Abstract
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.
