title: Engineering bacterial phenylalanine 4-hydroxylase for microbial synthesis of human neurotransmitter precursor 5-hydroxytryptophan.
abstract: 5-Hydroxytryptophan (5-HTP) is a drug that is clinically effective against depression, insomnia, obesity, chronic headaches, etc. It is only commercially produced by the extraction from the seeds of Griffonia simplicifolia because of a lack of synthetic methods. Here, we report the efficient microbial production of 5-HTP via combinatorial protein and metabolic engineering approaches. First, we reconstituted and screened prokaryotic phenylalanine 4-hydroxylase activity in Escherichia coli. Then, sequence- and structure-based protein engineering dramatically shifted its substrate preference, allowing for efficient conversion of tryptophan to 5-HTP. Importantly, E. coli endogenous tetrahydromonapterin (MH4) could be utilized as the coenzyme, when a foreign MH4 recycling mechanism was introduced. Whole-cell bioconversion allowed the high-level production of 5-HTP (1.1-1.2 g/L) from tryptophan in shake flasks. On this basis, metabolic engineering efforts were further made to achieve the de novo 5-HTP biosynthesis from glucose. This work not only holds great scale-up potential but also demonstrates a strategy for expanding the native metabolism of microorganisms.
authors.affiliation: College of Engineering, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Yuheng
authors.initials: Y
authors.lastname: Lin
authors.forename: Xinxiao
authors.initials: X
authors.lastname: Sun
authors.forename: Qipeng
authors.initials: Q
authors.lastname: Yuan
authors.forename: Yajun
authors.initials: Y
authors.lastname: Yan
mesh_terms: D006916:5-Hydroxytryptophan / Q000096:biosynthesis* / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D060847:Metabolic Engineering; D018377:Neurotransmitter Agents / Q000096:biosynthesis* / Q000737:chemistry; D010651:Phenylalanine Hydroxylase / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D015202:Protein Engineering; D011549:Pseudomonas / Q000201:enzymology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
chemical_list: D018377:Neurotransmitter Agents; D011994:Recombinant Proteins; D014364:Tryptophan; D006916:5-Hydroxytryptophan; D010651:Phenylalanine Hydroxylase