Rational Design of Flavonoid Production Routes Using Combinatorial and Precursor-Directed Biosynthesis

Abstract

Combinatorial biosynthesis has great potential for designing synthetic circuits and amplifying the production of new active compounds. Studies on multienzyme cascades are extremely useful for improving our knowledge on enzymatic catalysis. In particular, the elucidation of enzyme substrate promiscuity can be potentially used for bioretrosynthetic 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 multigene expression system that leads to precursor-directed biosynthesis of 21 flavonoid derivatives. When the vast knowledge from protein databases is exploited, 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.

GenoChemetic Strategy for Derivatization of the Violacein Natural Product Scaffold

“…To this end, we adapted a two-step protocol reported for the synthesis of violacein analogues. 23 The condensation of the 3-acylated intermediate (characterized with 1 H and 13 C NMR in Figure S4) with 5-bromo-isatin proved challenging due to the formation of a mixture of products. However, the desired product 6 (characterized with 1 H NMR in Figure S5) was obtained in 15% yield after two difficult chromatographic purification steps, further highlighting the benefits of the engineered biosynthesis.…”

“…A similar rationally designed precursor-directed biosynthetic strategy has also showed considerable success in generating analogues of flavonoids. 13 Other examples of using altered biosynthetic pathways for analogue generation include the use of various enzyme homologues from bisindole pathways, 14 , 15 interconversion of the biosynthetically related novobiocin and clorobiocin aminocoumarin antibiotics, 16 introduction of the halogenase from the hormaomycin pathway to generate clorobiocin analogues, 17 and generation of chlorinated monoterpene indole alkaloids. 18 A prominent strategy in generating natural product analogues is GenoChemetics, where reactive halogen handles are installed via engineered biosynthetic pathways on natural products, which are subsequently derivatized via living-culture-optimized cross-coupling reactions, resulting in analogues of the antibiotic pacidamycin.…”

“…A study on oxyviolacein, a hydroxylated analogue of violacein generated by feeding exogenous 5-hydroxy- l -tryptophan, showed bioactivity against a collection of pathogenic bacteria and fungi strains, suggesting that violacein analogues may be a good starting point for developing more potent antibiotics. A similar rationally designed precursor-directed biosynthetic strategy has also showed considerable success in generating analogues of flavonoids . Other examples of using altered biosynthetic pathways for analogue generation include the use of various enzyme homologues from bisindole pathways, , interconversion of the biosynthetically related novobiocin and clorobiocin aminocoumarin antibiotics, introduction of the halogenase from the hormaomycin pathway to generate clorobiocin analogues, and generation of chlorinated monoterpene indole alkaloids .…”

PCPD: Plant cytochrome P450 database and web-based tools for structural construction and ligand docking

“…Collecting all functionally characterized P450s not only helps to predict the catalytic function of other P450s based on sequence similarity but also facilitates the design of new pathways to biosynthesize natural and non-natural products by synthetic biology [ [19] , [20] , [21] ]. Totally 181 plant P450s with known functions were collected from the KEGG database, the BRENDA database, and published literatures ( Fig.…”

Exploiting Spermidine N-Hydroxycinnamoyltransferase Diversity and Substrate Promiscuity to Produce Various Trihydroxycinnamoyl Spermidines and Analogues in Engineered Yeast

“…In addition to dihydrohydroxycinnamic acids, the 4CLs were also demonstrated to catalyze the synthesis of CoA esters form various analogues and unnatural hydroxycinnamic acids. ,, Therefore, less common hydroxycinnamic acids were assessed to feed 4CL/SHT recombinant yeasts. Among acyl donors tested, o -coumaric and m -coumaric acids led to N 1 , N 5 , N 10 -tri- o -coumaroyl spermidine and N 1 , N 5 , N 10 -tri- m -coumaroyl spermidine synthesis respectively in yeast harboring At4CL2 and NtSHT, and 4-methylcinnamic acid led to N 1 , N 5 , N 10 -tri-4-methylcinnamoyl spermidine synthesis in yeast harboring At4CL5 and MdSHT (Table ), whereas 4-nitrocinnamic acid was not incorporated in THCSpd.…”