Simultaneous KRED and NHase/amidase activity

Developing a Biocascade Process: Concurrent Ketone Reduction-Nitrile Hydrolysis of 2-Oxocycloalkanecarbonitriles

Elisa Liardo, Nicolás Ríos-Lombardía, Francisco Morís, Javier González-Sabín, and Francisca Rebolledo

Org. Lett., 2016, 18 (14), pp 3366–3369

DOI: 10.1021/acs.orglett.6b01510

 

 A stereoselective bioreduction of 2-oxocycloalkanecarbonitriles was concurrently coupled to a whole cell-catalyzed nitrile hydrolysis in one-pot. The first step, mediated by ketoreductases, involved a dynamic reductive kinetic resolution, which led to 2-hydroxycycloalkanenitriles in very high enantio- and diastereomeric ratios. Then, the simultaneous exposure to nitrile hydratase and amidase from whole cells of Rhodococcus rhodochrous provided the corresponding 2-hydroxycycloalkanecarboxylic acids with excellent overall yield and optical purity for the all-enzymatic cascade.

Sequence alignment of Nit6803 from Syechocystis sp. PCC6803 with other nitrilases

Secondary structure elements are drawn on the basis of structures of Nit6803 and shown at the top of the aligned sequences. b-Sheets are shown as arrows in yellow, whereas a-helices are shown as bars in red. Residues involved in enzymatic catalysis are indicated are highlighted in red rectangle, whereas the proposed key residue involved in substrate preference is highlighted in blue rectangle. Nit6803, PaNit, PH0642, DNCAase and RrNit indicate Syechocystis sp. PCC6803 nitrilase (GI: 16331918), hyperthermophilic nitrilase (GI: 14521598), Pyrococcus horikoshii hypothetical protein (GI: 14590532), N-carbamoyl-D-amino-acid amidohydrolase (GI: 34921541) and Rhodococcus rhodochrous ATCC 33278 nitrilase (GI: 417384).

 from this paper by Yuan, Wei and co-workers.

NHase expression in Corynebacterium glutamicum

Industrial scale biocatalytic production of acrylamide relies on the use of engineered strains of Rhodococcus. There is a newly accepted manuscript in Applied Microbiology and Biotechnology from three Korean groups lead by J-H Lee and H-S Kim where the NHase from a Rhodococcus strain is expressed in Corynebacterium glutamicum (a cell factory already used commercially in amino acid biosynthesis) and tested for its ability to hydrate acrylonitrile. Whilst it didnt have the same activity as the NHase in the homologous system, the advantage the authors propose is that the rate of growth and hence enzyme production is higher with C. glutamicum.