Characterization of Type IV Carboxylate Reductases (CARs) for Whole Cell‐Mediated Preparation of 3‐Hydroxytyrosol

Abstract

Fragrance and flavor industries could not imagine business without aldehydes. Processes for their commercial production raise environmental and ecological concerns. The chemical reduction of organic acids to aldehydes is challenging. To fulfill the demand of a mild and selective reduction of carboxylic acids to aldehydes, carboxylic acid reductases (CARs) are gaining importance. We identified two new subtype IV fungal CARs from Dichomitus squalens CAR (DsCAR) and Trametes versicolor CAR (Tv2CAR) in addition to literature known Trametes versicolor CAR (TvCAR). Expression levels were improved by the co‐expression of GroEL‐GroES with either the trigger factor or the DnaJ‐DnaK‐GrpE system. Investigation of the substrate scope of the three enzymes revealed overlapping substrate‐specificities. Tv2CAR and DsCAR showed a preferred pH range of 7.0 to 8.0 in bicine buffer. TvCAR showed highest activity at pH 6.5 to 7.5 in MES buffer and slightly reduced activity at pH 6.0 or 8.0. TvCAR appeared to tolerate a wider pH range without significant loss of activity. Type IV fungal CARs optimal temperature was in the range of 25–35 °C. TvCAR showed a melting temperature (Tm) of 55 °C indicating higher stability compared to type III and the other type IV fungal CARs (Tm 51–52 °C). Finally, TvCAR was used as the key enzyme for the bioreduction of 3,4‐dihydroxyphenylacetic acid to the antioxidant 3‐hydroxytyrosol (3‐HT) and gave 58 mM of 3‐HT after 24 h, which correlates to a productivity of 0.37 g L−1 h−1.

In Vivo Reduction of Medium‐ to Long‐Chain Fatty Acids by Carboxylic Acid Reductase (CAR) Enzymes: Limitations and Solutions

“…Expression analysis of CAR enzymes, cell disruption and Ni‐affinity chromatography (IMAC) purification of enzymes : Methods were conducted as described in Horvat et al …”

“…Specific activity of CAR via NADPH‐depletion monitoring : The NADPH depletion assay was used to determine initial rate activities for the reduction of aliphatic carboxylates as described, however, carboxylic acids were dissolved in DMSO.…”

“…coli K-12 MG1655 RARE (DE3) cells were transfected with the plasmids listed in Table S4. Cultivation and expression was performed as described in Strohmeier et al [31] Expression analysis of CAR enzymes, cell disruption and Niaffinity chromatography (IMAC) purification of enzymes: Methods were conducted as described in Horvat et al [68] Heat purification of GkPPTase: Cell-free extract (CFE) was prepared by resuspending the cell pellet of E. coli K-12 MG1655 RARE pMS470:GkPPTase in 200 mM MES, pH 7.5. Cells were disrupted by ultrasonication and CFE prepared by centrifugation for 35 min at 65,563 rcf and 4°C.…”

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

“…130 kDa) and structurally flexible enzymes [24] . Especially fungal CARs tend to low expression levels in the bacterial expression host E. coli , [25] and their production could potentially be improved in a eukaryotic host.…”

Meet the Women of Catalysis

“…Margit is fascinated by enzymes and cellular systems as means to catalyze chemical transformations. One important goal is to reach beyond the limitations of a natural enzyme by applying protein engineering to develop it towards desired characteristics. A further strong research line is the discovery and detailed study of enzymes that are not (well) known yet, in order to understand them in depth and to determine whether and how they could become applicable for synthesis of desired compounds in satisfying amounts and qualities …”