(188ae) Overcome the Challenges of Balancing Complex Rosmarinic Acid Biosynthetic Pathway By Utilizing Microbial Co-Cultures

Rosmarinic acid (RA) is an important natural product with demonstrated biological activities and nutraceutical values. The RA biosynthesis involves a complex convergent pathway: two parallel upstream modules are responsible for formation of pathway precursors, caffeic acid and salvianic acid A, respectively; one downstream module is dedicated to combining the two precursors for formation of rosmarinic acid. Current efforts for heterologous RA biosynthesis only resulted in sub-optimal production performance, as balancing the bioconversion strength of three separate pathway modules is highly challenging.

Here, we constructed microbial co-cultures composed of multiple metabolically engineered E. coli strains for improving RA biosynthesis. In the two-member co-culture design, two E. coli strains were employed to accommodate the RA pathway. The first strain harbored one upstream module, and the second strain harbored the other upstream module and the downstream module. Co-cultivation of these two strains led to the desired RA production. Moreover, the inoculation ratio of the engineered strains was varied to optimize the relative population size of the co-culture strains and thus coordinate the biosynthetic capabilities of the corresponding pathway modules for pathway balancing. We showed that, compared with the production by the mono-culture of a single strain harboring the entire pathway, this strategy greatly improved the RA biosynthesis.

In the three-member co-culture design, the RA pathway modules were accommodated in three specialized E. coli strains, respectively, for more flexible pathway regulation. This allowed for direct coordination of the biosynthetic efforts between three E. coli strains through manipulating the strain-to-strain ratio in the co-culture population and thus facilitated straightforward balancing of the designated modules. As a result, the achieved RA biosynthesis was considerably higher than that of the two-member co-cultures. The findings of this work demonstrate the strong potentials of modular co-culture engineering for addressing the challenges of engineering complex biosynthetic pathways for microbial biosynthesis.