JP2021529515A - 有機酸を産生するための微生物細胞株の単離 - Google Patents
有機酸を産生するための微生物細胞株の単離 Download PDFInfo
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Abstract
Description
本願は、2018年6月18日提出の米国特許出願第62/686463号に対する優先権を主張するものであり、その内容全体を本明細書に援用する。
技術分野
本開示は一般に、有機酸を過剰産生する微生物細胞株及びその作製方法に関する。
1つの観点では、微生物細胞株を作製及び単離する方法を提供するものであり、単離された微生物細胞株は、親微生物細胞株と比較して有機酸を過剰産生する。該方法では、有機酸が添加されたpH制御培地における親株の、好ましくは非固定化培養での連続継代が用いられ、pHは、有機酸のpKa値を上回る値に制御される。いくつかの態様では、pHは、好ましくは約5.5〜約7.5の範囲、より好ましくは中性又はその付近である約6.0〜約7.0、最も好ましくは約7.0である。いくつかの態様では、培地は固化されている。いくつかの態様では、培地は、例えば倍加速度又は増殖速度が、例えば少なくとも5%、10%、20%、30%、40%、50%、又はそれより多く低下するように、正常な微生物細胞増殖を阻害するのに十分な量の有機酸が添加されている。いくつかの態様では、有機酸は、連続継代の逐次反復において漸増量で添加される。いくつかの態様では、有機酸は、連続継代の逐次反復において同一量で添加される。いくつかの態様では、有機酸は、プロピオン酸、乳酸、酢酸、又は酪酸である。いくつかの態様では、有機酸はプロピオン酸であり、例えば、培地には、約1.0%〜3.0%のプロピオン酸、例えば約3.0%のプロピオン酸が添加される。いくつかの態様では、親細胞株は野生型生物である。いくつかの態様では、親細胞株は、単細胞微生物に由来する微生物細胞株である。
pH = pKa + log10([A−]/[HA])
[式中、[HA]は、プロトン化した解離していない非荷電弱酸の濃度であり、[A−]は、脱プロトン化し、解離した負荷電共役塩基の濃度である]。典型的な発酵プロセスでは、有機酸が最大濃度に達したときの微生物培養物のpHは、緩衝剤の使用なしでの有機酸のpKaに近似する。pHが約4〜5の溶液は、有機酸産生細菌の公知のpH許容性と比較して酸性というわけではない。これらの細菌の大半はこの範囲のpH値において増殖するが、細胞増殖に最適なpHは典型的には約6〜7である。
菌株3−1の単離
10mLのM24+2.0%グルコース培地にて、プロピオン酸菌(ATCC 25562)を高細胞密度になるまで増殖させた。その後、この培養物の連続希釈物(100〜10−3)を、1.0%、2.0%、及び3.0%(W/V)PAを添加し、水酸化ナトリウムを用いて全てpH7.0に中和した、寒天で固化した固体M24+2.0%グルコース培地上にプレーティングした。PAを追加していない固体M24+2.0%グルコース培地上にも細胞をプレーティングした。
実施例2
菌株3−1及び野生型プロピオン酸菌によるPA産生
野生型プロピオン酸菌(ATCC 255562)及び菌株3−1を、2%グルコースを添加したM24培地において、嫌気性条件下30℃にて、凍結保存物から培養した。細胞を、10mLで開始し、次いで50mLの新鮮M24培地に48時間ごとに継代培養し、1Lバイオリアクター容器用の種として用いた。
実施例3
菌株3−1のゲノム解析
菌株3−1のゲノム再配列決定を用いて、高濃度の有機酸に対する遺伝的耐性をもたらす変異変化を介して重要遺伝子を同定した。
参考文献
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2. Zhu Y., J. Li, M. Tan, L. Liu, L. Jiang, J. Sun, P. Lee, G. Du, J. Chen. 2010. Optimization and scale-up of propionic acid production by propionic acid-tolerant Propionibacterium acidipropionici with glycerol as the carbon source. Bioresource Technology 101:8902-8906.
3. Wang Z., S.-T. Yang. 2013. Propionic acid production in glycerol/glucose co-fermentation by Propionibacterium freudenreichii subsp. shermanii. Bioresource Technology 137:116-123.
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6. Coral J. 2008. Propionic acid production by Propionibacteriumsp. using low-cost carbon sources in submerged fermentation. Dissertation. Federal University of Parana.
7. Zhang A., J. Sun, Z. Wang, S.-T. Yang, H. Zhou. 2015. Effects of carbon dioxide on cell growth and propionic acid production from glycerol and glucose by Propionibacterium acidipropionici. Bioresource Technology 175:374-381.
8. Wang Z., M. Lin, L. Wang, E. M. Ammar, S.-T. Yang. 2015. Metabolic engineering of Propionibacterium freudenreichii subsp. shermanii for enhanced propionic acid fermentation: Effects of overexpressing three biotin-dependent carboxylases. Process Biochemistry 50:194-204.
9. Suwannakham S., Y. Huang, S.-T. Yang. 2006. Construction and characterization of ack knock-out mutants of Propionibacterium acidipropionici for enhanced propionic acid fermentation. Biotechnology and Bioengineering 94:383-95.
10. Suwannakham S., S.-T. Yang. 2005. Enhanced propionic acid fermentation by Propionibacterium acidipropionici mutant obtained by adaptation in a fibrous‐bed bioreactor. Biotechnology and Bioengineering 91:325-337.
11. Suwannakham S. 2005. Metabolic engineering for enhanced propionic acid fermentation by Propionibacterium acidipropionici. Dissertation. Ohio State University.
12. Tufvesson P., A. Ekman, R. R. R. Sardari, K. Engdahl, L. Tufvesson. 2013. Economic and environmental assessment of propionic acid production by fermentation using different renewable raw materials. Bioresource Technology 149:556-564.
13. Thierry A., S.-M. Deutsch, H. Falentin, M. Dalmasso, F. J. Cousin, G. Jan. 2011. New insights into physiology and metabolism of Propionibacterium freudenreichii. International Journal of Food Microbiology 149:19-27.
14. Scholz C.F.P., M. Kilian. 2016. The natural history of cutaneous propionibacteria, and reclassification of selected species within the genus Propionibacteriumto the proposed novel genera Acidipropionibacteriumgen. nov., Cutibacterium gen. nov. and Pseudopropionibacterium gen. nov. International Journal of Systematic and Evolutionary Microbiology 66:4422-4432.
15. Rehberger J.L., B.A. Glatz. 1998. Response of cultures of Propionibacterium to acid and low pH: tolerance and inhibition. Journal of Food Production 61:211-216.
16. Ahmadi N., K. Khosravi-Darani, A.M. Mortazavian.2017. An overview of biotechnological production of propionic acid: From upstream to downstream processes. Electronic Journal of Biotechnology 28:67-75.
17. Guan N., L. Liu, X. Zhug, Q. Xu, J. Li, G. Du, J. Chen. 2012. Genome-shuffling improves acid tolerance of Propionibacterium acidipropionici and propionic acid production. Advances in Chemistry Research 15:143-152.
18. Guan N., H. Shin, R. R. Chen, J. Li, L. Liu, G. Du, J. Chen. 2014. Understanding of how Propionibacterium acidipropionici respond to propionic acid stress at the level of proteomics. Scientific Reports 4:6951.
19. Guan N., H.D. Shin, G. Du, J. Chen, L. Liu. 2016. Metabolic engineering of acid resistance elements to improve acid resistance and propionic acid production of Propionibacterium jensenii. Biotechnology and Bioengineering 113:1294-304.
20. Jiang L., H. Cui, L. Zhu, Y. Hu, X. Xu, S. Li, H. Huang. 2015. Enhanced propionic acid production from whey lactose with immobilized Propionibacterium acidipropionici and the role of trehalose synthesis in acid tolerance. Green Chemistry 15:250-259.
21. EP 0141642
22. WO 85/04901
23. US 2011/0151529 A1
24. WO 2012/064883 A2
25. WO 2017/055932 A2
Claims (21)
- 有機酸を産生する微生物細胞株を作製及び単離する方法であって、正常な微生物細胞増殖を阻害するのに十分な量の有機酸が添加された培地における親細胞株の非固定化培養での連続継代を含み、培地のpHは、有機酸のpKa値を上回る値に制御され、単離された微生物細胞株は、親細胞株と比較して有機酸を過剰産生する、前記方法。
- 培地が固化されている、請求項1に記載の方法。
- 有機酸が、連続継代の逐次反復において漸増量で添加される、請求項1に記載の方法。
- 有機酸が、連続継代の逐次反復において同一量で添加される、請求項1に記載の方法。
- 培地のpHが約5.5〜7.5の範囲内の値に制御される、請求項1に記載の方法。
- 培地のpHが約6.0〜7.0の範囲内の値に制御される、請求項5に記載の方法。
- 培地のpHが約7.0に制御される、請求項6に記載の方法。
- 親細胞株が野生型である、請求項1に記載の方法。
- 微生物細胞株が単細胞微生物に由来する、請求項1〜8のいずれか1項に記載の方法。
- 微生物細胞株が、プロピオニバクテリウム属、ラクトバチルス属、アセトバクター属、グルコノバクター属、又はクロストリジウム属に由来する、請求項9に記載の方法。
- 微生物細胞株がプロピオン酸菌に由来する、請求項9に記載の方法。
- 有機酸が、プロピオン酸、乳酸、酢酸、又は酪酸である、請求項1〜8のいずれか1項に記載の方法。
- 培地に約1.0%〜3.0%のプロピオン酸が添加されている、請求項12に記載の方法。
- 培地に約3.0%のプロピオン酸が添加されている、請求項13に記載の方法。
- 請求項1〜14のいずれか1項に記載の方法に従って産生される、微生物細胞株。
- 表3に同定される少なくとも2つの変異を有する微生物細胞株であって、好ましくはプロピオニバクテリウム属、ラクトバチルス属、アセトバクター属、グルコノバクター属、又はクロストリジウム属に由来する、前記微生物細胞株。
- 表3に同定される変異の全てを有する、請求項16に記載の微生物細胞株。
- 表3に同定されるタンパク質又はそれらの相同体をコードする少なくとも2つの遺伝子において機能喪失型変異を有する、微生物細胞株。
- 表3に同定されるタンパク質又はそれらの相同体をコードする全ての遺伝子において機能喪失型変異を有する、請求項18に記載の微生物細胞株。
- (1)O抗原リガーゼドメイン含有タンパク質、(2)M18ファミリーのアミノペプチダーゼ、(3)アミノ酸パーミアーゼ、及び(4)アデニングリコシラーゼから成る群より選択されるタンパク質をコードする遺伝子において少なくとも1つの機能喪失型変異を有する、微生物細胞株。
- 少なくとも1つの機能喪失型変異が、O抗原リガーゼドメイン含有タンパク質をコードする遺伝子にある、請求項20に記載の微生物細胞株。
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