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A versatile terpene synthase (LcTPS2) producing unconventional macrocyclic terpenoids was characterized from Leucosceptrum canum. Engineered E. coli and Nicotiana benthamiana expressing LcTPS2 produced six 18-/14-membered sesterterpenoids including five new ones and two 14-membered diterpenoids, representing the first macrocyclic sesterterpenoids from plants and the largest sesterterpenoid ring system identified to date. Surprisingly, none of these macrocyclic terpenoids were detected in L. canum. Two variants F516A and F516G producing approximately 3.3- and 2.5- folds respectively more sesterterpenoids than the wild type enzyme was engineered. Both 18- and 14-membered ring sesterterpenoids displayed significant inhibitory activity on the IL-2 and IFN-I³ production of T cells via inhibition of MAPK pathway. The findings will contribute to the development of efficient biocatalysts to create bioactive macrocyclic sesterterpenoids, and also herald a new potential in the well-trodden territory of plant terpenoid biosynthesis.

Here we report the one-pot, cell-free enzymatic synthesis of the plant monoterpene nepetalactol starting from the readily available geraniol. A pair of orthogonal cofactor regeneration systems permitted NAD(+)-dependent geraniol oxidation followed by NADPH-dependent reductive cyclization without isolation of intermediates. The orthogonal cofactor regeneration system maintained a high ratio of NAD(+) to NADH and a low ratio of NADP(+) to NADPH. The overall reaction contains four biosynthetic enzymes, including a soluble P450; and five accessory and cofactor regeneration enzymes. Furthermore, addition of a NAD(+)-dependent dehydrogenase to the one-pot mixture led to ~1 g/L of nepetalactone, the active cat- attractant in catnip.

Class I terpene synthase (TPS) generates bioactive terpenoids with diverse backbones. Sesterterpene synthase (sester-TPS, C25), a branch of class I TPSs, was recently identified in Brassicaceae. However, the catalytic mechanisms of sester-TPSs are not fully understood. Here, we first identified three nonclustered functional sester-TPSs (AtTPS06, AtTPS22, and AtTPS29) in Arabidopsis thaliana. AtTPS06 utilizes a type-B cyclization mechanism, whereas most other sester-TPSs produce various sesterterpene backbones via a type-A cyclization mechanism. We then determined the crystal structure of the AtTPS18–FSPP complex to explore the cyclization mechanism of plant sester-TPSs. We used structural comparisons and site-directed mutagenesis to further elucidate the mechanism: (1) mainly due to the outward shift of helix G, plant sester-TPSs have a larger catalytic pocket than do mono-, sesqui-, and di-TPSs to accommodate GFPP; (2) type-A sester-TPSs have more aromatic residues (five or six) in their catalytic pocket than classic TPSs (two or three), which also determines whether the type-A or type-B cyclization mechanism is active; and (3) the other residues responsible for product fidelity are determined by interconversion of AtTPS18 and its close homologs. Altogether, this study improves our understanding of the catalytic mechanism of plant sester-TPS, which ultimately enables the rational engineering of sesterterpenoids for future applications., Sesterterpenoids, a relatively new group of terpenoids, are structurally diverse and widely distributed in bacteria, fungi, and the plant kingdom. However, the enzymatic mechanism of sesterterpene synthase (sester-TPS), the first committed biosynthesis step, remains obscure. This study reveals the critical amino acids controlling the substrate and product specificity of plant sester-TPS by determining the first crystal structure of the sester-TPS–FSPP complex.

Catalyzing stereo- and regio-specific oxidation of inert hydrocarbon backbones, and a range of more exotic reactions inherently difficult in formal chemical synthesis, cytochromes P450 (P450s) offer outstanding potential for biotechnological engineering. Plants and their dazzling diversity of specialized metabolites have emerged as rich repository for functional P450s with the advances of deep transcriptomics and genome wide discovery. P450s are of outstanding interest for understanding chemical diversification throughout evolution, for gaining mechanistic insights through the study of their structure–function relationship, and for exploitation in Synthetic Biology. In this review, we highlight recent developments and examples in the discovery of plant P450s involved in the biosynthesis of industrially relevant monoterpenoids, sesquiterpenoids, diterpenoids and triterpenoids, throughout 2016 and early 2017. Examples were selected to illustrate the spectrum of value from commodity chemicals, flavor and fragrance compounds to pharmacologically active terpenoids. We focus on a recently emerging theme, where P450s control metabolic bifurcations and chemical diversity of the final product profile, either within a pathway, or through neo-functionalization in related species. The implications may inform approaches for rational assembly of recombinant pathways, biotechnological production of high value terpenoids and generation of novel chemical entities. Graphical Abstract

Epistasis, the interaction between mutations and the genetic background, is a pervasive force in evolution that is difficult to predict yet derives from a simple principle − biological systems are interconnected. Therefore, one effect may be intimately linked to another, hence interdependent. Untangling epistatic interactions between and within genes is a vibrant area of research. Deriving a mechanistic understanding of epistasis is a major challenge. Particularly, elucidating how epistasis can attenuate the effects of otherwise dominant mutations that control phenotypes. Using the emergence of terpene cyclization in specialized metabolism as an excellent example, this review describes the process of discovery and interpretation of dominance and epistasis in relation to current efforts. Specifically, we outline experimental approaches to isolating epistatic networks of mutations in protein structure, formally quantifying epistatic interactions, then building biochemical models with chemical mechanisms in efforts to achieve an understanding of the physical basis for epistasis. From these models we describe informed conjectures about past evolutionary events that underlie the emergence, divergence and specialization of terpene synthases to illustrate key principles of the constraining forces of epistasis in enzyme function.

Research toward renewable and sustainable energy has identified specific terpenes capable of supplementing or replacing current petroleum-derived fuels. Despite being naturally produced and stored by many plants, there are few examples of commercial recovery of terpenes from plants because of low yields. Plant terpene biosynthesis is regulated at multiple levels, leading to wide variability in terpene content and chemistry. Advances in the plant molecular toolkit, including annotated genomes, high-throughput omics profiling, and genome editing, have begun to elucidate plant terpene metabolism, and such information is useful for bioengineering metabolic pathways for specific terpenes. We review here the status of terpenes as a specialty biofuel and discuss the potential of plants as a viable agronomic solution for future terpene-derived biofuels.

Wherein we learn that when working with plant terpene synthases, sometimes it is not the isotopes that get scrambled.

Bemisia tabaci, the whitefly vector of Tomato yellow leaf curl virus (TYLCV), seriously reduces tomato production and quality. Here, we report the first evidence that infection by TYLCV alters the host preferences of invasive B. tabaci B (Middle East-Minor Asia 1) and Q (Mediterranean genetic group), in which TYLCV-free B. tabaci Q preferred to settle on TYLCV-infected tomato plants over healthy ones. TYLCV-free B. tabaci B, however, preferred healthy tomato plants to TYLCV-infected plants. In contrast, TYLCV-infected B. tabaci, either B or Q, did not exhibit a preference between TYLCV-infected and TYLCV-free tomato plants. Based on gas chromatography-mass spectrometry (GCMS)analysis of plant terpene volatiles, significantly more β-myrcene, thymene, β-phellandrene, caryophyllene, (+)-4-carene, and α-humulene were released from the TYLCV-free tomato plants than from the TYLCV-infected ones. The results indicate TYLCV can alter the host preferences of its vector Bemisia tabaci B and Q.

Plants produce a diversity of volatile organic compounds that have important ecological functions. When emitted from plants upon herbivore damage, volatiles serve as direct defense compounds or attract natural enemies of herbivores, a response known as “indirect defense.” Terpene metabolites constitute a predominant group of plant volatiles with recognized function in plant defense. The possibility of developing alternative pest control strategies based on these natural defenses has fueled novel attempts to metabolically engineer volatile emissions and enhance their effectiveness in plant defense. We address recent advances in this area with an emphasis on homoterpene volatiles. We show that the Arabidopsis model serves as an important resource, which provides molecular and genetic tools to investigate and manipulate the biosynthesis of volatile terpenes, especially homoterpenes, and to decipher their roles in plant defense responses.

The distribution of eggs of the Scolytidae predator Medetera aldrichii Wheeler on oviposition grids arranged on a prey-infested tree, showed that selection of predator oviposition sites was influenced by proximity of the prey's gallery entrance and ventilation holes. Adult and larval response to commercial D-alpha-pinene in laboratory ol-factometric studies indicated that volatiles emanating from prey galleries at these sites release appetitive ovipositional behavior from the gravid female and guide the bark-surface orientation of the newly eclosed predator larva from oviposition sites toward prey gallery openings.