Enhanced synthesis of 5-Hydroxy-L-tryptophan through tetrahydropterin regeneration

Ryotaro Hara, Kuniki Kino

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

5-Hydroxy-L-tryptophan (5-HTP) is a naturally occurring aromatic amino acid present in the seeds of the African plant Griffonia simplicifolia. Although 5-HTP has therapeutic effects in various symptoms, efficient method of producing 5-HTP has not been established. In this study, we developed a novel cofactor regeneration process to achieve enhanced synthesis of 5-HTP by using modified L-phenylalanine 4-hydroxylase of Chromobacterium violaceum. For the synthesis of 5-HTP using Escherichia coli whole cell bioconversion, L-tryptophan and 5-HTP degradation by E. coli endogenous catabolic enzymes should be considered. The tryptophanase gene was disrupted using the λ red recombination system, since tryptophanase is postulated as an initial enzyme for the degradation of L-tryptophan and 5-HTP in E. coli. For regeneration of the cofactor pterin, we screened and investigated several key enzymes, including dihydropteridine reductase from E. coli, glucose dehydrogenase from Bacillus subtilis, and pterin-4α-carbinolamine dehydratase from Pseudomonas syringae. Genes encoding these three enzymes were overexpressed in an E. coli tryptophanase-deficient host, resulting in the synthesis of 0.74 mM 5-HTP in the presence of 0.1 mM pterin and the synthesis of 0.07 mM 5-HTP in the absence of regeneration of pterin. These results clearly indicated the successful regeneration of pterin. Following optimization of the reaction conditions, 2.5 mM 5-HTP was synthesized with cofactor regeneration, while 0.8 mM 5-HTP was recovered without cofactor regeneration under the same reaction conditions, suggesting that the principle described here provides a new method for cofactor regeneration.

Original languageEnglish
Article number70
Pages (from-to)1-7
Number of pages7
JournalAMB Express
Volume3
DOIs
Publication statusPublished - 2013 Jan 1

Keywords

  • 5-hydroxy-L-tryptophan
  • Cofactor regeneration
  • L-phenylalanine 4-hydroxylase
  • NAD(P)H
  • Tetrahydropterin

ASJC Scopus subject areas

  • Biophysics
  • Applied Microbiology and Biotechnology

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