Substrate‐Independent High‐Throughput Assay for the Quantification of Aldehydes

Ressmann, Anna K.; Schwendenwein, Daniel; Leonhartsberger, Simon; Mihovilovič, Marko D.; Bornscheuer, Uwe T.; Winkler, Margit; Rudroff, Florian

doi:10.1002/adsc.201900154

Cited by 34 publications

(33 citation statements)

References 36 publications

(38 reference statements)

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“…As described in our recent publication, aldehyde formation by CAR Ni was investigated using an engineered E. coli strain with Reduced Aldehyde Reduction capability. [14] Product yields determined by the ABAO‐assay were confirmed by GC and HPLC measurements and indicated the assay's utility as a quantitative analytical tool to be used in the presence of resting cells.…”

Section: Resultsmentioning

confidence: 99%

“…[14] Product yields determined by the ABAO‐assay were confirmed by GC and HPLC measurements and indicated the assay's utility as a quantitative analytical tool to be used in the presence of resting cells. Sample preparation steps such as centrifugation, dilution, pH adjustment and on‐plate calibration were adapted to fit the needs of a high‐throughput validation of CAR Ni variants …”

Section: Resultsmentioning

confidence: 99%

“…Phenylacetic acid ( 1 a ) was used as the substrate for assay control. All dihydroquinazoline products 3 a – j were synthesized on preparative scale and analyzed by NMR …”

Section: Methodsmentioning

confidence: 99%

“…Due to low sequence identities (<26%) to other subgroups, the majority of CAR engineering endeavors still rely on random mutagenesis. To enable efficient identification of new CAR variants, we developed an amino benzamidoxime (ABAO)‐based assay for screening mutant libraries in a whole cell system . CARs may thus be developed faster into a broadly applicable and widely used enzyme class.…”

Section: Introductionmentioning

confidence: 99%

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Random Mutagenesis‐Driven Improvement of Carboxylate Reductase Activity using an Amino Benzamidoxime‐Mediated High‐Throughput Assay

et al. 2019

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Carboxylic acid reductases (CARs) catalyze the direct adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH) dependent reduction of carboxylic acids to their corresponding aldehydes. The identification and improvement of CARs by protein engineering is, however, severely limited by the lack of fast and generic methods to quantify aldehydes. Within this study, we applied a convenient high-throughput assay (HTA) based on amino benzamidoxime (ABAO) that allows the substrateindependent and chemoselective quantification of aldehydes. Random mutagenesis of the well-known CAR from Nocardia iowensis (CAR Ni ) to improve its activity for sterically demanding 2-substituted benzoic acid derivatives was conducted in a K M -dependent fashion, and the HTA applied in the presence of microbial cells. The study identified a hot spot in the active site of CAR Ni that increased the affinity to 2-methoxybenzoic acid 9-fold upon mutation from glutamine to proline (Q283P). The catalytic performance of CAR NiQ283P appeared to be significantly improved also for other substrates such as 2-substituted (2-Cl, 2-Br) as well as 3-and 4substituted benzoic acids (3-OMe, 4-OMe), and even aliphatic octanoic acid.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…Phenylacetic acid ( 1 a ) was used as the substrate for assay control. All dihydroquinazoline products 3 a – j were synthesized on preparative scale and analyzed by NMR …”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Random Mutagenesis‐Driven Improvement of Carboxylate Reductase Activity using an Amino Benzamidoxime‐Mediated High‐Throughput Assay

et al. 2019

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“…Unsubstituted amino benzamidoxime (ABAO) was synthesized as described. [21] The BDP library was purchased from bisy GmbH (Hofstätten, Austria).…”

Section: Methodsmentioning

confidence: 99%

Amino Benzamidoxime (ABAO)‐Based Assay to Identify Efficient Aldehyde‐Producing Pichia pastoris Clones

et al. 2020

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The chemoselective synthesis of aldehydes is a challenging task. Nature provides carboxylic acid reductases (CARs) as elegant tools for the direct reduction of carboxylic acids to their respective aldehydes. The discovery of new CARs and strains that efficiently produce these enzymes necessitates a robust high-throughput assay with selectivity for aldehydes. We recently reported a simple assay that allows the substrate independent and chemoselective quantification of aldehydes (irrespective of their chemical structure). The assay utilized amino benzamidoxime (ABAO), which forms UV-active and fluorescent dihydroquinazolines. In this study, we adapted the ABAO-assay for the identification and comparison of Pichia pastoris clones with the ability to produce aldehydes from carboxylic acids. Specifically, CAR and PPTase from Mycobacterium marinum (MmCAR and MmPPTase) were co-expressed using different bidirectional promoters (BDPs). A library of 598 clones was screened for piperonal production with the ABAO assay and the results were validated by HPLC quantification. 1 OD unit of the best Pichia pastoris clone 2.A7, regulating MmCAR and MmPPTase expression by two strong constitutive promoters, fully converted 5 mM of piperonylic acid within 2 h.

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A High‐Throughput in Vitro Assay System for the Detection of the Enzymatic Dihydroxylation of Aliphatic Olefins

Rutkowski,

Talley,

Combs

et al. 2024

Adv Synth Catal

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Rieske dioxygenase enzymes can perform the cis‐dihydroxylation of aliphatic olefins, representing a potential green alternative to established methods of performing this important transformation. However, the activity of the natural enzymes in this context is low relative to their more well‐known activity in the cis‐dihydroxylation of aromatics. To enable the engineering of dioxygenase enzymes for improved activity in the dihydroxylation of aliphatic olefins, we have developed an assay system to detect the relevant diol metabolites produced from whole‐cell fermentation cultures. Optimization studies were carried out to maximize the sensitivity of the assay system, and its utility in the in vitro screening of enzyme variant libraries was demonstrated. The assay system was utilized in screening studies that identified Rieske dioxygenase variants with significantly improved activity in the dihydroxylation of aliphatic olefins relative to the wild‐type enzyme.

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Substrate‐Independent High‐Throughput Assay for the Quantification of Aldehydes

Cited by 34 publications

References 36 publications

Random Mutagenesis‐Driven Improvement of Carboxylate Reductase Activity using an Amino Benzamidoxime‐Mediated High‐Throughput Assay

Random Mutagenesis‐Driven Improvement of Carboxylate Reductase Activity using an Amino Benzamidoxime‐Mediated High‐Throughput Assay

Amino Benzamidoxime (ABAO)‐Based Assay to Identify Efficient Aldehyde‐Producing Pichia pastoris Clones

A High‐Throughput in Vitro Assay System for the Detection of the Enzymatic Dihydroxylation of Aliphatic Olefins

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