Bioproduction of a Large-Scale Library of Tryptamine Derivatives for Neuropsychiatric Drug Screening

Drug screening programs targeting novel indolethylamines with pharmacological properties suitable for the treatment of psychiatric and central nervous system disorders benefit from the availability of large compound libraries normally prepared using synthetic chemistry. Bioproduction strategies based on microbial metabolic engineering and fermentation generally fail to achieve the throughput, scale, or versatility of synthetic chemistry owing, in part, to a lack of efficient and promiscuous enzymes. Moreover, synthetic biology rarely extends to the purification of targeted products, which is an essential component of synthetic chemistry and drug screening regimes. A lattice of biosynthetic routes beginning with endogenous tryptophan or exogenous indole derivatives were engineered in <i>Escherichia coli</i> using heterologous genes encoding enzymes sourced from plants, mushrooms, microbes and animals. Twelve tryptophan decarboxylase candidates were screened and highly versatile top-performers from <i>Bacillus atrophaeus</i> and the gut microbiome species <i>Clostridium sporogenes</i> were identified. Seven halogenases, three tryptophan synthase β-subunits, six <i>N</i>-methyltransferases, five regioselective prenyltransferases, a cytochrome P450 oxidoreductase 5-hydroxylase, an <i>N</i>-acetyltransferase, a 4-<i>O</i>-kinase and various accessory proteins were also tested. These enzymes were used in various combinations and permutations to build <i>E. coli</i> strains capable of 344 putative biotransformations, which resulted in the formation of 279 products with only 63 targeted compounds not detected. A set of 17 novel <i>N</i>-acetylated derivatives were selected for upscaled culturing and purification to ≥95% from 0.5 to 1 L of the fermentation broth, which yielded ∼6-80 mg of each molecule. The potential of each compound for bioactivity at 14 different receptors or transporters with established or purported involvement in neuropsychiatric diseases was tested using a single ligand concentration. Nearly all the <i>N</i>-acetylated compounds showed interaction with the melatonin (MT₁) receptor, and several molecules showed interaction with serotonergic receptors 5-HT_2B, 5-HT_2C, and 5-HT₇. Overall, we show that bio-fermentation is useful in the large-scale screening of molecules with potential in drug development.