(228b) Molecular Basis of 5-Hydroxytryptophan Synthesis in Saccharomyces Cerevisiae

5-Hydroxytryptophan (5-HTP) is an intermediate in the biosynthesis of the neurotransmitter serotonin1 and has been used in the clinic for over 30 years. 5-HTP is mainly extracted from the seeds of the African plant Griffonia simplicifolia, which, however, is largely limited by the season- and region-dependent supply of the raw materials. Recently, microbial production of 5-HTP has been achieved successfully in Escherichia coli^1,2, which offers an alternative route for producing this small molecule drug. Encouraged by the pioneering studies on de novo synthesis of 5-HTP in E. coli, a model prokaryotic microorganism, in this study we aim to explore the possibility of using eukaryotic organisms such as Saccharomyces cerevisiae to synthesize 5-HTP and uncover the molecular basis of 5-HTP synthesis in eukaryotic cells. We report that 5-hydroxytryptophan can be synthesized in S. cerevisiaeby heterologously expressing prokaryotic phenylalanine 4-hydroxylase or eukaryotic tryptophan 3/5-hydroxylase, together with enhanced synthesis of MH4 or BH4 cofactors. The results showed that the activities of OcT5H and SmT5H were 50-75% higher than that of XcP4H and BpP4H when using BH4 as the cofactor. We also find that DFR1, a native enzyme that catalyze reactions for folate acid synthesis, can also catalyze the reaction for MH4 synthesis. It is the first time, to the best of our knowledge, that the molecular basis of 5-HTP synthesis was revealed in a eukaryotic organism, which paves the ways for future metabolic engineering (e.g., increasing the supply of tryptophan precursor and cofactors) to promote yeast-based production of 5-HTP.

1 Lin, Y., Sun, X., Yuan, Q. & Yan, Y. Engineering bacterial phenylalanine 4-hydroxylase for microbial synthesis of human neurotransmitter precursor 5-hydroxytryptophan. ACS Synth Biol 3, 497-505, doi:10.1021/sb5002505 (2014).

2 Sun, X., Lin, Y., Yuan, Q. & Yan, Y. Precursor-Directed Biosynthesis of 5-Hydroxytryptophan Using Metabolically Engineered E. coli. ACS Synth Biol 4, 554-558, doi:10.1021/sb500303q (2015).