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.

 

Chiral intermediate for cilastatin by nitrilase hydratase/amidase combo

There is a new accepted manuscript in the Journal of Molecular Catalysis B from Yu-Guo Zheng and co-workers entitled "Industrial production of chiral intermediate of cilastatin by nitrile hydratase and amidase catalyzed one-pot, two-step biotransformation" which describes their research into using a chiral nitrile to carboxylic acid conversion which results from a cascade of NHase from (Rhodococcus boritolerans FW815) then amidase (from Delftia tsuruhatensis ZJB-05174). The compound they are aiming to hydrolyze is rac 2,2-dimethylcyclopropanecarbonitrile to the S-2,2-dimethylcyclopropanecarboxamide which is an intermediate in the synthesis of cilastatin (a booster component to some antibiotics which prolongs their activity by blocking the kidney’s dehydropeptidase activity). This is achieved using a NHase which is notably active but not chirally selective, and an amidase which is only avid for the R enantiomer of the amide. The R acid and S amide are separated using macroporous resin adsorption chromatography and the R acid is converted to the acid chloride. This can than have its chirality scrambled with a bit of heat such that its conversion to amide by addition of ammonia gives a fresh racemic batch of amide to challenge the R selective amidase with. The conditions described are to get it to work on the 100kg scale. Considering that this is a report on a process which has been undergoing continuous optimization since 2005, I guess the choice of amidase to do the chiral resolution was “baked in” before there was literature evidence of significantly stereoselective nitrile hydratases which might have prompted an enzyme discovery effort in that direction. Having said that racemization using the wrong acid chloride and heat rather than on the wrong isomer of the nitrile is a neat touch.

Nitrilase from sequence to 50 litre scale

We have been working on developing a specific nitrilase reaction in a consortium with Chemoxy and Biocatalysts Ltd with funding assistance from the UK Technology Strategy Board. In nine months, we have gone from the selection of amino acid sequences to expressed active proteins to assessment of substrate preference using a novel greener assay method which works with cell-free extracts to chemistry on the one litre scale, and finally at the end of last month to doing the reaction in the 50 litre plant at CPI's National Industrial Biotechnology Facility. Just to finish the boasting, the outcome of that reaction was over a kilo of product with great conversion.

We are now going into a TSB-funded collaborative research and development project with the same partners to really scale this process and bring it nearer to market by optimizing the enzyme, its use and reuse. It's going to be fun.