This study engineers Escherichia coli for the biosynthesis of six rosmarinic acid (RA) derivatives (IA, isorinic acid; CPLA, caffeoyl phenyllactic acid; PSA, p-coumaroyl salvianic acid; P4PLA, p-coumaroyl 4-hydroxyphenyllactic acid; PPLA, p-coumaroyl phenyllactic acid). Three process strategies: single-cell, stepwise, and coculture synthesis are evaluated to optimize production. Results show that the optimal bioprocess is compound-dependent: single-cell for PPLA/P4PLA, stepwise for CPLA/PSA/RA, and coculture for IA.

Rosmarinic acid (RA) is a phytochemical that exhibits a variety of biological activities. The biological synthesis pathway of RA has been well-established in plants. In this study, the shikimate pathway is engineered to enhance the availability of precursor substrates and the biosynthetic pathway is reconstructed to produce six RA derivatives: p-coumaroyl phenyllactic acid (PPLA), p-coumaroyl 4-hydroxyphenyllactic acid (P4PLA), caffeoyl phenyllactic acid (CPLA), p-coumaroyl salvinic acid (PSA), rosmarinic acid (RA), and isorinic acid (IA). To identify the optimal method for maximizing the production of the target compounds while minimizing the formation of unwanted byproducts, single-cell, stepwise, and coculture approaches are employed. Using the single-cell method, 145.1 mg L⁻¹ of PPLA and 361.6 mg L⁻¹ of P4PLA are synthesized. The stepwise method yields 28.0 mg L⁻¹ of CPLA, 23.4 mg L⁻¹ of PSA (which contains synthesized IA), and 10.9 mg L⁻¹ of RA. Finally, the coculture method results in the synthesis of 40.2 mg L⁻¹ of IA (which contains synthesized PSA).