Combinatorial Design of Biosynthetic Pathways for the Generation of Beta-Arylamines

β-arylamines are a class of small-molecule pharmaceuticals with a wide array of effector functions, they act as stimulants, appetite suppressants, antidepressants, vasoconstrictors, and migraine prophylactics, among others. The class also includes a number of endocrine signaling molecules that are naturally derived from aromatic amino acids in eukaryotes and their associated microbiota. Because β-arylamines are ubiquitous, there are many enzymes that are known or thought to modify the aromatic amino acids and their derivatives. We have mined metagenomics databases to identify over 300 such enzymes, which were then sequence optimized for expression in E. coli and synthesized. The aim of this study is to generate libraries of pathways consisting of many classes of enzymes from all domains of life. It is our goal to create not only novel biosynthetic pathways, but also new β-arylamines.

In initial experiments, panels of decarboxylases and N-methyltransferases were assayed for their ability to modify phenylalanine, tyrosine, and tryptophan in vivo. While most of the enzymes converted the amino acids to products at levels detectable by LC-MS, 14% were able to robustly catalyze these transformations in the E. coli cytosol. By co-expressing two enzymes, the human indole-N-methyltransferase and a microbiome-derived decarboxylase, we synthesized N,N-dimethyltryptamine in E. coli, demonstrating the ability to combine enzymes from disparate species into a single biosynthetic pathway. Additionally, the enzymes have shown activity on non-canonical amino acids, such as meta-tyrosine and p-amino-phenylalanine, further supporting our ability to generate novel pathways.

In this study, we add another seven catalytic classes into our collection of enzymes: acetyl transferases, β-hydroxylases, monooxygenases, N-hydroxylases, O-methyltransferases, ring-halogenases, and ring-hydroxylases. Data obtained by individual enzyme assays will be used to inform the design of pathway libraries, through which we will vary both the included enzymes and their expression levels. LC-MS and NMR will then be used to determine the structure, titer, and novelty of the synthesized compounds.