Synthetic pathways and processes for effective production of 5-hydroxytryptophan and serotonin from glucose in Escherichia coli

Abstract

Background: Tryptophan derivatives such as 5-hydroxytryptophan (5HTP) and serotonin are valuable molecules with pharmaceutical interest. 5HTP is presently mainly obtained by extraction from the plant Griffonia simplicifolia and serotonin is produced by chemical synthesis. A simple biotechnological method for the production of these compounds is desired.

Results: In a first attempt to synthesize serotonin from glucose, we used a single engineered Escherichia coli strain and observed a low production of maximal 0.8 ± 0.2 mg/L of serotonin, probably due to the undesired site-reaction of direct decarboxylation of tryptophan and the consequent decrease of the precursor 5HTP. To circumvent this problem, we have constructed a stepwise system in which the 5HTP production and the serotonin conversion are separated. 962 ± 58 mg/L of 5HTP was produced in the first step using a recombinant strain with a semi-rationally engineered aromatic amino acid hydroxylase, the highest concentration reported so far. In a subsequent step of 5HTP bioconversion using a recombinant strain harboring a tryptophan decarboxylase, 154.3 ± 14.3 mg/L of serotonin was produced.

Conclusions: We present results of a two-stage fermentation process for the production of 5HTP and serotonin. The first strain is a highly efficient 5HTP producer, and after fermentation the supernatant is separated and used for the production of serotonin. This is the first report for the microbial production of serotonin from glucose.

Keywords: 5-hydroxytryptophan; Aromatic amino acid hydroxylase; Protein engineering; Serotonin; Synthetic pathway.

Fig. 1

Novel artificial pathway for the biosynthesis of 5-hydroxytryptophan and serotonin in E. coli. PEP, phosphoenolpyruvate; E4P, erythrose 4-phosphate; CtAAAH, aromatic amino acid hydroxylase from Cupriavidus taiwanensis; TDC, tryptophan decarboxylase from Catharanthus roseus; PCD, pterin-4 alpha-carbinolamine dehydratase from human; DHPR, dihydropteridine reductase from human

Fig. 2

5HTP production in different E. coli strains. a tryptophan production and (b) 5HTP production of TrpD-Gi and TrpD-Pl strains carrying the CtAAAH-F gene under control of the P_trc and P_J23110 promoters respectively. c Tryptophan and 5HTP production of TrpD-Pl / pACJ23-CtAAAH-F over time

Fig. 3

Protein engineering of CtAAAH-F. a CtAAAH-F binding pocket predicted: the atoms of the cofactor and 2-His-1-caboxylate facial triad are present in gray, iron atom in pink; tryptophan surface is shown in blue, F197 and E219 atoms are present in orange and their surfaces are shown in green. b CtAAAH-LC binding pocket predicted: as in a., but L197 and C219 are present in orange, as well as their surfaces. c Lineweaver-Burk plot of CtAAAH-F and CtAAAH-LC. d tryptophan and 5HTP production after batch fermentation. Values in the graph are the average of triplicates, error bars correspond to the standard error of the mean (SEM)

Fig. 4

Serotonin production using a two-stage strategy. a and d growth curves; (b and e) production/consumption of tryptophan, 5HTP and serotonin. Side reaction (tryptophan conversion to tryptamine) is also indicated in panel e. with dashed lines; c and f HPLC retention pattern of tryptophan, 5HTP and serotonin; (a, b and c) panels correspond to the TrpD-Pl / pACJ23-CtAAAH-LC fermentation for the production of 5HTP from glucose; panels (d, e and f) correspond to the BL21(DE3) ΔtnaA / pCOLAJ23-TDC fermentation for the production of serotonin. Values in the graph are the average of triplicates, error bars correspond to the standard error of the mean (SEM)

Fig. 5

Library construction and screening strategies. a Plasmid and part of the CtAAAH-F gene were amplified with phosphorothioate primers and (b) cleavage with I₂/EtOH solution leaving sticky ends. The amplicon was hybridized with (c) synthetic phosphorylated oligos and (d) transformed into an E. coli strain which contains an intracellular tryptophan fluorescent sensor. e Tryptophan consumers can be distinguished by the lack or diminished formation of fluorescence