Protein Engineering for the Production 5-Hydroxytrypophan and Serotonin in Escherichia coli

5-Hydroxytryptophan (5HTP) is a non-canonical amino acid with therapeutic effect over various symptoms (e.g. depression, insomnia, chronic headaches, etc.). It is also the precursor of serotonin which, unlike 5HTP, does not cross the blood-brain barrier. Both 5HTP and serotonin are interesting for the pharmaceutical industry and currently they are either chemically synthesized or extracted from seeds of the African plant Griffonia simplicifolia. Current efforts are focused on their biotechnological production, and in our group we aim to link their production with the glucose central metabolism in order to obtain valuable metabolites from inexpensive carbon sources.

So far, we have developed a strain that is capable to hydroxylate tryptophan and produce 5HTP by using rational design approaches. To this end, phenylalanine hydroxylase (PAH) from Cupriavidus taiwanensis (CtPAH) was chosen as a starting point. Molecular modeling and docking analyses were conducted and prominent residues that may lead to an increase of the preference of tryptophan as a substrate were identified. Modeling analyses also suggested the need to eliminate part of the N-terminal region of the enzyme. Point mutations in five out of six residues showed higher activity toward tryptophan both in vitro and in vivo. The engineered CtPAHs were then introduced into an E. coli strain that is capable of producing tryptophan with a high productivity. To the date, we are comparing the effects of different promoters that may control the expression of CtPAH for a fine tuning.

To further increase the enzyme activity, we also endeavor to move from a rational design to a semi-rational evolutionary approach. For this regard, we are working in the generation of libraries, where we can combine different targeted sites with restriction enzyme and/or ligase independent methods. In order to screen the collection of mutants, a colorimetric method has been established that allows us to detect hydroxylated aromatic rings directly in agar plates. In parallel, we are working with in vitro cell free expression systems with the goal of developing multistep-single tube reactions that can facilitate the detection of the metabolites and therefore speeding up the screening of the library.

Regarding to the serotonin production, we are using a combination of three enzymes: one decarboxylase and a combination of P450 from Oryza sativa and its respective NADPH-cytochrome P450 reductase. So far, we can detect serotonin in cells supplemented with tryptophan, but low titers were detected when the corresponding genes were transformed into the tryptophan producer. To overcome this, we intent to follow the same approaches described above in order to increase the production either in vivo or in vitro.