微氧条件下F420前体的中试规模生产

IF 3.2 3区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS
Annika Lenić, Bettina Bardl, Florian Kloss, Gundela Peschel, Ivan Schlembach, Gerald Lackner, Lars Regestein, Miriam A. Rosenbaum
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引用次数: 0

摘要

氧化还原辅助因子的功能研究对于许多潜在的生物催化过程是重要的,然而对这些分子的有限获取往往阻碍了有效的研究活动。脱氮黄素依赖酶介导原核生物的一系列生化氧化还原反应。辅酶F420依赖性酶对不对称氧化还原生物催化和其他具有挑战性的反应很感兴趣,但迄今为止,从天然生产者和工程宿主菌株中获得的低F420滴度限制了对这些酶的深入研究。FO是F420的天然前体,它具有许多氧化还原特性,并且在某些酶中被证实为F420的替代品。在这里,我们重点研究了从30 l中试规模的搅拌槽生物工艺到优化的下游纯化工艺的整体工艺流程的开发,以从工程大肠杆菌宿主菌株中获得纯FO。在异种fbiC表达过程中,我们能够通过限氧工艺条件将辅助因子合成从核黄素转移到FO,并在发酵液中达到5.05 mg L−1 FO的最终滴度,这首次允许为后续实验纯化相关量。生物反应器系统内的在线荧光测量使得监测FO形成和确认与生长相关的FO生物合成成为可能。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Pilot Scale Production of a F420 Precursor Under Microaerobic Conditions

Pilot Scale Production of a F420 Precursor Under Microaerobic Conditions

The functional investigation of redox cofactors is important for many potential biocatalytic processes, yet limited access to these molecules is often hampering efficient research activities. Deazaflavin-dependent enzymes mediate a range of biochemical redox reactions in prokaryotes. Coenzyme F420-dependent enzymes are interesting for asymmetric redox biocatalysis and other challenging reactions, but low F420 titers harvested from natural producers and engineered host strains so far limit intensive investigation of these enzymes. FO is a natural precursor of F420, which already shares many of the redox properties and was previously confirmed as a surrogate for F420 in certain enzymes. Here, we focused our research on the development of an overall process workflow from a 30-L pilot scale stirred tank bioprocess to an optimized downstream purification process to obtain pure FO from an engineered Escherichia coli host strain. We were able to shift the cofactor synthesis from riboflavin to FO via the implementation of oxygen-limited process conditions during heterologous fbiC expression and reached a final titer of 5.05 mg L−1 FO in our fermentation broth, which for the first time allows the purification of relevant amounts for subsequent experiments. Online fluorescence measurement within the bioreactor system made it possible to monitor FO formation and confirmed growth-associated FO biosynthesis.

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来源期刊
Biotechnology Journal
Biotechnology Journal Biochemistry, Genetics and Molecular Biology-Molecular Medicine
CiteScore
8.90
自引率
2.10%
发文量
123
审稿时长
1.5 months
期刊介绍: Biotechnology Journal (2019 Journal Citation Reports: 3.543) is fully comprehensive in its scope and publishes strictly peer-reviewed papers covering novel aspects and methods in all areas of biotechnology. Some issues are devoted to a special topic, providing the latest information on the most crucial areas of research and technological advances. In addition to these special issues, the journal welcomes unsolicited submissions for primary research articles, such as Research Articles, Rapid Communications and Biotech Methods. BTJ also welcomes proposals of Review Articles - please send in a brief outline of the article and the senior author''s CV to the editorial office. BTJ promotes a special emphasis on: Systems Biotechnology Synthetic Biology and Metabolic Engineering Nanobiotechnology and Biomaterials Tissue engineering, Regenerative Medicine and Stem cells Gene Editing, Gene therapy and Immunotherapy Omics technologies Industrial Biotechnology, Biopharmaceuticals and Biocatalysis Bioprocess engineering and Downstream processing Plant Biotechnology Biosafety, Biotech Ethics, Science Communication Methods and Advances.
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