Assembly of dynamic P450-mediated metabolons - order versus chaos

PURPOSE OF REVIEW: We provide an overview of the current knowledge on cytochrome P450-mediated metabolism organized as metabolons and factors that facilitate their stabilization. Essential parameters will be discussed including those that are commonly disregarded using the dhurrin metabolon from Sorghum bicolor as a case study. RECENT FINDINGS: Sessile plants control their metabolism to prioritize their resources between growth and development, or defense. This requires fine-tuned complex dynamic regulation of the metabolic networks involved. Within the recent years, numerous studies point to the formation of dynamic metabolons playing a major role in controlling the metabolic fluxes within such networks. SUMMARY: We propose that P450s and their partners interact and associate dynamically with POR, which acts as a charging station possibly in concert with Cytb5. Solvent environment, lipid composition, and non-catalytic proteins guide metabolon formation and thereby activity, which have important implications for synthetic biology approaches aiming to produce high-value specialized metabolites in heterologous hosts

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

RNA-seq, de novo transcriptome assembly and flavonoid gene analysis in 13 wild and cultivated berry fruit species with high content of phenolics

This research was funded by the European Union Framework Program 7, Project BacHBerry [FP7–613793]. The authors also acknowledge support from the Institute Strategic Programmes ‘Designing Future Wheat’ (BB/P016855/1), ‘Understanding and Exploiting Plant and Microbial Secondary Metabolism’ (BB/J004596/1) and ‘Molecules from Nature’ (BB/P012523/1) from the UK Biotechnology and Biological Sciences Research Council to the John Innes Centre and the European funded COST ACTION FA1106 QualityFruit. VT, PV and CM have also received funding from the European Union’s Horizon 2020 research and innovation programme through the TomGEM project under grant agreement No. 679796. The funding bodies had no role in the design of the study, collection, analysis and interpretation of data nor in writing the manuscript.Background: Flavonoids are produced in all flowering plants in a wide range of tissues including in berry fruits. These compounds are of considerable interest for their biological activities, health benefits and potential pharmacological applications. However, transcriptomic and genomic resources for wild and cultivated berry fruit species are often limited, despite their value in underpinning the in-depth study of metabolic pathways, fruit ripening as well as in the identification of genotypes rich in bioactive compounds. Results: To access the genetic diversity of wild and cultivated berry fruit species that accumulate high levels of phenolic compounds in their fleshy berry(-like) fruits, we selected 13 species from Europe, South America and Asia representing eight genera, seven families and seven orders within three clades of the kingdom Plantae. RNA from either ripe fruits (ten species) or three ripening stages (two species) as well as leaf RNA (one species) were used to construct, assemble and analyse de novo transcriptomes. The transcriptome sequences are deposited in the BacHBerryGEN database (http://jicbio.nbi.ac.uk/berries) and were used, as a proof of concept, via its BLAST portal (http://jicbio.nbi.ac.uk/berries/blast.html) to identify candidate genes involved in the biosynthesis of phenylpropanoid compounds. Genes encoding regulatory proteins of the anthocyanin biosynthetic pathway (MYB and basic helix-loop-helix (bHLH) transcription factors and WD40 repeat proteins) were isolated using the transcriptomic resources of wild blackberry (Rubus genevieri) and cultivated red raspberry (Rubus idaeus cv. Prestige) and were shown to activate anthocyanin synthesis in Nicotiana benthamiana. Expression patterns of candidate flavonoid gene transcripts were also studied across three fruit developmental stages via the BacHBerryEXP gene expression browser (http://www.bachberryexp.com) in R. genevieri and R. idaeus cv. Prestige. Conclusions: We report a transcriptome resource that includes data for a wide range of berry(-like) fruit species that has been developed for gene identification and functional analysis to assist in berry fruit improvement. These resources will enable investigations of metabolic processes in berries beyond the phenylpropanoid biosynthetic pathway analysed in this study. The RNA-seq data will be useful for studies of berry fruit development and to select wild plant species useful for plant breeding purposes.publishersversionpublishe

Percutaneous transhepatic mitral commissurotomy

A novel, transhepatic approach to mitral valvuloplasty is described in a patient with an inferior vena caval filter. After transhepatic transseptal puncture, an Inoue dilatation catheter was passed through the hepatic parenchyma and across the atrial septum. Balloon mitral valvuloplasty was performed without complications. This approach should be considered when femoral venous access is restricted or is not feasible. © 1996 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38224/1/22_ftp.pd

最近の經濟學界

Direct assembly of multiple linear DNA fragments via homologous recombination, a phenomenon known as in vivo assembly or transformation associated recombination, is used in biotechnology to assemble DNA constructs ranging in size from a few kilobases to full synthetic microbial genomes. It has also enabled the complete replacement of eukaryotic chromosomes with heterologous DNA. The moss Physcomitrella patens, a non-vascular and spore producing land plant (Bryophyte), has a well-established capacity for homologous recombination. Here, we demonstrate the in vivo assembly of multiple DNA fragments in P. patens with three examples of effective genome editing: we (i) efficiently deleted a genomic locus for diterpenoid metabolism yielding a biosynthetic knockout, (ii) introduced a salt inducible promoter, and (iii) re-routed endogenous metabolism into the formation of amorphadiene, a precursor of high-value therapeutics. These proof-of-principle experiments pave the way for more complex and increasingly flexible approaches for large-scale metabolic engineering in plant biotechnology