Cyanide-free Nitrile Synthesis and “Metabolic Tuning” of Molecular Parts

Asano, Y., Toyama Prefectural University

We are exploiting new enzymes from microorganisms, plants and animals and utilizing them for the synthesis of various useful chemicals, and L-amino acid determination, etc. In this presentation, I will describe how we are learning from Nature to get the molecular parts and combine them to synthesize chemicals and improve the biocatalysts for applications.

1. Cyanide-free nitrile synthesis: In microorganisms, we have been characterizing the â??aldoxime-nitrile pathwayâ?, comparing with those found in plants and animals. We have shown that aldoxime dehydratase from Bacillus sp. strain OxB-1catalyzes not only the dehydration reaction of α-substituted β-arylaldoximes to from the corresponding chiral nitriles (1), but also â??Kemp eliminationâ? reaction as the first natural enzyme. In plants, we identified two cytochrome P450s CYP79D16 (L-Phe to phenylacetaldoxime) and CYP71AN24 (phenylacetaldoxime to mandelonitrile) in Prunus mume (2). We also identified a new P450 from a plant Fallopia sachalinensis, catalyzing the formation of phenylacetonitrile (PAN) from phenylacetaldoxime (the same reaction as aldoxime dehydratase) (3). Millipedes use the â??aldoxime-nitrile pathwayâ? to synthesize poisonous HCN precursor cyanohydrin in a quite elegant way (4). Nitriles as a new target of metabolic synthesis: An artificial hybrid biosynthetic pathway for phenylacetonitrile (PAN) production was constructed in E. coli. In the pathway, L-Phe was converted to PAN by a combination of CYP79A21 from Arabidopsis thaliana and Bacillusaldoxime dehydratase (5).

2. â??Metabolic tuningâ? of molecular parts: (i) Growth-dependent molecular selection of stable L-tryptophan dehydrogenase: A mutant enzyme TrpDH L59F/D168G/A234D/I296N with thermal stability was simply obtained by cultivating E. coli transformants harboring various mutant genes in an L-tryptophan auxotroph of E. coli (6). (ii) Soluble expression by directed evolution using a fusion reporter method: For the heterologous production of L-lysine ε-oxidase (LodA), a plasmid carrying LodA gene fused in-frame with a phleomycine resistant gene was randomly mutated and the plasmids were transformed into E. coli BL21 (DE3) harboring lodB, which acts in the posttranslational modification of LodA. A soluble LodA variant isolated by this method contained six silent mutations and one missense mutation (7). These enzymes are used for selective determination of L-amino acids.

1. R. Metzner et al, ChemCatChem, 6, 3105 (2014).
2. T. Yamaguchi et al, Plant Molec. Biol., 86, 215 (2014).
3. T. Yamaguchi et al, Plant Molec. Biol., published online (2016).
4. M. Dadashipour et al, Proc. Natl. Acad. Sci. USA, 112, 10605 (2015).
5. Y. Miki and Y. Asano, Appl. Environ. Microbiol., 80, 6828 (2014)
6. D. Matsui et al, J. Biotechnol., 196-197, 27-32 (2015).
7. D. Matsui and Y. Asano, Biosci. Biotechnol. Biochem., 79, 11473 (2015).