Enhanced synthesis of 5-hydroxy-l-tryptophan through tetrahydropterin regeneration

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.

Engineering Bacterial Phenylalanine 4-Hydroxylase for Microbial Synthesis of Human Neurotransmitter Precursor 5-Hydroxytryptophan

“…As we were writing this manuscript, another study also reported the 287 5-HTP production in E. coli with a mutant P4H from C. violaceum 35 . However, its function 288 had to completely rely on the supplementation of exogenous pterin coenzyme 289 6,7-dimethyl-5,6,7,8-tetrahydropterine hydrochloride 35 , which is disadvantageous in terms of 290 economic viability as we discuss below. 291 292 Self-supply or regeneration of cofactors (including co-enzymes and co-substrates) is one of 293 the greatest advantages for whole-cell biosynthesis and biocatalysis.…”

Building microbial factories for the production of aromatic amino acid pathway derivatives: From commodity chemicals to plant-sourced natural products

“…Tryptophan 5-hydroxylase in humans and animals uses 5,6,7,8-tetrahydrobiopterin BH4and Fe 2+ as cofactors and O 2 as a co-substrate. During Trp hydroxylation, BH4 is oxidized to pterin-4α-carbinolamine (BH3OH) and is regenerated through pterin-4α-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) (Hara and Kino, 2013). In humans, BH4 is converted from GTP via three steps, involving GTP cyclohydrolase I (GCHI), 6pyruvate-tetrahydropterin synthase (PTPS), and sepiapterin reductase (SPR) (Yamamoto et al, 2003) (Figure 3b).…”

Overview on Multienzymatic Cascades for the Production of Non-canonical α-Amino Acids

“…values > 99%; Parmeggiani et al, 2015; Figure 7D). Labeling of L-Tyr with carbon and hydrogen isotopes was achieved by coupling of PAL and L-phenylalanine 4 aminooxygenase (Pająk et al, 2018); this strategy might be expanded for the production of L-DOPA using p-hydroxyphenylacetate 3-hydroxylase (Min et al, 2015), including tetrahydropterin-and NADH-recycling systems (Hara and Kino, 2013) (Supplementary Figure S4).…”

“…(G) 5-Hydroxy-tryptophan synthesis through a L-phenylalanine 4-hydroxylase coupled to cofactor regeneration. PAH, L-phenylalanine 4-hydroxylase; PCD, pterin-4α-carbinolamine dehydratase; DPR, dihydropteridine reductase; GDH, glucose dehydrogenase TetraH, tetrahydropterin; 4 α C, 4α-carbinolamine; DhP, dihydropteridine (Hara and Kino, 2013).…”