Production of ribosomally synthesized and posttranslationally modified peptides (RiPPs) has rarely been reported in fungi, even though organisms of this kingdom have a long history as a prolific source of natural products. Here we report an investigation of the phomopsins, antimitotic mycotoxins. We show that phomopsin is a fungal RiPP and demonstrate the widespread presence of a pathway for the biosynthesis of a family of fungal cyclic RiPPs, which we term dikaritins. We characterize PhomM as an S-adenosylmethioninedependent α-N-methyltransferase that converts phomopsin A to an N,N-dimethylated congener (phomopsin E), and show that the methyltransferases involved in dikaritin biosynthesis have evolved differently and likely have broad substrate specificities. Genome mining studies identified eight previously unknown dikaritins in different strains, highlighting the untapped capacity of RiPP biosynthesis in fungi and setting the stage for investigating the biological activities and unknown biosynthetic transformations of this family of fungal natural products.RiPP | mycotoxin | posttranslational modification | methyltransferase | biosynthesis

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Biocatalytic cascade reactions have become increasingly important and useful for chemical synthesis. However, biocatalysts are often incompatible with organic solvents, which prohibits many cascade reactions involving nonpolar substrates. In this study, we used cell-free protein synthesis (CFPS) to express enzymes in an aqueousorganic biphasic system for the construction of an artificial enzymatic pathway. CFPS-expressed enzymes without purification performed efficiently to convert styrene (below 20 mM) to (S)-1-phenyl-1,2-ethanediol (two steps in one pot) with 100% conversion. In addition, our CFPS system showed great tolerance to different organic solvents, and, importantly, the entire biocatalytic system can be consistently scaled up without a reduction of the substrate conversion rate. We, therefore, anticipate that our cell-free approach will make a possible cost-effective, high-yielding synthesis of valuable chemicals.

Cell-free systems have been used to synthesize chemicals by reconstitution of in vitro expressed enzymes. However, coexpression of multiple enzymes to reconstitute long enzymatic pathways is often problematic due to resource limitation/competition (e.g., energy) in the one-pot cell-free reactions. To address this limitation, here we aim to design a modular, cell-free platform to construct long biosynthetic pathways for tunable synthesis of value-added aromatic compounds, using (S)-1-phenyl-1,2-ethanediol ((S)-PED) and 2-phenylethanol (2-PE) as models. Initially, all enzymes involved in the biosynthetic pathways were individually expressed by an E. coli-based cell-free protein synthesis (CFPS) system and their catalytic activities were confirmed. Then, three sets of enzymes were coexpressed in three cell-free modules and each with the ability to complete a partial pathway. Finally, the full biosynthetic pathways were reconstituted by mixing two related modules to synthesize (S)-PED and 2-PE, respectively. After optimization, the final conversion rates for (S)-PED and 2-PE reached 100 and 82.5%, respectively, based on the starting substrate of l-phenylalanine. We anticipate that the modular cell-free approach will make a possible efficient and high-yielding biosynthesis of value-added chemicals.