A single oxidosqualene cyclase produces the seco-triterpenoid α-onocerin

8,14-seco-Triterpenoids are characterized by their unusual open C-ring. Their distribution in nature is rare and scattered in taxonomically unrelated plants. The 8,14-seco-triterpenoid alpha-onocerin is only known from the evolutionarily distant clubmoss genus Lycopodium and the leguminous genus Ononis, which makes the biosynthesis of this seco-triterpenoid intriguing from an evolutionary standpoint. In our experiments with Ononis spinosa, alpha-onocerin was detected only in the roots. Through transcriptome analysis of the roots, an oxidosqualene cyclase, OsONS1, was identified that produces alpha-onocerin from squalene-2,3; 22,23-dioxide when transiently expressed in Nicotiana bethamiana. In contrast, in Lycopodium clavatum, two sequential cyclases, LcLCC and LcLCD, are required to produce alpha-onocerin in the N. benthamiana transient expression system. Expression of OsONS1 in the lanosterol synthase knockout yeast strain GIL77, which accumulates squalene-2,3; 22,23-dioxide, verified the alpha-onocerin production. A phylogenetic analysis predicts that OsONS1 branches off from specific lupeol synthases and does not group with the known L. clavatum alpha-onocerin cyclases. Both the biochemical and phylogenetic analyses of OsONS1 suggest convergent evolution of the alpha-onocerin pathways. When OsONS1 was coexpressed in N. benthamiana leaves with either of the two O. spinosa squalene epoxidases, OsSQE1 or OsSQE2, alpha-onocerin production was boosted, most likely because the epoxidases produce higher amounts of squalene-2,3; 22,23-dioxide. Fluorescence lifetime imaging microscopy analysis demonstrated specific protein-protein interactions between OsONS1 and both O. spinosa squalene epoxidases. Coexpression of OsONS1 with the two OsSQEs suggests that OsSQE2 is the preferred partner of OsONS1 in planta. Our results provide an example of the convergent evolution of plant specialized metabolism

Metabolic engineering of cucurbitacins in Cucurbita pepo hairy roots

In this paper we show that metabolic engineering in Cucurbita pepo hairy roots can be used to both effectively increase and modify cucurbitacins. Cucurbitacins are highly-oxygenated triterpenoids originally described in the Cucurbitaceae family, but have since been found in 15 taxonomically distant plant families. Cucurbitacin B, D, E and I are the most widespread amongst the Cucurbitaceae and they have both important biological and pharmacological activities. In this study C. pepo hairy roots were used as a platform to boost production and alter the structures of the afore mentioned cucurbitacins by metabolic engineering to potentially provide new or more desirable bioactivities. We report that the ability to induce cucurbitacin biosynthesis by basic Helix-Loop-Helix transcription factors is partially conserved within the Cucurbitaceae and therefore can potentially be used as a biotechnological tool to increase cucurbitacins in several genera of this family. Additionally, overexpression of a novel acyltransferase from cucurbitacin producing Iberis amara generates a hitherto undescribed acetylation at the C3-hydroxyl group of the cucurbitadienol backbone. While overexpression of the cytochromes P450 CsCYP88L2 and McCYP88L7 from Cucumis sativus and Momordica charantia (respectively), results in accumulation of new spectral feature as revealed by High resolution liquid chromatography mass spectroscopy analysis; the m/z of the new peak supports it might be a cucurbitacin hydroxylated at the C19 position in C. pepo hairy roots. Finally, this paper is a case study of how hairy roots can be used to metabolically engineer and introduce novel modifications in metabolic pathways that have not been fully elucidated

The seco-iridoid pathway from Catharanthus roseus

The (seco)iridoids and their derivatives, the monoterpenoid indole alkaloids (MIAs), form two large families of plant-derived bioactive compounds with a wide spectrum of high-value pharmacological and insect-repellent activities. Vinblastine and vincristine, MIAs used as anticancer drugs, are produced by Catharanthus roseus in extremely low levels, leading to high market prices and poor availability. Their biotechnological production is hampered by the fragmentary knowledge of their biosynthesis. Here we report the discovery of the last four missing steps of the (seco)iridoid biosynthesis pathway. Expression of the eight genes encoding this pathway, together with two genes boosting precursor formation and two downstream alkaloid biosynthesis genes, in an alternative plant host, allows the heterologous production of the complex MIA strictosidine. This confirms the functionality of all enzymes of the pathway and highlights their utility for synthetic biology programmes towards a sustainable biotechnological production of valuable (seco)iridoids and alkaloids with pharmaceutical and agricultural applications

Surface Thermodynamics, Viscosity, Activation Energy of N-Methyldiethanolamine Aqueous Solutions Promoted by Tetramethylammonium Arginate

The surface tension and viscosity values of N-methyldiethanolamine (MDEA) aqueous solutions promoted by tetramethylammonium arginate ([N1111][Arg]) were measured and modeled. The experimental temperatures were 303.2 to 323.2 K. The mass fractions of MDEA (wMDEA) and [N1111][Arg] (w[N1111][Arg]) were 0.300 to 0.500 and 0.025 to 0.075, respectively. The measured surface tension and viscosity values were satisfactorily fitted to thermodynamic models. With the aid of experimentally viscosity data, the activation energy (Ea) and H2S diffusion coefficient (DH2S) of MDEA-[N1111][Arg] aqueous solution were deduced. The surface entropy and surface enthalpy of the solutions were calculated using the fitted model of the surface tension. The quantitative relationship between the calculated values (surface tension, surface entropy, surface enthalpy, viscosity, activation energy, and H2S diffusion coefficient) and the operation conditions (mass fraction and temperature) was demonstrated

Plant triterpenoids with bond-missing skeletons: biogenesis, distribution and bioactivity

Covering: up to December 2018The polycyclic ABCD(E) framework of triterpenoids can miss a single endocyclic C-C bond as a result of a modification of the cyclization cascade that triggers their formation (interrupted- or diverted cascades), or can be the result of post-cyclization ring cleavage by late-stage oxidative modifications (seco-triterpenoids). Because of mechanistic and biogenetic differences, ring opening associated with loss of a skeletal fragment, as in nor-seco-triterpenoids (limonoids, quassinoids), will not be covered, nor will compounds where ring opening is part of a fragmentation cascade or of a multiple diversion from it. Even with these limitations, 342 bond-missing triterpenoids could be retrieved from the literature, with transversal distribution in the plant kingdom. Their structural diversity translates into a variety of biological targets, with dominance of potential applications in the realm of cancer, neuroprotection, and anti-infective therapy. In addition to the bioactivity and chemotaxonomic relevance of bond-missing triterpenoids, current knowledge on the genetic basis of interrupted- and diverted oxidosqualene cyclases will be summarized. This untapped source of enzymes could be useful to selectively modify triterpenoids by metabolic engineering, circumventing the bottlenecks of their isolation (poor yield or inadequate supply chain) to explore new areas of their chemical space

Plant triterpenoids with bond-missing skeletons:biogenesis, distribution and bioactivity

Covering: up to December 2018The polycyclic ABCD(E) framework of triterpenoids can miss a single endocyclic C-C bond as a result of a modification of the cyclization cascade that triggers their formation (interrupted- or diverted cascades), or can be the result of post-cyclization ring cleavage by late-stage oxidative modifications (seco-triterpenoids). Because of mechanistic and biogenetic differences, ring opening associated with loss of a skeletal fragment, as in nor-seco-triterpenoids (limonoids, quassinoids), will not be covered, nor will compounds where ring opening is part of a fragmentation cascade or of a multiple diversion from it. Even with these limitations, 342 bond-missing triterpenoids could be retrieved from the literature, with transversal distribution in the plant kingdom. Their structural diversity translates into a variety of biological targets, with dominance of potential applications in the realm of cancer, neuroprotection, and anti-infective therapy. In addition to the bioactivity and chemotaxonomic relevance of bond-missing triterpenoids, current knowledge on the genetic basis of interrupted- and diverted oxidosqualene cyclases will be summarized. This untapped source of enzymes could be useful to selectively modify triterpenoids by metabolic engineering, circumventing the bottlenecks of their isolation (poor yield or inadequate supply chain) to explore new areas of their chemical space

Independent evolutionary origin for insect deterrent cucurbitacins

The cucurbitacins are highly oxygenated triterpenoid, occurring widespread in cucurbitaceous species and in a few other plant families. Iberis spp. are Brassicaceae contain unique feeding inhibitors, cucurbitacins, that are free from insect Pieris rapae and Phyllotreta nemorum which specialized in Brassicaceae. Using de-novo assembled transcriptomics from Iberis amara, gene co-expression analysis and comparative genomics, we unraveled the evolutionary origin of the insect deterrent cucurbitacins in I. amara. Phylogenetic analysis of five oxidosqualene cyclases and heterologous expression allowed us to identify the first committed enzyme in cucurbitacin biosynthesis in I. amara, cucurbitadienol synthase. In addition, two species-specific cytochrome P450s (CYP708A16 and CYP708A15) were identified that catalyse the unique C16 and C22 hydroxylation of the cucurbitadienol backbone, enzymatic steps that have not been reported before. Furthermore, the unlinked genomic position of the cucurbitacin biosynthetic genes in I. amara contrasts with the highly organized gene cluster in the cucurbits. These results reveal that cucurbitacin biosynthesis has evolved convergently via different biosynthetic routes in different families rather than through divergence from an ancestral pathway. This investigation thus provides new insight into the mechanism of recurrent defensive chemical evolution and diversification

P starvation in tomato

To study the transcriptional changes of plants under P starvation, We performed the time series P starvation in tomato Solanum lycopersicum L. cv Craigella. To investigate the role of strigolactone in these P starvation responses, we also included its SlCCD8 knock down line 16