4.2.1 Conceptual design of the pathway for the production of 5HTP and serotonin
Two possible routes lead to the production of serotonin from tryptophan:
tryptophan can be hydroxylated into 5HTP and further converted into sero-tonin by decarboxylation. Alternatively, if tryptophan is first decarboxylated into tryptamine, this can be then converted into serotonin (Fig. 10). In both cases, the decarboxylation step can be performed by an aromatic amino acid decarboxylase (AADC). AADCs are present in animals, insects, and plants. In contrast to animal and insects AADC, which accept a broad range of aromatic amino acids. Plant AADC exhibits a narrow substrate specificity. Tryptophan decarboxylase from Catharanthus roseus (CrTDC), can only accept trypto-phan and 5HTP as natural substrates, and consequently produce tryptamine or serotonin (Noé et al., 1984).
Hydroxylation of tryptamine in E. coli was first shown by Park et al. (2011) using tryptamine 5-hydroxylase from rice (OsT5H), they reported a maximum production of 24 mg/L of serotonin when the media was supplied with 2 mM (408 mg/L) of tryptophan. Serotonin concentration did not increase with the substrate concentration which suggests that either OsT5H catalytic activity is low in E. coli or that serotonin product could have an inhibitory effect on OsT5H enzyme activity. Later this same group reported an increase in the enzyme activity when OsT5H was coexpressed with its respective NADPH-cytochrome P450 reductase (OsCPR) (Park et al., 2013).
Efforts have also been made to engineer E. coli strains capable of converting tryptophan to 5HTP. Tryptophan 5-hydroxylase (TPH), which is only present in eukaryotes, is capable of synthesizing 5HTP. Mammalian TPH has been expressed in E. coli, but it has low activity and poor stability when expressed in prokaryotes (Wang et al., 2002). Prokaryotic phenylalanine hydroxylases
Figure 10: Novel artificial pathways for the biosynthesis of serotonin via tryptamine and 5-hydroxytryptophan in E. coli. CtAAAH, aromatic amino acid hydroxylase from Cupriavidus taiwanensis; CrTDC, tryptophan decarboxylase from Catharanthus roseus; OsT5H, tryptamine 5-hydroxylase from rice – Oryza sativa;OsCPR, NADPH-cytochrome P450 reductase from rice.
from different species have been engineered to change the substrate preference from phenylalanine to tryptophan (PAH) (Hara and Kino, 2013; Lin et al., 2014). Lin et al. (2014) screened out and engineered PAH fromXanthomonas campestris, and they were able to produce 152.9 mg/L of 5HTP from glucose.
4.2.2 Bioconversion of tryptophan for the production of serotonin production in E. coli: proof of the concept
The potential of tryptophan and 5HTP bioconversion by CrTDC was ex-plored. An E. coli culture harboring plasmid pCOLADuet-1-TDC was con-centrated (OD600 15-20) and supplied with 1 mM of respective substrates. As shown in (Fig. 11), the initial conversion rate was higher when tryptophan is used as a substrate. Nevertheless, the production efficiency was similar af-ter 3 h of incubation at 30 ◦C. After 5 h incubation, there was a 93 % and 89 % bioconversion of tryptophan and 5HTP into tryptamine and serotonin, respectively. A higher affinity of CrTDC toward tryptophan (Km 0.75 mM), when compared with 5HTP (Km 1.3 mM), was previously reported, in this same study, both, tryptophan and tryptamine, were found to be inhibitors of the decarboxylation reaction (tryptamine Ki 3.1 mM) (Noé et al., 1984).
Hence, TDC inhibition with 5HTP have not been reported, an inhibition as-say using crude extracts and different 5HTP concentrations were conducted.
A slight decrease in decarboxylation activity was observed when the 5HTP concentration was 3 mM, and at 10 mM 50 % of the activity the protein activity was detected (Fig. 11). 5HTP has been reported to have strong in-hibition in decarboxylase activity of human and pig aromatic L-amino acid decarboxylase where 5HTP is the natural substrate (Bertoldi et al., 2008;
Verbeek et al., 2007). On the other hand, according to the Brenda Enzyme Database (http://www.brenda-enzymes.de) tryptamine inhibits plant related TDCs (C. roseus andPhalaris aquatica), in which tryptamine is an important building block for the formation of other secondary metabolites (alkaloids) (Facchini et al., 2000).
Aromatic amino acid hydroxylase from Cuprividus taiwanensis (CtAAAH) and tryptamine 5-hydroxylase from rice - Oryza sativa (OsT5H) with its re-spective NADPH-cytochrome P450 reductase (OsCPR) were used to com-pare the hydroxylase activity of tryptophan and tryptamine. In vivo bio-conversion assays were performed with cells (OD600 15-20) carrying plasmids pCOLADuet-1-GST∆37T5H-OsCPR2 or pCtAAAH in defined media
sup-Figure 11: Tryptophan decarboxylase activity from Catharanthus roseus (CrTDC) with tryptophan and 5HTP: a. Whole cell bioconversion efficiency ofCrTDC using E. coli (OD600 15-20) in media supplied with 1 mM tryptophan or 5HTP as substrate; b. relative activity of crude extracts withCrTDC protein with different 5HTP concentrations.
plied with 4 mM of tryptamine or tryptophan as substrates. Corresponding cofactors were also added to the media. After 5 h reaction around 10-12 % of the substrate was hydroxylated: 0.41 mM of 5HTP was detected in the supernatant of cells with plasmid pCtAAAH, and 0.48 mM of serotonin was produced in cells supplied with tryptamine (Fig. 12). These results were ex-pected and consistent with other values reported in the literature (Lin et al., 2014; Park et al., 2013). Despite a slightly lower conversion efficiency in cells
with plasmid pCtAAAH, great potential lies in this enzyme due that to pre-vious proteins engineering reports have been done with other AAAH (Kino et al., 2009; Lin et al., 2014). In silico analysis regarding this issue will be discussed in the next section.
Figure 12: Whole cell bioconversion assay for the hydroxylation of tryptamine and tryptophan. 5HTP was produced using strain BL21(DE3)∆tnaA harbor-ing plasmid pCtAAAH, serotonin was produced with plasmid GST∆37T5H-OsCPR2. Cell growth is represented by dashed lines, product formation with solid lines.
4.2.3 In silico evaluation of the serotonin synthetic pathway Computational tools can be used to evaluate predicted metabolic pathways.
Proposed conversion of tryptophan into serotonin via 5HTP was evaluated in silico using RetroPath (Carbonell et al., 2014) in order to estimate the feasibility of the synthetic path and to determine possible bottlenecks during the production in E. coli.
The tool predicts four steps or reactions during the whole pathway. Gibbs‘ free energy of the system was estimated: ∆G= -26.43. This negative value sug-gests favorable thermodynamic conditions for the set of bioconversions. Thein
silico analysis predicts the consumption of the endogenous cofactor tetrahy-dromonapterin (MH4) by the hydroxylase enzyme. Experimental evidence related to MH4 participating in hydroxylation reactions have been published by Lin et al. (2014) and Satoh et al. (2012). The computational analysis also predicted the consumption of a NADH during the regeneration of MH4 and the production of one hydroxyl radical (HO.) molecule. Two hydrogen protons would be consumed during the production of serotonin, the first one is used during the regeneration of cofactor, the second in the decarboxylation step (Fig. 13).