Cell‐Free Synthesis of Dopamine and Serotonin in Two Steps with Purified Enzymes

Abstract

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...

Construction of Cell Factory Capable of Efficiently Converting L-tryptophan Into 5-hydroxytryptamine

“…However, 5-HT was released within 4 h by HaDDC with 5-HTP as a substrate. These results showed that HaDDC exhibited strict substrate speci city similar to that displayed by drDDC from D. melanogaster [7,15]. According to the results shown in Table 1, HaDDC exhibited a high e ciency on 5-HTP, and the conversion rate was 88.45% at 4 h, which was higher than that of TDC reported previously [6,11].…”

“…The in vitro cascade of SmTPH and HaDDC was conducted in a reaction system (2 mL) that contained 7 mM DTT, 0.1 M ferrous ammonium sulfate, 0.1 mg/mL catalase, 0.6 mM BH 4 , 0.1 mM PLP, 0.5 mM L-Trp, 100 µL puri ed SmTPH (1.0 g/L), 100 µL puri ed HaDDC (1.0 g/L) and 50 mM HEPES (pH 7.0), and incubated at 30℃ [1,7]. In addition to these cofactors, reactions were supplemented with catalase and dithiothreitol to protect against oxidative degradation [18].…”

“…Currently, the main supply of 5-HT depends on the extraction from mammals (e.g. buffalo, rat, and pig) or plants (sa ower, sea buckthorn, and Moringa Oleifera seeds) [7]. However, the low 5-HT yield from natural extraction and the insu cient supply of raw materials result in demands that exceed the supply.…”

Construction of cell factory capable of efficiently converting l-tryptophan into 5-hydroxytryptamine

“…In addition, previous study reported that DDC can catalyse 5-HTP into 5-HT without the ability of converting l-Trp [13,14]. However, almost studies have focused on the characterization of the enzyme involved in the biosynthesis of dopamine, and systematic studies for the production of 5-HT have not been conducted [7,15].…”

“…Currently, the main supply of 5-HT depends on the extraction from mammals (e.g. buffalo, rat, and pig) or plants (safflower, sea buckthorn, and Moringa Oleifera seeds) [7]. However, the low 5-HT yield from natural extraction and the insufficient supply of raw materials…”