Microbial Synthesis of Human-Hormone Melatonin at Gram Scales

Abstract

Melatonin is a commercially attractive tryptophan-derived hormone. Here we describe a bioprocess for the production of melatonin using Escherichia coli to high titers. The first engineered strain produced 0.13 g/L of melatonin from tryptophan under fed-batch fermentation conditions. A 4-fold improvement on melatonin titer was further achieved by (1) protein engineering of rate-limiting tryptophan hydroxylase to improve 5-hydroxytryptophan biosynthesis and (2) chromosomal integration of aromatic-amino-acid decarboxylase to limit byproduct formation and to minimize gene toxicity to the host cell. Fermentation optimization improved melatonin titer by an additional 2-fold. Deletion of yddG, a tryptophan exporter, exhibited an additive beneficial effect. The final engineered strain produced ∼2.0 g/L of melatonin with tryptophan supplemented externally and ∼1.0 g/L with glucose as the sole carbon source for tryptophan supply. This study lays the foundation for further developing a commercial melatonin-producing E. coli strain.

Microbial production of beneficial indoleamines (serotonin and melatonin) with potential application to biotechnological products for human health

“…In this sense, several strategies of metabolic engineering and synthetic biology, such as homologous recombination and CRISPR-Cas9 technologies, can be applied to improve serotonin and melatonin biosynthesis in microorganisms. Microbial cell factories should be optimized through successive design-build-test-learn cycles (Luo et al 2020). These cycles can include enzyme and promoter engineering, metabolic flux control, pathway optimization, and genome-scale metabolic models, among other strategies (Guan et al 2020).…”

“…Currently, little is known about the genetic background involved in the microbial biosynthetic and catabolic pathways of serotonin and melatonin. Nevertheless, it has been suggested that the expression of the genes encoding the biosynthetic enzymes tryptophan 5hydroxylase and serotonin N-acetyltransferase is one of the most relevant factors during melatonin production (Tan et al 2015;Luo et al 2020). Recently, several genes from bacterial origin were tested to construct a melatonin-producing Escherichia coli strain (Luo et al 2020).…”

“…At an industrial level, melatonin is commonly produced by chemical synthesis, using solvents and toxic catalysts to avoid the viral contamination related to extraction in animal tissues (Sun et al 2016). In contrast, microbial production can be an "eco-friendly" and more sustainable alternative to conventional chemical processes (Germann et al 2016;Luo and Foster, 2019;Luo et al 2020).…”

“…This invention is based on recombinant microbial cells of E. coli with heterologous nucleic acid sequences encoding a monoxygenase for producing the oxidation of an amino acid in order to obtain melatonin and related compounds. Recently, Luo et al (2020) described a bioprocess to improve the microbial production of melatonin using E. coli recombinant cells. After some engineering modifications and optimization of fermentation conditions, the maximum melatonin titer achieved after fedbatch fermentations was 2000 mg L -1 (20 fold-times improvement in comparison with the initial Accepted Article strain) using external supplementation of tryptophan, and ∼1000 mg L -1 with only glucose as carbon source for tryptophan supply.…”

“…The development of foods or pharmaceuticals including purified biomolecules from microbial origin is an eco-friendly alternative to products containing active ingredients obtained from chemical synthesis. In this sense, in the last years, few studies have described the melatonin production, mainly by genetically modified microbes (Germann et al 2016;Luo and Foster, 2019;Luo et al 2020). On the other hand, wild-type isolates from fermented foods, plants and environmental samples have been described as melatonin and serotonin producers (Al-Hassan et al 2011;Jiao et al 2016;Fernandez-Cruz et al 2019;Fracassetti et al 2020).…”