Efficient biotechnological production of N-(Hydroxycinnamoyl)tyramines by utilizing plant-derived enzymes in a bench-scale bioreactor with in situ product precipitation.

The efficient recovery of plant polyphenolic bioactives is facing technological challenges of low concentrations and complex mixtures starting from plant tissue. In the search of alternatives, we developed a high-yield biotechnological production process for hydroxycinnamic acid-tyramine conjugates in Escherichia coli by converting soluble substrates into insoluble products. A recombinant plant-enzyme cascade was established by co-expression of Arabidopsis thaliana 4-coumarate:CoA ligase and Solanum tuberosum hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase. We overcame initial enzymatic limitations by developing a temperature adapted and sequentially phased bioprocess on a 1 L reactor scale. With 5 mM/h substrate supply rate we achieved 31.9 g/L (102 mM) of feruloyltyramine (FerT), 14.4 g/L (50.7 mM) coumaroyltyramine, and 14.6 g/L (46.5 mM) isoferuloyltyramine with a space time yield of 1.75 g *L-1 *h-1 FerT. The vast majority of the product was obtained as filterable precipitate, simplifying the downstream process compared to classical procedures. Washed filter cake from a FerT production after filtration via 16-40 µm glass filter, showed 99% mass content of FerT. Product identity was confirmed by NMR after recrystallization. The newly developed process is robust, cost-effective and scalable, paving the way for commercial applications of biotechnologically produced hydroxycinnamic acid-tyramines. KEY POINTS: • Plant derived enzymes expressed at low temperature enable high productivity • In situ precipitated product is readily filtered from fermentation broth • Carbon transfer rate mirrors toxicity of phenolics and guide process optimization.