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

A novel type III fungal CAR was identified from the organism Thermothelomyces thermophila. High expression levels were observed in E. coli using the pETDuet-1 plasmid system in combination with an autoinduction protocol. A broad substrate scope ranging from aromatic to aliphatic carboxylic acids was tested and TtCAR showed activity for all substrates. High specific activities for aromatic substrates and short chain aliphatic substrates were observed, comparable to those of NcCAR, the first type III fungal CAR. TtCAR's pH and temperature optima were at 6.5 and 30 degrees C, respectively. Up to 20% (v/v) cosolvents did not show a decrease in specific activity of TtCAR using (E)-cinnamic acid as a substrate. Its half-life at 40 degrees C was determined to be 8.25 h and its melting temperature (T-m.) was 56 degrees C. In vitro reactions with TtCAR reduced 95.2% of 10mM vanillic acid, which correlated to a titer of 1.4 g L-1 of vanillin. The space time yield of 0.029 g L-1 h(-1) indicates that further improvements would be necessary for an industrially relevant application. This would be especially important when competing against de novo synthesis of bio vanillin by microbial strains producing > 30 g L-1. In de novo and in vivo biosynthesis systems, by-products are fairly common. By contrast, we were pleased to observe less than 0.7% of vanillyl alcohol formed, making the cell-free acid reduction in the envisaged sequential two-step bioconversion from eugenol to vanillin very attractive.