Anthocyanins are ubiquitous plant pigments used in a variety of technological applications. Yet, after over a century of research, the penultimate biosynthetic step to anthocyanidins attributed to the action of leucoanthocyanidin dioxygenase has never been efficiently reconstituted outside plants, preventing the construction of heterologous cell factories. Through biochemical and structural analysis, here we show that anthocyanin-related glutathione transferases, currently implicated only in anthocyanin transport, catalyse an essential dehydration of the leucoanthocyanidin dioxygenase product, flavan-3,3,4-triol, to generate cyanidin. Building on this knowledge, introduction of anthocyanin-related glutathione transferases into a heterologous biosynthetic pathway in baker’s yeast results in >35-fold increased anthocyanin production. In addition to unravelling the long-elusive anthocyanin biosynthesis, our findings pave the way for the colourants’ heterologous microbial production and could impact the breeding of industrial and ornamental plants. [Figure not available: see fulltext.]

Enzymatic late-stage diversification of small molecules has the potential to rapidly generate diversity in compound libraries dedicated to drug discovery. In this context, freestanding Fe(II)/α-ketoglutarate-dependent halogenases have raised particular interest as this enzyme family allows the otherwise difficult regio- and stereoselective halogenation of unactivated C(sp³)−H bonds. Here, we report the development of two engineered variants of the halogenase WelO5* for the racemic resolution of a mixture of stereoisomers generated in the synthesis of a bioactive martinelline-derived fragment. By screening a 3-site combinatorial variant library, we could identify two variants exhibiting exquisite substrate selectivity towards the desired enantiomers. Strikingly, the inversion of substrate stereopreference between the halogenase variants was achieved by varying only three residues in the active site. Protein crystallization and subsequent structure elucidation of the wildtype enzyme and a WelO5* variant shed light on the factors governing substrate acceptance and selectivity.

Squalene–hopene cyclases (SHCs) have great potential for the industrial synthesis of enantiopure cyclic terpenoids. A limitation of SHC catalysis has been the enzymes’ strict (S)-enantioselectivity at the stereocenter formed after the first cyclization step. To gain enantio-complementary access to valuable monocyclic terpenoids, an SHC-wild-type library including 18 novel homologs was set up. A previously not described SHC (AciSHC) was found to synthesize small amounts of monocyclic (R)-γ-dihydroionone from (E/Z)-geranylacetone. Using enzyme and process optimization, the conversion to the desired product was increased to 79 %. Notably, analyzed AciSHC variants could finely differentiate between the geometric geranylacetone isomers: While the (Z)-isomer yielded the desired monocyclic (R)-γ-dihydroionone (>99 % ee), the (E)-isomer was converted to the (S,S)-bicyclic ether (>95 % ee). Applying the knowledge gained from the observed stereodivergent and enantioselective transformations to an additional SHC-substrate pair, access to the complementary (S)-γ-dihydroionone (>99.9 % ee) could be obtained.