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.

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.

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.

Chemoenzymatic synthesis of non-natural terpenes using the promiscuous activity of terpene synthases allows for the expansion of the chemical space of terpenoids with potentially new bioactivities. In this report, we describe protocols for the preparation of a novel aphid attractant, (S)-14,15-dimethylgermacrene D, by exploiting the promiscuity of (S)-germacrene D synthase from Solidago canadensis and using an engineered biocatalytic route to convert prenols to terpenoids. The method uses a combination of five enzymes to carry out the preparation of terpenoid semiochemicals in two steps: (1) diphosphorylation of five or six carbon precursors (prenol, isoprenol and methyl-isoprenol) catalyzed by Plasmodium falciparum choline kinase and Methanocaldococcus jannaschii isopentenyl phosphate kinase to form DMADP, IDP and methyl-IDP, and (2) chain elongation and cyclization catalyzed by Geobacillus stearothermophilus (2E,6E)-farnesyl diphosphate synthase and S. canadensis (S)-germacrene D synthase to produce (S)-germacrene D and (S)-14,15-dimethylgermacrene D. Using this method, new non-natural terpenoids are readily accessible and the approach can be adopted to produce different terpene analogs and terpenoid derivatives with potential novel applications.

The step catalyzed by terpene synthases is a well-recognized and significant bottleneck in engineered terpenoid bioproduction. Consequently, substantial efforts have been devoted towards increasing metabolic flux catalyzed by terpene synthases, employing strategies such as gene overexpression and protein engineering. Notably, numerous studies have demonstrated remarkable titer improvements by applying translational fusion, typically by fusing the terpene synthase with a prenyl diphosphate synthase that catalyzes the preceding step in the pathway. The main appeal of the translational fusion approach lies in its simplicity and orthogonality to other metabolic engineering tools. However, there is currently limited understanding of the underlying mechanism of flux enhancement, owing to the unpredictable and often protein-specific effects of translational fusion. In this chapter, we discuss practical considerations when engineering translationally fused terpene synthases, drawing insights from our experience and existing literature. We also provide detailed experimental workflows and protocols based on our previous work in budding yeast (Saccharomyces cerevisiae). Our intention is to encourage further research into the translational fusion of terpene synthases, anticipating that this will contribute mechanistic insights not only into the activity, behavior, and regulation of terpene synthases, but also of other enzymes.

CONCLUSIONS: CaTPS2 and CaTPS3 were significantly expressed in flowers of Curcuma alismatifolia \'Shadow\' and demonstrated bifunctional enzyme activity, CaTPS2 generated linalool and nerolidol as products, and CaTPS3 catalyzed β-myrcene and β-farnesene formation. This study presents the discovery and functional characterization of floral terpene synthase (TPS) genes in Curcuma alismatifolia \'Shadow\', a cultivar renowned for its unique fragrance. Addressing the gap in understanding the genetic basis of floral scent in this species, we identified eight TPS genes through comprehensive transcriptome sequencing. Among these, CaTPS2 and CaTPS3 were significantly expressed in floral tissues and demonstrated bifunctional enzyme activity corresponding to the major volatile compounds detected in \'Shadow\'. Functional analyses, including in vitro assays complemented with rigorous controls and alternative identification methods, elucidated the roles of these TPS genes in terpenoid biosynthesis. In vitro studies were conducted via heterologous expression in E. coli, followed by purification of the recombinant protein using affinity chromatography, enzyme assays were performed with GPP/FPP as the substrate, and volatile products were inserted into the GC-MS for analysis. Partially purified recombinant protein of CaTPS2 catalyzed GPP and FPP to produce linalool and nerolidol, respectively, while partially purified recombinant protein of CaTPS3 generated β-myrcene and β-farnesene with GPP and FPP as substrates, respectively. Real-time quantitative PCR further validated the expression patterns of these genes, correlating with terpenoid accumulation in different plant tissues. Our findings illuminate the molecular mechanisms underpinning floral fragrance in C. alismatifolia and provide a foundation for future genetic enhancements of floral scent in ornamental plants. This study, therefore, contributes to the broader understanding of terpenoid biosynthesis in plant fragrances, paving the way for biotechnological applications in horticulture plant breeding.

Photosynthetic microalgae like Nannochloropsis hold enormous potential as sustainable, light-driven biofactories for the production of high-value natural products such as terpenoids. Nannochloropsis oceanica is distinguished as a particularly robust host with extensive genomic and transgenic resources available. Its capacity to grow in wastewater, brackish, and sea waters, coupled with advances in microalgal metabolic engineering, genome editing, and synthetic biology, provides an excellent opportunity. In the present work, we demonstrate how N. oceanica can be engineered to produce the diterpene casbene-an important intermediate in the biosynthesis of pharmacologically relevant macrocyclic diterpenoids. Casbene accumulated after stably expressing and targeting the casbene synthase from Daphne genkwa (DgTPS1) to the algal chloroplast. The engineered strains yielded production titers of up to 0.12 mg g-1 total dry cell weight (DCW) casbene. Heterologous overexpression and chloroplast targeting of two upstream rate-limiting enzymes in the 2-C-methyl- d-erythritol 4-phosphate pathway, Coleus forskohlii 1-deoxy- d-xylulose-5-phosphate synthase and geranylgeranyl diphosphate synthase genes, further enhanced the yield of casbene to a titer up to 1.80 mg g-1 DCW. The results presented here form a basis for further development and production of complex plant diterpenoids in microalgae.

Studying the independent evolution of similar traits provides valuable insights into the ecological and genetic factors driving phenotypic evolution.1 The transition from outcrossing to self-fertilization is common in plant evolution2 and is often associated with a reduction in floral attractive features such as display size, chemical signals, and pollinator rewards.3 These changes are believed to result from the reallocation of the resources used for building attractive flowers, as the need to attract pollinators decreases.2,3 We investigated the similarities in the evolution of flower fragrance following independent transitions to self-fertilization in Capsella.4,5,6,7,8,9 We identified several compounds that exhibited similar changes in different selfer lineages, such that the flower scent composition reflects mating systems rather than evolutionary history within this genus. We further demonstrate that the repeated loss of β-ocimene emission, one of the compounds most strongly affected by these transitions, was caused by mutations in different genes. In one of the Capsella selfing lineages, the loss of its emission was associated with a mutation altering subcellular localization of the ortholog of TERPENE SYNTHASE 2. This mutation appears to have been fixed early after the transition to selfing through the capture of variants segregating in the ancestral outcrossing population. The large extent of convergence in the independent evolution of flower scent, together with the evolutionary history and molecular consequences of a causal mutation, suggests that the emission of specific volatiles evolved as a response to changes in ecological pressures rather than resource limitation.

Terpenoids are one of the largest class of natural products with diverse structures and activities. This enormous diversity is embedded in enzymes called terpene synthases (TSs), which generate diverse terpene skeletons via sophisticated cyclization cascades. In addition to the many highly selective TSs, there are many promiscuous TSs that accept multiple prenyl substrates, or even noncanonical ones, with 6, 7, 8, 11, and 16 carbon atoms, synthesized via chemical approaches, C-methyltransferases, or engineered lepidopteran mevalonate pathways. The substrate promiscuity of TSs not only expands the structural diversity of terpenes but also highlights their potential for the discovery of novel terpenoids via combinatorial biosynthesis. In this review, we focus on the current knowledge on multisubstrate terpene synthases (MSTSs) and highlight their potential applications.

We report the complete telomere-to-telomere genome assembly of Oldenlandia diffusa which renowned in traditional Chinese medicine, comprising 16 chromosomes and spanning 499.7 Mb. The assembly showcases 28 telomeres and minimal gaps, with a total of only five. Repeat sequences constitute 46.41% of the genome, and 49,701 potential protein-coding genes have been predicted. Compared with O. corymbosa, O. diffusa exhibits chromosome duplication and fusion events, diverging 20.34 million years ago. Additionally, a total of 11 clusters of terpene synthase have been identified. The comprehensive genome sequence, gene catalog, and terpene synthase clusters of O. diffusa detailed in this study will significantly contribute to advancing research in this species\' genetic, genomic, and pharmacological aspects.