Selective Enzymatic Transformation to Aldehydes in vivo by Fungal Carboxylate Reductase from Neurospora crassa

Abstract The enzymatic reduction of carboxylic acids is in its infancy with only a handful of biocatalysts available to this end. We have increased the spectrum of carboxylate‐reducing enzymes (CARs) with the sequence of a fungal CAR from Neurospora crassa OR74A (NcCAR). NcCAR was efficiently expressed in E. coli using an autoinduction protocol at low temperature. It was purified and characterized in vitro, revealing a broad substrate acceptance, a pH optimum at pH 5.5–6.0, a T m of 45 °C and inhibition by the co‐product pyrophosphate which can be alleviated by the addition of pyrophosphatase. The synthetic utility of NcCAR was demonstrated in a whole‐cell biotransformation using the Escherichia coli K‐12 MG1655 RARE strain in order to suppress overreduction to undesired alcohol. The fragrance compound piperonal was prepared from piperonylic acid (30 mM) on gram scale in 92 % isolated yield in >98% purity. This corresponds to a productivity of 1.5 g/L/h.


Selective enzymatic transformation to aldehydes in vivo by fungal carboxylate reductase from Neurospora crassa
Daniel Schwendenwein, Giuseppe Fiume, Hansjörg Weber, Florian Rudroff, Margit Winkler*  Figure S1. Illustration of the expression vector pETDuet1-EcPPTaseHTNcCAR. The plasmid encodes one T7 promoter in front of both expression cassettes, the T7 terminator behind the second expression cassette, an ampicillin resistance gene, the lacI gene for the two lac operators in front of every gene, a his tag at the 5' end of the NcCAR and a pBR322-derived high copy origin of replication. The copy number is downregulated by the product of the rop gene and reaches a medium copy number. The two genes encoding EcPPTase and NcCAR were cloned in the two consecutive multiple cloning sites with NcoI and HindIII for EcPPTase and NdeI and XhoI for NcCAR.

Determination of kinetic parameters
Cinnamic acid concentration [mM]

Reaction monitoring by HPLC a. Determination of Cinnamic acid 1a reduction by HPLC/UV
The analysis of 1a, 1b and 1c was carried out with a Kinetex 2.6µ Biphenyl 100A HPLC column (Phenomenex) with a Phenylhexyl Security Guard ULTRA cartridge (Phenomenex). The mobile phases were ammonium acetate (5 mM) and 0.5% v/v acetic acid in water and ACN at a flow-rate of 0.26 mL min -1 . A stepwise gradient was used: 25-55% ACN (5 min), 55-70% ACN (5.0-7.2 min) 70-90% ACN (7.2-7.5 min). After 90 s, the column was re-equilibrated to starting conditions. The compounds were detected at 254 nm (DAD). For 1a, 1b and 1c, calibration with authentic standards was done at 254 nm and linear interpolation used for their quantification.

b. Effect of pyrophosphatase addition on cinnamic acid conversion
Inhibition by pyrophosphate was studied by comparison of the NcCAR mediated conversion of cinnamic acid 1a with or without the addition of commercial inorganic pyrophosphatase from baker's yeast (Sigma). NcCAR preparation after gel-filtration with a concentration of 0.35 mg mL -1 was used for these experiments (0.0175 mg mL -1 final concentration). The standard spectrophotometric assay was used and the time course of the reaction was monitored by analysis of duplicate samples that were taken after 30, 60, 90, 120 and 240 minutes. In parallel, the reactions were carried out in the presence of pyrophosphatase with a final concentration of 0.0175 mg mL -1 . The results are depicted in Figure  S4.

Reaction monitoring and product confirmation by NMR a. 1 H and 13 C NMR
When 1a reduction did not proceed any further as judged by 31 P NMR, an 1 H NMR was measured to assess the progress of cinnamic acid 1a reduction. Figure S5 shows both cinnamic acid 1a and the respective aldehyde 1b in approximately 2:1 ratio.
Piperonal was analyzed by 1 H and 13 C NMR, respectively ( Figure S6 and Figure S7).