Four-Step Pathway from Phenylpyruvate to Benzylamine, an Intermediate to the High-Energy Propellant CL-20.

Abstract

Benzylamine is a commodity chemical used in the synthesis of motion-sickness treatments and anticonvulsants, in dyeing textiles, and as a precursor to the high-energy propellant CL-20. Because chemical production generates toxic waste streams, biosynthetic alternatives have been explored, recently resulting in a functional nine-step pathway from central metabolism (phenylalanine) in E. coli. We report a novel four-step pathway for benzylamine production, which generates the product from cellular phenylpyruvate using enzymes from different sources: a mandelate synthase (Amycolatopsis orientalis), a mandelate oxidase (Streptomyces coelicolor), a benzoylformate decarboxylase (Pseudomonas putida), and an aminotransferase (Salicibacter pomeroyi). This pathway produces benzylamine at 24 mg/L in 15 h (4.5% yield) in cultures of unoptimized cells supplemented with phenylpyruvate. Because the yield is low, supplementation with pathway intermediates is used to troubleshoot the design. This identifies conversion inefficiencies in the mandelate synthase-mediated synthesis of (S)-mandelic acid, and subsequent genome mining identifies a new mandelate synthase (Streptomyces sp. 1114.5) with improved yield. Supplementation experiments also reveal native redirection of ambient phenylpyruvate away from the pathway to phenylalanine. Overall, this work illustrates how retrosynthetic design can dramatically reduce the number of enzymes in a pathway, potentially reducing its draw on cellular resources. However, it also shows that such benefits can be abrogated by inefficiencies of individual conversions. Addressing these barriers can provide an alternative approach to green production of benzylamine, eliminating upstream dependence on chlorination chemistry.