Hydroxytyrosol (HT) is a valuable natural phenolic compound with strong antioxidant activity and various physiological and pharmaceutical functions. In this study, we established an artificial pathway for HT biosynthesis. First, efficient enzymes were selected to construct a tyrosol biosynthetic pathway. Aro10 from Saccharomyces cerevisiae was shown to be a better ketoacid decarboxylase than Kivd from Lactococcus lactis for tyrosol production. While knockout of feaB significantly decreased accumulation of the byproduct 4-hydroxyphenylacetic acid, overexpression of alcohol dehydrogenase ADH6 further improved tyrosol production. The titers of tyrosol reached 1469 ± 56 mg/L from tyrosine and 620 ± 23 mg/L from simple carbon sources, respectively. The pathway was further extended for HT production by overexpressing Escherichia coli native hydroxylase HpaBC. To enhance transamination of tyrosine to 4-hydroxyphenylpyruvate, NH4Cl was removed from the culture media. To decrease oxidation of HT, ascorbic acid was added to the cell culture. To reduce the toxicity of HT, 1-dodecanol was selected as the extractant for in situ removal of HT. These efforts led to an additive increase in HT titer to 1243 ± 165 mg/L in the feeding experiment. Assembly of the full pathway resulted in 647 ± 35 mg/L of HT from simple carbon sources. This work provides a promising alternative for sustainable production of HT, which shows scale-up potential.
In this study, we designed an artificial pathway composed of tyramine β-hydroxylase (TBH) and phenylethanolamine N-methyltransferase (PNMT) for the biosynthesis of both octopamine and synephrine. As most TBH and PNMT originate from eukaryotic animals and plants, the heterologous expression and identification of functional TBH and PNMT are critical for establishing the pathway in mode microorganisms like Escherichia coli. Here, three TBHs were evaluated, and only TBH from Drosophila melanogaster was successfully expressed in the soluble form in E. coli. Its expression was promoted by evaluating the effects of different expression strategies. The specific enzyme activity of TBH was optimized up to 229.50 U·g–1, and the first step in the biosynthetic pathway was successfully established and converted tyramine to synthesize 0.10 g/L of octopamine. Furthermore, the second step to produce synephrine from octopamine was developed by screening PNMT, enhancing enzyme activity, and optimizing reaction conditions, with a maximum synephrine production of 2.02 g/L. Finally, based on the optimization of the reaction conditions for each individual reaction, the one-pot cascade reaction for synthesizing synephrine from tyramine was constructed by combining the TBH and PNMT. The synthetic synephrine reached 30.05 mg/L with tyramine as substrate in the two-step enzyme cascade system. With further optimization and amplification, the titers of octopamine and synephrine were increased to 0.45 and 0.20 g/L, respectively, with tyramine as substrate. This work was the first achievement of the biosynthesis of octopamine and synephrine to date.
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