초록
<P>5-Hydroxy-<SMALL>L</SMALL>-tryptophan (5-HTP) is a naturally occurring aromatic amino acid present in the seeds of the African plant <I>Griffonia simplicifolia</I>. 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 <SMALL>L</SMALL>-phenylalanine 4-hydroxylase of <I>Chromobacterium violaceum</I>. For the synthesis of 5-HTP using <I>Escherichia coli</I> whole cell bioconversion, <SMALL>L</SMALL>-tryptophan and 5-HTP degradation by <I>E. coli</I> 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 <SMALL>L</SMALL>-tryptophan and 5-HTP in <I>E. coli</I>. For regeneration of the cofactor pterin, we screened and investigated several key enzymes, including dihydropteridine reductase from <I>E. coli</I>, glucose dehydrogenase from <I>Bacillus subtilis</I>, and pterin-4α-carbinolamine dehydratase from <I>Pseudomonas syringae</I>. Genes encoding these three enzymes were overexpressed in an <I>E. coli</I> 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.</P>