(670h) Microbial Production of a Multi-Functional Drug 5-Hydroxytryptophan Via Combined Metabolic and Protein Engineering Approaches

Depression is a mental disorder affecting 350 million people over the world. According to the data released by World Health Organization (WHO), less than 50% of the patients on the globe (less than 10% in some regions) have received medical treatment. Deficiency of the neurotransmitter serotonin (5-hydroxytryptamine) in the central nervous system is thought to be a related physiological factor for bad mood. 5-Hydroxytryptophan (5-HTP) is the direct biosynthetic precursor to serotonin in human and animals. It has been proved to be clinically effective in treating depression, as well as insomnia, fibromyalgia, obesity, etc. Currently, commercial production of 5-HTP is merely achieved by the extraction from the seeds of an African plant Griffonia simplicifolia due to lack of (bio-)synthetic methods. Here we demonstrate a novel biotechnological production process via combined protein and metabolic engineering approaches. Instead of using the unstable animal tryptophan 5-hydroxylases, we reconstituted, screened, and modified bacterial phenylalanine 4-hydroxylase (P4H) activity in the microbial host Escherichia coli. Sequence and structure-based protein engineering dramatically reversed its substrate preference from phenylalanine to tryptophan, leading to high catalytic activity in converting tryptophan to 5-HTP. Most strikingly, the E. coli endogenous tetrahydromonapterin (MH4) can be utilized as an efficient coenzyme when a heterologous MH4 regeneration system is reconstituted. Whole cell bioconversion enabled the high-level production of 5-HTP from tryptophan in shake flasks. Furthermore, metabolic engineering efforts were made to achieve the total biosynthesis of 5-HTP from glucose by grafting the 5-hydroxlation reaction into the tryptophan overproducing strains. This approach does not require the addition of precursors or expensive pterin coenzymes into the medium but only utilizes abundant renewable carbon sources. This microbial platform holds great potential for scale-up production of 5-HTP.