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

Abstract

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.

Engineering carboxylic acid reductase for selective synthesis of medium-chain fatty alcohols in yeast

“…Protein engineering has been applied as a powerful tool for fine-tuning enzyme activities (38), often through the modification of amino acid sequences that are found in nature to expand the borders of biocatalysis. Conferred by protein engineering strategies, such as incorporation of mutations and domain modifications, it was shown that the property of CARs can be improved toward desired functions (39,40).…”

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

“…Recently, we described a high‐throughput assay, which takes advantage of the reactive nature of the CAR‐produced aldehyde to form UV‐detectable and fluorescent dihydroquinazolines with amino benzamidoxime (ABAO) [21] . Its first application was subject to a mutagenesis study of the CAR from Nocardia iowensis ( Ni CAR), expressed in E. coli K‐12 MG1655 RARE (DE3), [22] to enhance activity for the poor substrate 2‐methoxybenzoic acid [23] …”

“…They were grown in YPD media at 28°C and 320 rpm (DWP cultivation) or 150 rpm (flask cultivation) for 60 h. For each construct, approximately 46 clones were picked and screened, except for construct #9 (184 clones, Table 2). Each DWP containing P. pastoris clones also contained 24 single clones of the empty vector control (EVC) for on-plate calibration as described by Schwendenwein et al [23] Main cultures were cultivated and induced with either BMGY (1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 × 10 À 5 % biotin, 10 mM MgSO 4 and 1% glycerol) or BMMY (1% yeast extract, 2% peptone, 100 mM potassium phosphate, pH 6.0, 1.34% YNB, 4 × 10 À 5 % biotin, 10 mM MgSO 4 and 1% methanol) at 28°C for 48 h. 1% Methanol or glycerol was added every 12 h. OD 600 measurements in microtiter plates were performed in a Synergy Mx Plate reader (BioTek, Winooski, USA). P. pastoris cells were harvested by centrifugation at 4,000 rpm (3,220 x g) for 10 min in an Eppendorf tabletop centrifuge 5810R for expression analysis, biotransformations or ABAO-screening.…”

“…[21] Its first application was subject to a mutagenesis study of the CAR from Nocardia iowensis (NiCAR), expressed in E. coli K-12 MG1655 RARE (DE3), [22] to enhance activity for the poor substrate 2-methoxybenzoic acid. [23] CAR proteins are rather large (approx. 130 kDa) and structurally flexible enzymes.…”

Production of Aldehydes by Biocatalysis

“…Measurement of the concentration of various aldehydes can be accomplished by the addition of the 2-amino benzamidoxime derivates (ABAO) assay ( Figure 15 ). The resulting dihydroquinazoline product can be used for quantification at 380 [ 95 ] or 405 nm [ 96 ] in µM range and semi-quantification at nM range using fluorescence. This method can be used for high-throughput screening and is chemo-selective for aldehydes [ 95 , 96 ].…”