Discovery of carboxylic acid reductase (CAR) from Thermothelomyces thermophilus and its evaluation for vanillin synthesis

A novel type III fungal CAR was identified from the organismThermothelomyces thermophila. High expressionlevels were observed inE. coliusing the pETDuet-1 plasmid system in combination with an autoinductionprotocol. A broad substrate scope ranging from aromatic to aliphatic carboxylic acids was tested andTtCARshowed activity for all substrates. High specific activities for aromatic substrates and short chain aliphaticsubstrates were observed, comparable to those ofNcCAR, the first type III fungal CAR.TtCAR’s pH and tem-perature optima were at 6.5 and 30 °C, respectively. Up to 20% (v/v) cosolvents did not show a decrease inspecific activity ofTtCAR using (E)-cinnamic acid as a substrate. Its half-life at 40 °C was determined to be 8.25 hand its melting temperature (Tm) was 56 °C.Invitroreactions withTtCAR reduced 95.2% of 10 mM vanillic acid,which correlated to a titer of 1.4 g L−1of vanillin. The space time yield of 0.029 g L-1h-1indicates that furtherimprovements would be necessary for an industrially relevant application. This would be especially importantwhen competing againstde novosynthesis of bio vanillin by microbial strains producing > 30 g L-1. Inde novoandin vivobiosynthesis systems, by-products are fairly common. By contrast, we were pleased to observe lessthan 0.7% of vanillyl alcohol formed, making the cell-free acid reduction in the envisaged sequential two-stepbioconversion from eugenol to vanillin very attractive