Enzyme-catalyzed reductions have been studied for decades and have been introduced in more than 10 industrial processes for production of various chiral alcohols, alpha-hydroxy acids and alpha-amino acids. The earlier hurdle of expensive cofactors was taken by the development of highly efficient cofactor regeneration methods. In addition, the accessible number of suitable dehydrogenases and therefore the versatility of this technology is constantly increasing and currently expanding beyond asymmetric production of alcohols and amino acids. Access to a large set of enzymes for highly selective C=C reductions and reductive amination of ketones for production of chiral secondary amines and the development of improved D-selective amino acid dehydrogenases will fuel the next wave of industrial bioreduction processes.

Nylon-6 is a bulk polymer used for many applications. It consists of the non-natural building block 6-aminocaproic acid, the linear form of caprolactam. Via a retro-synthetic approach, two synthetic pathways were identified for the fermentative production of 6-aminocaproic acid. Both pathways require yet unreported novel biocatalytic steps. We demonstrated proof of these bioconversions by in vitro enzyme assays with a set of selected candidate proteins expressed in Escherichia coli. One of the biosynthetic pathways starts with 2-oxoglutarate and contains bioconversions of the ketoacid elongation pathway known from methanogenic archaea. This pathway was selected for implementation in E. coli and yielded 6-aminocaproic acid at levels up to 160 mg/L in lab-scale batch fermentations. The total amount of 6-aminocaproic acid and related intermediates generated by this pathway exceeded 2 g/L in lab-scale fed-batch fermentations, indicating its potential for further optimization toward large-scale sustainable production of nylon-6.

BACKGROUNDIt is widely accepted that the poor thermostability of Baeyer–Villiger monooxygenases limits their use as biocatalysts for applied biocatalysis in industrial applications. The goal of this study was to investigate the biocatalytic oxidation of 3,3,5‐trimethylcyclohexanone using a thermostable cyclohexanone monooxygenase from Thermocrispum municipale (TmCHMO) for the synthesis of branched ϵ‐caprolactone derivatives as building blocks for tuned polymeric backbones. In this multi‐enzymatic reaction, the thermostable cyclohexanone monooxygenase was fused to a phosphite dehydrogenase (PTDH) in order to ensure co‐factor regeneration.RESULTSUsing reaction engineering, the reaction rate and product formation of the regio‐isomeric branched lactones were improved and the use of co‐solvents and the initial substrate load were investigated. Substrate inhibition and poor product solubility were overcome using continuous substrate feeding regimes, as well as a biphasic reaction system with toluene as water‐immiscible organic solvent. A maximum volumetric productivity, or space–time‐yield, of 1.20 g L‐1 h‐1 was achieved with continuous feeding of substrate using methanol as co‐solvent, while a maximum product concentration of 11.6 g L‐1 was achieved with toluene acting as a second phase and substrate reservoir.CONCLUSIONThese improvements in key process metrics therefore demonstrate progress towards the up‐scaled Baeyer–Villiger monooxygenase‐biocatalyzed synthesis of the target building blocks for polymer application. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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