Engineering bacterial phenylalanine 4-hydroxylase for microbial synthesis of human neurotransmitter precursor 5-hydroxytryptophan.
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| 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.
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| Authors | Yuheng Lin, Xinxiao Sun, Qipeng Yuan, Yajun Yan |
| Journal | ACS synthetic biology (ACS Synth Biol) Vol. 3 Issue 7 Pg. 497-505 (Jul 18 2014) ISSN: 2161-5063 [Electronic] United States |
| PMID | 24936877 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't) |
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