Methods for the detection and analysis of dioxygenase catalyzed dihydroxylation in mutant derived libraries

Wissner, Julian L.; Escobedo‐Hinojosa, Wendy; Heinemann, Peter M.; Hunold, Andreas; Hauer, Bernhard

doi:10.1016/bs.mie.2020.04.022

Cited by 7 publications

(3 citation statements)

References 42 publications

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“…cis -Dihydodiendiols and their corresponding catechols can be analyzed via HPLC-DAD due to their conjugated π-system at 262 nm. Extracted samples were analyzed by HPLC-DAD [ 3 ]. An Agilent 1260 Infinity II system (Santa Clara, US), equipped with a C18-column (Agilent Eclipse XDB-C18, 5 µm, 4.6 × 150 mm, Santa Clara, US) and a diode array detector (Agilent 1260 Infinity II DAD HS, Santa Clara, US) was operated isothermally at 30 °C.…”

Section: Methodsmentioning

confidence: 99%

Strategy for identification of cis-dihydrodiendiol-degrading dehydrogenases in E. coli BW25113

et al. 2020

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cis -Dihydrodiendiols are valuable compounds, finding multiple application as chiral synthons in organic chemistry. The biotechnological route for the generation of cis -dihydrodiendiols involves the dihydroxylation of aromatic compounds, catalyzed by Rieske non-heme iron dioxygenases. To date, numerous examples of recombinant E. coli , harboring such dioxygenases, can be found in the literature. Nevertheless, there is only a minor number of publications, addressing the E. coli catalyzed degradation of cis -dihydrodiendiols into catechols via dehydrogenases. Identification and elimination of such dehydrogenase catalyzed degradation is key for the establishment of enhanced recombinant E. coli platforms pursuing the production of cis -dihydrodiendiols. Here, we provide a fast and easy strategy for the identification of promiscuous alcohol dehydrogenases in E. coli BW25113, catalyzing the degradation of cis -dihydrodiendiols into catechols. This approach is based on the screening of dehydrogenase deficient KEIO strains, regarding their incapability of degrading a cis -dihydrodiendiol of choice. Novel screening strategy for E. coli BW25113 dehydrogenase knock-outs, incapable of degrading cis -dihydrodiendiols was validated for cis -1,2-dihydrocatechol as substrate Corresponding knock-outs can be used for recombinant production of cis -dihydrodiendiols Simple analysis based on liquid chromatography with diode array detector (HPLC-DAD)

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Section: Methodsmentioning

confidence: 99%

Strategy for identification of cis-dihydrodiendiol-degrading dehydrogenases in E. coli BW25113

et al. 2020

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“…Also, undesired side reactions, such as product degradation by dehydrogenases, occur in whole cell systems. This was countered by the knockout of the responsible dehydrogenases and optimization of E. coli strains. ,− In a recent study, the glycerol dehydrogenase GldA in E. coli BW25113 catalyzing the degradation of cis -1,2-dihydrocatechol ( 44 ) was identified. By using the knockout strain E. coli BW25113 GldA from the KEIO collection, the degradation of cis -1,2-dihydrocatechol ( 44 ) produced from the TDO-catalyzed dihydroxylation from benzene ( 43 ) was dramatically reduced.…”

Section: Vestige Of Bernhard′s Phdselective Oxidation Of Olefins Aro...mentioning

confidence: 99%

A Career in Catalysis: Bernhard Hauer

Nebel¹,

Breuer

Schneider

et al. 2023

ACS Catal.

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On the occasion of Professor Bernhard Hauer′s (partial) retirement, we reflect on and highlight his distinguished career in biocatalysis. Bernhard, a biologist by training, has greatly influenced biocatalysis with his vision and ideas throughout his four-decade career. The development of his career went hand in hand with the evolution of biocatalysis and the application and development of enzymes for chemical processes. In this Account, we present selected examples of his early work on the development of enzymes and their application in an industrial setting, with a focus on his specific contributions to harnessing the catalytic power of enzymes for novel reactions and the understanding and engineering of flexible loops and channels on catalysis.

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“…In research laboratories, biocatalytic reactions are often operated on analytical scale generating low amounts of product, only detectable by HPLC, GC or indirect assays [1]. Thereby, products are identified by comparison of retention times and fragmentation patterns with commercially available standards, self-synthesized standards or by comparison with databases.…”

Section: Introductionmentioning

confidence: 99%

Enhanced Semi‐Preparative Biotransformation of Cumene Dioxygenase: From Analytical Scale to Product Isolation

Schelle

Lepoittevin

Hauer

2023

Chemie Ingenieur Technik

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Scale‐up of oxygenase catalyzed reactions is often challenging due to the limited oxygen mass transfer in aqueous solutions. To overcome such limitation, we studied different scale‐up conditions using recombinant resting cells of E. coli JM109(DE3), harboring the cumene dioxygenase of Pseudomonas fluorescens IP01, for the dihydroxylation of naphthalene to (1R,2S)‐cis‐1,2‐dihydro‐1,2‐naphthalenediol. Thereby, vigorous stirring of the biotransformation in a 2 L round bottom flask in combination with an oxygen‐enriched headspace exhibited outstanding product formation after 1 h. Furthermore, the enhanced setup was used for the cumene dioxygenase catalyzed biosynthesis of 240 mg of valuable (+)‐trans‐carveol from (R)‐(+)‐limonene, demonstrating the application of our workflow for volatile compounds.

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Methods for the detection and analysis of dioxygenase catalyzed dihydroxylation in mutant derived libraries

Cited by 7 publications

References 42 publications

Strategy for identification of cis-dihydrodiendiol-degrading dehydrogenases in E. coli BW25113

Strategy for identification of cis-dihydrodiendiol-degrading dehydrogenases in E. coli BW25113

A Career in Catalysis: Bernhard Hauer

Enhanced Semi‐Preparative Biotransformation of Cumene Dioxygenase: From Analytical Scale to Product Isolation

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