For example, the intermediates of the synthesis of dopamine and serotonin are L-DOPA (L-dihydroxy-phenylalanine) and 5-HTP (5-hydroxytryptophan), respectively, and have found use as administered precursors that are capable of crossing the blood-brain barrier. Although several studies have focused on the characterization of the separate enzymes involved in the biosynthesis of serotonin and dopamine, [3-5] surprisingly little effort has been put into developing enzymatic methods for the production of the final neurotransmitters starting from their proteinogenic amino acids precursors, i.e., L-tryptophan (Trp) and L-tyrosine (Tyr). If developed, such a system may open up new opportunities to build nanofactories and artificial cells for the treatment of neurological disorders. [6-8] To produce the monoamine neurotransmitters serotonin and dopamine in vitro, hydroxylases specific to the amino acid substrate were exploited. The catalytic domain of human tryptophan hydroxylase isoform 2 (TPH) was chosen for the synthesis of 5-HTP from Trp, [9] because this enzyme was previously shown to not be inhibited by high concentrations of the substrate. [10] Two versions of the catalytic domain of rat tyrosine hydroxylase were tested for the production of L-DOPA, including a recombinant, wild type version (rTH) and a truncated construct (ΔTH) that was not inhibited by substrate. [11] Additionally, a hydroxylase from the bacterium Chlamydia pneumoniae, Cpn1046, was tested. Cpn1046 has broader substrate specificity compared to TH and is homologous to eukaryotic aromatic amino acid hydroxylases. [12] Since the aromatic amino acid decarboxylase from Drosophila melanogaster (AADC) is active on both 5-HTP and L-DOPA, [13] a single enzyme was used for the decarboxylation step for the synthesis of both serotonin and dopamine. We found that TPH and ΔTH completely hydroxylated Trp and Tyr to produce the intermediates 5-HTP and L-DOPA, respectively. When TPH and ΔTH were coupled with AADC, serotonin and dopamine were efficiently produced in vitro. Each enzyme (TPH, rTH, ΔΤΗ, AADC, and Cpn1046) expressed well when fused to maltose binding protein (MBP), and each protein was purified in a single step with amylose resin (Figure S1, Supporting Information). However, multiple bands were observed on a SDS-PAGE of Cpn1046. Typically, 50 mg of purified protein was obtained per liter of bacterial culture expressing each construct. Conversely, the use of The synthesis of serotonin and dopamine with purified enzymes is described. Both pathways start from an amino acid substrate and synthesize the monoamine neurotransmitter in two enzymatic steps. The enzymes human tryptophan hydroxylase isoform 2, Rattus norvegicus tyrosine hydroxylase, Chlamydia pneumoniae Cpn1046, and aromatic amino acid decarboxylase from Drosophila melanogaster are recombinantly expressed, purified, and shown to be functional in vitro. The hydroxylases efficiently convert L-DOPA (L-dihydroxy-phenylalanine) and 5-HTP (5-hydroxytryptophan) from L-tyrosine and L-tryptophan, respectiv...

Metabolic dysfunction arising from missing or impaired enzymes comprising the branched chain amino acid (BCAA) degradation pathway, especially those involving leucine, can result in the accumulation of toxic metabolic intermediates and cause severe metabolic disease. Removal of dietary BCAAs via their degradation by engineered microbes could be a viable approach to prevent BCAA-mediated disease sequelae. In this article, we describe the design and construction of an engineered leucine degrading strain of E. coli Nissle, the improvement of the degradation pathway through high throughput screening, and the demonstration of strain activity in animal models monitored by disease and strain-specific biomarkers. This work provides a path for the development of engineered probiotic bacterial strains as a treatment for BCAA-related metabolic disorders in humans.