Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis

Enzymes with high activity are readily produced through protein engineering, but intentionally and efficiently engineering enzymes for an expanded scope is a contemporary challenge. Measuring reaction outcomes on mixtures of substrates, called here SUbstrate Multiplexed Screening (SUMS), has long been used to rigorously quantitate enzyme specificity. Despite the potential utility of SUMS to guide engineering of promiscuous enzymes, this approach has not found widespread adoption in biocatalysis. Here, we develop principles of how to design robust SUMS methods that, rather than assess absolute specificity, use heuristic readouts of substrate promiscuity to identify hits for further investigation. This rich information enables engineering of activity for multiple substrates simultaneously and identifies enzyme variants with altered promiscuity, even when overall activity is lower. We demonstrate the effectiveness of SUMS by engineering two enzymes to produce pharmacologically active tryptamines from simple indole precursors in a biocatalytic cascade. These advances leverage common laboratory equipment and represent a highly accessible and customizable method for enzyme engineering.

Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis

Enzymes with high activity are readily produced through protein engineering, but intentionally and efficiently engineering enzymes for an expanded scope is a contemporary challenge. Measuring reaction outcomes on mixtures of substrates, called here SUbstrate Multiplexed Screening (SUMS), has long been used to rigorously quantitate enzyme specificity. Despite the potential utility of SUMS to guide engineering of promiscuous enzymes, this approach has not found widespread adoption in biocatalysis. Here, we develop principles of how to design robust SUMS methods that, rather than assess absolute specificity, use heuristic readouts of substrate promiscuity to identify hits for further investigation. This rich information enables engineering of activity for multiple substrates simultaneously and identifies enzyme variants with altered promiscuity, even when overall activity is lower. We demonstrate the effectiveness of SUMS by engineering two enzymes to produce pharmacologically active tryptamines from simple indole precursors in a biocatalytic cascade. These advances leverage common laboratory equipment and represent a highly accessible and customizable method for enzyme engineering.

Structural and Data Science-Driven Analysis to Assess Substrate Specificity of Diketopiperazine Reverse Prenyltransferase NotF: Cascade Biocatalytic Synthesis of (–)-Eurotiumin A

Prenyltransfer is an early-stage carbon–hydrogen bond (C–H) functionalization prevalent in the biosynthesis of a diverse array of biologically active bacterial, fungal, plant, and metazoan diketopiperazine (DKP) alkaloids. Towards the development of a unified strategy for biocatalytic construction of prenylated DKP indole alkaloids, we sought to identify and characterize a substrate-permissive C2 reverse prenyltransferase (PT). In the biosynthesis of cytotoxic notoamide metabolites, PT NotF is responsible for catalyzing the first tailoring event of C2 reverse prenyltransfer of brevianamide F (cyclo(L-Trp-L-Pro)). Obtaining a high-resolution crystal structure of NotF (in complex with native substrate and prenyl donor mimic dimethylallyl S-thiolodiphosphate (DMSPP)) revealed a large, solvent exposed substrate binding site, intimating NotF may possess significant substrate promiscuity. To assess the full potential of NotF’s broad substrate selectivity, we synthesized a panel of 30 tryptophanyl DKPs with a suite of sterically and electronically differentiated amino acids, which were selectively prenylated by NotF in often synthetically useful conversions (2 to >99%). Quantitative representation of this substrate library enabled the development of a descriptive statistical model that provided insight into the origins of NotF’s substrate promiscuity. Through this unique approach for understanding enzyme scope, we identified key substrate descriptors such as electrophilicity, size, and flexibility, that govern enzymatic turnover by NotF. Additionally, we demonstrated the ability to couple NotF-catalyzed prenyltransfer with oxidative cyclization using recently characterized flavin monooxygenase, BvnB, from the brevianamide biosynthetic pathway. This one-pot, in vitro biocatalytic cascade proceeds with exceptional substrate recognition, and enabled the first chemoenzymatic synthesis of the marine fungal natural product, (–)-eurotiumin A, in three steps and 60% overall yield.

Amorpha-4,11-diene synthase: a key enzyme in artemisinin biosynthesis and engineering

Amorpha-4,11-diene synthase (ADS) catalyzes the first committed step in the artemisinin biosynthetic pathway, which is the first catalytic reaction enzymatically and genetically characterized in artemisinin biosynthesis. The advent of ADS in Artemisia annua is considered crucial for the emergence of the specialized artemisinin biosynthetic pathway in the species. Microbial production of amorpha-4,11-diene is a breakthrough in metabolic engineering and synthetic biology. Recently, numerous new techniques have been used in ADS engineering; for example, assessing the substrate promiscuity of ADS to chemoenzymatically produce artemisinin. In this review, we discuss the discovery and catalytic mechanism of ADS, its application in metabolic engineering and synthetic biology, as well as the role of sesquiterpene synthases in the evolutionary origin of artemisinin.