Prenyltransferases are terpene synthases that combine 5-carbon precursor molecules into linear isoprenoids of varying length that serve as substrates for terpene cyclases, enzymes that catalyze fascinating cyclization reactions to form diverse terpene natural products. Terpenes and their derivatives comprise the largest class of natural products and have myriad functions in nature and diverse commercial uses. An emerging class of bifunctional terpene synthases contains both prenyltransferase and cyclase domains connected by a disordered linker in a single polypeptide chain. Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is one of the most well-characterized members of this subclass and serves as a model system for the exploration of structure-function relationships. PaFS has been structurally characterized using a variety of biophysical techniques. The enzyme oligomerizes to form a stable core of six or eight prenyltransferase domains that produce a 20-carbon linear isoprenoid, geranylgeranyl diphosphate (GGPP), which then transits to the cyclase domains for the generation of fusicoccadiene. Cyclase domains are in dynamic equilibrium between randomly splayed-out and prenyltransferase-associated positions; cluster channeling is implicated for GGPP transit from the prenyltransferase core to the cyclase domains. In this chapter, we outline the methods we are developing to interrogate the nature of cluster channeling in PaFS, including enzyme activity and product analysis assays, approaches for engineering the linker segment connecting the prenyltransferase and cyclase domains, and structural analysis by cryo-EM.

Structural biology research of terpene synthases (TSs) has provided a useful basis to understand their catalytic mechanisms in producing diverse terpene products with polycyclic ring systems and multiple chiral centers. However, compared to the large numbers of>95,000 terpenoids discovered to date, few structures of TSs have been solved and the understanding of their catalytic mechanisms is lagging. We here (i) introduce the basic catalytic logic, the structural architectures, and the metal-binding conserved motifs of TSs; (ii) provide detailed experimental procedures, in gene cloning and plasmid construction, protein purification, crystallization, X-ray diffraction data collection and structural elucidation, for structural biology research of TSs; and (iii) discuss the prospects of structure-based engineering and de novo design of TSs in generating valuable terpene molecules, which cannot be easily achieved by chemical synthesis.

Large terpene synthases (large-TSs) are a new family of TSs. The first large-TS discovered was from Bacillus subtilis (BsuTS), which is involved in the biosynthesis of a C35 sesquarterpene. Large-TSs are the only enzymes that enable the biosynthesis of sesquarterpenes and do not share any sequence homology with canonical Class I and II TSs. Thus, the investigation of large-TSs is promising for expanding the chemical space in the terpene field. In this chapter, we describe the experimental methods used for identifying large-TSs, as well as their functional and structural analyses. Additionally, several enzymes related to the biosynthesis of large-TS substrates have been described.

Vanadium-dependent haloperoxidases (VHPOs) are a unique family of enzymes that utilize vanadate, an aqueous halide ion, and hydrogen peroxide to produce an electrophilic halogen species that can be incorporated into electron rich organic substrates. This halogen species can react with terpene substrates and trigger halonium-induced cyclization in a manner reminiscent of class II terpene synthases. While not all VHPOs act in this capacity, several notable examples from algal and actinobacterial species have been characterized to catalyze regio- and enantioselective reactions on terpene and meroterpenoid substrates, resulting in complex halogenated cyclic terpenes through the action of single enzyme. In this article, we describe the expression, purification, and chemical assays of NapH4, a difficult to express characterized VHPO that catalyzes the chloronium-induced cyclization of its meroterpenoid substrate.

Pyr4-family terpene cyclases are noncanonical transmembrane class II terpene cyclases that catalyze a variety of cyclization reactions in the biosynthesis of microbial terpenoids, such as meroterpenoids. However, although these cyclases are widely distributed in microorganisms, their three-dimensional structures have not been determined, possibly due to the transmembrane locations of these enzymes. In this chapter, we describe procedures for the functional analysis of transmembrane terpene cyclases based on their model structures generated using AlphaFold2. We used AdrI, the Pyr4-family terpene cyclase required for the biosynthesis of andrastin A and its homologs, as an example.

Expression and purification of membrane-bound proteins remains a challenge and limits enzymology efforts, contributing to a substantial knowledge gap in the biochemical functions of many proteins found in nature. Accordingly, the study of bacterial UbiA terpene synthases (TSs) has been limited due to the experimental hurdles required to purify active enzymes for characterization in vitro. Previous work employed the use of microsomes or crude membrane fractions to test enzyme activity; however, these methods can be labor intensive, require access to an ultracentrifuge, or may not be suitable for all membrane-bound TSs. We detail here an alternative strategy for the in vivo expression and biochemical characterization of the membrane associated UbiA TSs by employing a precursor overproduction system in Escherichia coli.

Coral terpenes are important molecules with numerous applications. Here, we describe a robust and simple method to produce coral terpene scaffolds at scale. As an example of the approach, here we discover, express, and characterize further klysimplexin R synthases, expanding the known enzymology of soft coral terpene cyclases. We hope that the underlying method described will enable widespread basic research into the functions of coral terpenes and their biosynthetic genes, as well as the commercial development of biomedically and technologically important molecules.

Octocorals are the most prolific source of terpenoids in the marine environment, with more than 4000 different compounds known from the phylum to date. However, the biochemical and genetic origin of their production remained elusive until recent studies showed that octocorals encode genes responsible for the biosynthesis of terpenoids in their own chromosomal DNA rather than from microbial symbionts as originally proposed. The identified coral genes include those encoding a new group of class I terpene cyclases (TCs) clustered among other candidate classes of tailoring enzymes. Phylogenetic analyses established octocoral TCs as a monophyletic clade, distinct from TCs of plants, bacteria, and other organisms. The newly discovered group of TCs appears to be ubiquitous in octocorals and is evolutionarily ancient. Given the recent discovery of octocoral terpenoid biochemistry and only limited genomic data presently available, there is substantial potential for discovering new biosynthetic pathways from octocorals for terpene production. The following chapter outlines practical experimental procedures for octocoral DNA and RNA extraction, genome and transcriptome assembly and mining, TC cloning and gene expression, protein purification, and in vitro analyses.

Terpenes constitute one of the largest family of natural products with potent applications as renewable platform chemicals and medicines. The low activity, selectivity and stability displayed by terpene biosynthetic machineries can constitute an obstacle towards achieving expedient biosynthesis of terpenoids in processes that adhere to the 12 principles of green chemistry. Accordingly, engineering of terpene synthase enzymes is a prerequisite for industrial biotechnology applications, but obstructed by their complex catalysis that depend on reactive carbocationic intermediates that are prone to undergo bifurcation mechanisms. Rational redesign of terpene synthases can be tedious and requires high-resolution structural information, which is not always available. Furthermore, it has proven difficult to link sequence space of terpene synthase enzymes to specific product profiles. Herein, the author shows how ancestral sequence reconstruction (ASR) can favorably be used as a protein engineering tool in the redesign of terpene synthases without the need of a structure, and without excessive screening. A detailed workflow of ASR is presented along with associated limitations, with a focus on applying this methodology on terpene synthases. From selected examples of both class I and II enzymes, the author advocates that ancestral terpene cyclases constitute valuable assets to shed light on terpene-synthase catalysis and in enabling accelerated biosynthesis.

Plants are prolific producers of terpenoids. Terpenoid biosynthesis is initiated by terpene synthases (TPS). In plants, two types of terpenes synthase genes are recognized: typical plant TPS genes and microbial-terpene synthase like-genes (MTPSL). While TPS genes are ubiquitous in land plants, MTPSL genes appear to be restricted to non-seed land plants. Evolutionarily, TPS genes are specific to land plants, whereas MTPSL genes have related counterparts in other organisms, especially fungi and bacteria. The presence of microbial type TPS in plants, fungi and bacteria, with the latter two often being associated with plants, poses a challenge in accurately identifying bona fide MTPSL genes in plants. In this chapter, we present bioinformatic procedures designed to identify MTPSL genes in sequenced plant genomes and/or transcriptomes. Additionally, we outline validation methods for confirming the identified microbial-type TPS genes as genuine plant genes. The method described in this chapter can also be adopted to analyze microbial type TPS in organisms other than plants.