセッツ州)を購入し、製造者の指示通りに使用して、ギブソン法を実施することができる。
表1 遺伝子の名称および説明
同時の挿入なく標的染色体配列が欠失されることが意図される場合、上述の一般的な設計の第2の断片2)は省かれる。適正な組み込みを含有する形質転換体は、適正な期待されるサイズの上流および下流ジャンクション断片の両方を示す診断PCRによって識別される。第2のステップにおいて、選択マーカ遺伝子は、対抗選択、および、カセットの内側の「Down」配列と、組み込まれたカセットの下流に論理的に存在する染色体における「Down」と相同的である配列との間の相同組換えによって「ループアウト」される。
以下の化学ベースDNA形質転換法を、菌株MYR2297およびMYR2787のために改善されるように、Abdel-Banatら(Abdel-Banat、2010年)によって出版されているプロトコルから適合させた。菌株の多くは本明細書で説明される実施例において名づけられ、使用される。
表2 成長培地の組成。列挙されるすべての量は1リットル当たりである。ペトリ皿に関して、20g/Lの寒天を加えた。
出発株は、ウラシル栄養要求性酵母菌株、およびD-LAC生産が可能な野性型HAA1遺伝子、すなわちMYR2787 HAA1を有するK.マルシアナスであった。MYR2787 HAA1の構築を以下に説明する。EcldhA遺伝子を発現するように設計された3つの異なるカセットをプラスミド上に構築した。3つすべての場合において、KmPDC1プロモータからldhA遺伝子が発現された。3つのEcldhAカセットを設計し、相同組換えによって、KmPDC1、KmGPP1、およびKmNDE1遺伝子座である標的遺伝子座において挿入した。ScURA3遺伝子から選択することによって、ウラシルを除いたCM培地上で破壊株を選択した。次いで、5'-FOAを含有する培地上で下流フランクの直接反復間の相同組換えによってURA3遺伝子をループアウトし、後の形質転換のためにURA3遺伝子を再利用した。各形質転換ステップおよび各ループアウトステップで、単一コロニーを、必要に応じて1回または複数回、再画線し、背景細胞から適正な菌株を離し、ヘテロ接合二倍体を除去した。カセットの末端と、組み込まれたカセットのすぐ上流またはすぐ下流にある、標的遺伝子座での染色体配列との間の境界を挟む適切なプライマを使用するPCRによって、適正な挿入および適正なループアウトを識別した。PCR診断では、適正に組み込まれたホモ接合二倍体から、一倍体に組み込まれたカセットを適正に区別することができなかったため、この区別は構築のいかなるステップでもされなかった。菌株MYR2297で出発し、3つのldhAカセットを、上述で列挙した順序で1つずつ導入した。カセットの最初の組み込みが終わる毎に、5'-FOA対抗選択によってURA3遺伝子をループアウトした。このようにして第3のカセットが導入された後、テンプレートとしてのK.マルシアナス野性型株染色体DNAからPCRによって得られた線状DNA断片の形質転換によって、天然KmURA3遺伝子を再導入し、ウラシルを除いたCMプレート上で選択することによってウラシル原栄養株を得た。得られた菌株は、この時点で、組み込まれたldhA遺伝子を3コピー含有し、MYR2787 HAA1と名づけられた。
ldhA発現カセットに交換することによって、菌株MYR2287に組み込まれるようにHAA1過剰発現カセットを設計した。プラスミドは、以下の順序で、以下の特徴、1)強力な構成的プロモータ、2)HAA1コード領域、3)ターミネータ、4)選択マーカとしてのScURA3、5)ldhA遺伝子座の中間配列への相同性を有する。3つのldhA発現遺伝子座のうちのいずれか1つに組み込まれることが可能なようにカセットは設計されたが、GPP1遺伝子座でHAA1過剰発現カセットが交換された組み込みがさらなる考察のために選択された。得られたD-LAC生産株は、この時点で、GPP1遺伝子座において、ldhA発現カセットの代わりにHAA1発現カセットを含有し、MYR2787 2X HAA1と名づけられた。
低pH条件で上述のとおりに得られたすべての酵母菌株の成長能力を決定し、比較するために、MYR2297 HAA1、MYR2297 Δhaa1、MYR2787 HAA1、MYR2787 Δhaa1 、およびMYR2787 2X HAA1における菌株を、0.5%、1.0%、1.5%、および2.0%w/vのL-乳酸なしならびにありで、ウラシルを除いたCM培地において培養した。各培地における出発pHはそれぞれ、3.5、2.7、2.5、2.4、および2.3であった。すべての固形培地は、2%の寒天を含有した。同じ寒天プレート上で互いに横並びで107cellから出発し、30℃で2日間インキュベートして、段階10倍希釈をスポットすることによって、酵母菌株間での成長の差異の定量的比較を行った。
酵母の成長能力およびD-LAC生産を決定し、比較するために、MYR2787 HAA1、MYR2787 Δhaa1、およびMYR2787 2X HAA1のD-LAC生産株を、BioLectorミニ発酵槽でフラワープレートにおいて培養した。MYR2787 HAA1、MYR2787 Δhaa1、およびMYR2787 2X HAA1の酵母菌株の単一コロニーを、チューブに入った5mLのウラシルを除いたCMに個別に植菌し、速度率200rpmで12~18時間回転インキュベーションを用いることによって37℃でインキュベートした。その後、0.1mLの植菌材料を、0.1~0.2の出発OD600nmおよびpH6.0~6.2で、BioLector培養プレート中の0.9mLのSDM2培地(表2に示されるような)に植菌した。フラワープレートをそれぞれ、速度率1200rpmおよび37℃で48時間振盪させ、インキュベートした。実験の間、湿度を50~80%で制御して水分蒸発を除去した。発酵の間、pHは制御しなかった。したがって、D-LAC生産に起因して、各培養物のpHは発酵に沿って低下していった。培養が開始すると、30分毎にバイオマスをOD600nmで自動的に測定した。48時間後、実験を停止した。各菌株の細胞質量を遠心分離によって遠心沈殿させた。上清を回収し、乳酸含有量の量を分析した。
本発明の最良の形態は、詳細な説明において開示されているとおりである。
Claims (5)
- 遺伝子操作されたクルイベロミセス・マルシアナス酵母細胞であって、前記酵母細胞は、少なくとも、アミノ酸配列識別番号2にエンコードするヌクレオチド配列を不活性化または欠失させる遺伝子修飾を含む、遺伝子操作されたクルイベロミセス・マルシアナス酵母細胞。
- 前記アミノ酸配列識別番号2は、酸反応性転写因子Haa1である、請求項1に記載の遺伝子操作されたクルイベロミセス・マルシアナス酵母細胞。
- 前記遺伝子修飾は、親と比較して乳酸耐性を増大させる、請求項1または2に記載の遺伝子操作されたクルイベロミセス・マルシアナス酵母細胞。
- 前記遺伝子修飾は、親と比較して乳酸生産を増大させる、請求項1または2に記載の遺伝子操作されたクルイベロミセス・マルシアナス酵母細胞。
- 請求項1に記載の遺伝子操作されたクルイベロミセス・マルシアナス酵母細胞を使用する、乳酸生産のための方法であって、
請求項1に記載の遺伝子操作されたクルイベロミセス・マルシアナス酵母細胞を発酵培地で増殖させて乳酸を生成する段階、及び、
前記発酵培地から前記乳酸を任意に精製する段階を備える、方法。
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2009000124A (ja) | 2002-05-30 | 2009-01-08 | Natureworks Llc | 酵母におけるd−乳酸の生産方法およびその材料 |
JP2009517045A (ja) | 2005-11-23 | 2009-04-30 | ネイチャーワークス・エル・エル・シー | L−又はd−ラクタート:フェリチトクロームc酸化還元酵素遺伝子が機能しない乳酸産生酵母 |
JP2015528312A (ja) | 2012-09-14 | 2015-09-28 | ミリアント・コーポレイションMyriant Corporation | 低pH条件下での発酵による有機酸の生産 |
JP2019513408A (ja) | 2016-04-18 | 2019-05-30 | サイコニウム・ラクティック・アシッド・ゲーエムベーハー | 乳酸産生法 |
WO2019159011A2 (en) | 2018-02-16 | 2019-08-22 | Ptt Global Chemical Public Company Limited | Microorganisms and processes for lactic acid production |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9085781B2 (en) * | 2012-07-23 | 2015-07-21 | Edeniq, Inc. | Acetate resistance in yeast based on introduction of a mutant HAA1 allele |
CA2950221A1 (en) * | 2014-05-26 | 2015-12-03 | Vib Vzw | Causative genes conferring acetic acid tolerance in yeast |
WO2015194900A1 (ko) * | 2014-06-20 | 2015-12-23 | 한국생명공학연구원 | 젖산 분해 경로가 봉쇄된 클루이베로마이세스 막시아누스 및 이의 용도 |
KR101686899B1 (ko) * | 2014-06-20 | 2016-12-16 | 한국생명공학연구원 | 신규한 클루이베로마이세스 막시아누스 mj1 및 이의 용도 |
KR102219700B1 (ko) | 2014-06-23 | 2021-02-24 | 삼성전자주식회사 | Fps1의 활성이 감소된, 내산성을 갖는 효모 세포 및 그를 이용하여 락테이트를 생산하는 방법 |
US10612049B2 (en) | 2014-11-24 | 2020-04-07 | Vib Vzw | Causative genes conferring acetic acid tolerance in yeast |
KR102303832B1 (ko) | 2015-05-12 | 2021-09-17 | 삼성전자주식회사 | 내산성을 갖는 효모 세포, 상기 효모 세포를 제조하는 방법 및 이의 용도 |
KR102311681B1 (ko) | 2015-07-28 | 2021-10-12 | 삼성전자주식회사 | 내산성을 갖는 효모 세포, 그를 이용하여 유기산을 생산하는 방법 및 상기 내산성 효모 세포를 생산하는 방법 |
WO2018115251A1 (en) * | 2016-12-21 | 2018-06-28 | Vib Vzw | Xylose isomerases that confer efficient xylose fermentation capability to yeast |
-
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Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2009000124A (ja) | 2002-05-30 | 2009-01-08 | Natureworks Llc | 酵母におけるd−乳酸の生産方法およびその材料 |
JP2009517045A (ja) | 2005-11-23 | 2009-04-30 | ネイチャーワークス・エル・エル・シー | L−又はd−ラクタート:フェリチトクロームc酸化還元酵素遺伝子が機能しない乳酸産生酵母 |
JP2015528312A (ja) | 2012-09-14 | 2015-09-28 | ミリアント・コーポレイションMyriant Corporation | 低pH条件下での発酵による有機酸の生産 |
JP2019513408A (ja) | 2016-04-18 | 2019-05-30 | サイコニウム・ラクティック・アシッド・ゲーエムベーハー | 乳酸産生法 |
WO2019159011A2 (en) | 2018-02-16 | 2019-08-22 | Ptt Global Chemical Public Company Limited | Microorganisms and processes for lactic acid production |
Non-Patent Citations (4)
Title |
---|
ABBOTT, D. A. et al.,Physiological and Transcriptional Responses to High Concentrations of Lactic Acid in Anaerobic Chemostat Cultures of Saccharomyces cerevisiae,Applied and Environmental Microbiology,2008年,Vol. 74, Iss. 18,P. 5759-5768,https://doi.org/10.1128/AEM.01030-08 |
FERNANDES, A. R. et al.,Saccharomyces cerevisiae adaptation to weak acids involves the transcription factor Haa1p and Haa1p-regulated genes,Biochemical and Biophysical Research Communications,2005年,Vol. 337, Iss. 1,P. 95-103,https://doi.org/10.1016/j.bbrc.2005.09.010 |
LERTWATTANASAKUL, N. et al.,Genetic basis of the highly efficient yeast Kluyveromyces marxianus: complete genome sequence and transcriptome analyses,Biotechnology for Biofuels,2015年,Volume 8, Article number: 47,P. 1-14,DOI: 10.1186/s13068-015-0227-x |
PALMA, M. et al.,The Zygosaccharomyces bailii transcription factor Haa1 is required for acetic acid and copper stress responses suggesting subfunctionalization of the ancestral bifunctional protein Haa1/Cup2,BMC Genomics,2017年,Vol.18, Article No. 75,P. 1-22 |
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