De novo biosynthesis of synephrine in engineered Escherichia coli.

Synephrine, the predominant and active alkaloid in Citrus aurantium, is used in the treatment of various conditions, including bronchial asthma, hypotension, collapse, shock, and orthostatic hypotension. However, the extraction of synephrine from plant sources is hindered by several limitations, including low natural abundance, inefficient extraction processes, and complex purification steps. In this study, we developed a de novo biosynthetic platform for synephrine production. We first validated the catalytic activity of three key enzymes-TYDC, MwDβH, and hPNMT- in the synephrine synthesis pathway through in vitro biocatalytic assays. These enzymes were expressed in a tyrosine-producing Escherichia coli BAK11 strain, enabling de novo biosynthesis of synephrine from glucose. To enhance production, the β-hydroxylases were screened and engineered, resulting in a strain with a truncated variant of the β-hydroxylase that achieved a synephrine titer of 47.8  mg/L, representing a 2.7-fold increase compared to the initial strain. Using fed-batch fermentation with glucose as the sole carbon source, a synephrine titer of 88.1 mg/L was achieved in a 5-L fermenter. To our knowledge, this represents the first report of de novo microbial synthesis of synephrine, offering a novel, cost-effective, and sustainable strategy for the biobased production of synephrine.