Carboxylic acid reductase (CAR) catalyzes the ATP- and NADPH-dependent reduction of carboxylic acids to the corresponding aldehydes. The enzyme is related to the non-ribosomal peptide synthetases, consisting of an adenylation domain fused via a peptidyl carrier protein (PCP) to a reductase termination domain. Crystal structures of the CAR adenylation–PCP didomain demonstrate that large-scale domain motions occur between the adenylation and thiolation states. Crystal structures of the PCP–reductase didomain reveal that phosphopantetheine binding alters the orientation of a key Asp, resulting in a productive orientation of the bound nicotinamide. This ensures that reduction of the aldehyde product does not occur. Combining crystallography with small-angle x-ray scattering (SAXS), we propose that molecular interactions between initiation and termination domains are limited to competing PCP docking sites. This is supported by the fact that (R)-pantetheine can support CAR activity for mixtures of the isolated domains. Our model suggests directions for further development of CAR as a biocatalyst.
The construction of biocatalytic cascades for the production of chemical precursors is fast becoming one of the most efficient approaches to multi-step synthesis in modern chemistry. However, despite the use of low solvent systems and renewably-resourced catalysts in reported examples, many cascades are still dependent on petrochemical starting materials, which as of yet cannot be accessed in a sustainable fashion. Herein we report the production of the versatile chemical building block cinnamyl alcohol from the primary metabolite and fermentation product L-phenylalanine. Through the combination of three biocatalyst classes (phenylalanine ammonia lyase, carboxylic acid reductase and alcohol dehydrogenase) the target compound could be reached in high purity, demonstrable at 100 mg scale achieving 53 % yield using ambient temperature and pressure in aqueous solution. This system represents a synthetic strategy in which all components present at time zero are biogenic and thus minimising damage to the environment. Further we extend this biocatalytic cascade by its inclusion in a L-phenylalanine overproducing strain of Escherichia coli. This metabolically engineered strain produces cinnamyl alcohol in mineral media using a glycerol and glucose as carbon source. This study demonstrates the potential to establish green routes to the synthesis of cinnamyl alcohol from a waste stream such as glycerol derived, for example, from lipase treated biodiesel.
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