Production of plant-specific tyramine derivatives by dual expression of tyramine N-hydroxycinnamoyltransferase and 4-coumarate:coenzyme A ligase in Escherichia coli.

Plant-specific bioactive compounds including feruloyltyramine (FT), 4-coumaroyltyramine (CT), and caffeoyltyramine (CaT) were simultaneously produced in Escherichia coli by heterologous expression of two biosynthetic genes encoding 4-coumarate:coenzyme A ligase and tyramine N-hydroxycinnamoyltransferase (THT) cloned from Arabidopsis thaliana and pepper, respectively. Simultaneous supplementation of substrates to the recombinant E. coli resulted in the secretion of multiple tyramine derivatives into the medium at high yield: CT (189 mg l(-1)), FT (135 mg l(-1)), CaT (40 mg l(-1)).

Conversion of 5-hydroxytryptophan into serotonin by tryptophan decarboxylase in plants, Escherichia coli, and yeast.

The L-tryptophan decarboxylase (TDC) gene of rice was heterologously expressed in various organisms. Transgenic rice overexpressing TDC showed accumulation of serotonin upon 5-hydroxytryptophan treatment, which was consistent with the in vitro 5-hydroxytryptophan decarboxylase enzyme activity of purified recombinant rice TDC in a pyridoxal phosphate-dependent manner. Recombinant yeast harboring TDC produced serotonin at the expense of the endogenous 5-hydroxytryptophan levels.

Expression of serotonin derivative synthetic genes on a single self-processing polypeptide and the production of serotonin derivatives in microbes.

The plant-specific serotonin derivatives feruloylserotonin (FS) and 4-coumaroylserotonin (CS) are synthesized by the enzymes 4-coumarate:coenzyme A ligase (4CL) and serotonin N-hydroxycinnamoyltransferase (SHT). To express these genes coordinately, SHT was fused in-frame with the self-processing FDMV 2A sequence followed by 4CL in a single open reading frame and introduced into Escherichia coli or Saccharomyces cerevisiae. The transgenes were abundantly expressed in both recombinant microbes, but functional expression was achieved only in yeast, with cleavage at the 2A sequence yielding monomeric SHT-2A and 4CL as judged by immunoblot and product analyses.

Enzymatic features of serotonin biosynthetic enzymes and serotonin biosynthesis in plants.

Serotonin, a pineal hormone in mammals, is found in a wide range of plant species at detection levels from a few nanograms to a few milligrams, and has been implicated in several physiological roles, such as flowering, morphogenesis and adaptation to environmental changes. Serotonin synthesis requires two enzymes, tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H), with TDC serving as a rate-limiting step because of its high K(m) relation to the substrate tryptophan (690 microM) and its undetectable expression level in control plants. However, T5H and downstream enzymes, such as serotonin N-hydroxycinnamoyl transferase (SHT), have low K(m) values with corresponding substrates.

Endosperm-specific expression of serotonin N-hydroxycinnamoyltransferase in rice.

Serotonin N-hydroxycinnamoyltransferase (SHT) is a key enzyme in the synthesis of feruloylserotonin (FS) and 4-coumaroylserotonin (CS). These serotonin derivatives show strong antioxidant activity, making them valuable for both nutritional and pharmacological use in humans. Ectopic expression of SHT under the control of the endosperm specific-glutelin and prolamin promoters from rice was produced via Agrobacterium-mediated transformation.