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Rational design of flavonoid production routes using combinatorial and precursor-directed biosynthesis.

Combinatorial biosynthesis has great potential for designing synthetic circuits and amplifying the production of new active compounds. Studies on multi-enzyme cascades are extremely useful for improving our knowledge on enzymatic catalysis. In particular, the elucidation of enzyme substrate promiscuity can be potentially used for bio-retrosynthetic approaches, leading to the design of alternative and more convenient routes to produce relevant molecules. In this perspective, plant-derived polyketides are extremely adaptable to those synthetic biological applications. Here, we present a combination of an in vitro CoA ligase activity assay coupled with a bacterial multi-gene expression system that leads to precursor-directed biosynthesis of 21 flavonoid derivatives. By exploiting the large knowledge from protein databases, the herein presented procedure can be easily repeated with additional plant-derived polyketides. Lastly, we report an efficient in vivo expression system that can be further exploited to heterologously express pathways not necessarily related to plant polyketide synthases.