Identification of squalene epoxidase in triterpenes biosynthesis in Poria cocos by molecular docking and CRISPR-Cas9 gene editing.

BACKGROUND: Squalene epoxidase is one of the rate-limiting enzymes in the biosynthetic pathway of membrane sterols and triterpenoids. The enzyme catalyzes the formation of oxidized squalene, which is a common precursor of sterols and triterpenoids. RESULT: In this study, the squalene epoxidase gene (PcSE) was evaluated in Poria cocos. Molecular docking between PcSE and squalene was performed and the active amino acids were identified. The sgRNA were designed based on the active site residues. The effect on triterpene synthesis in P. cocos was consistent with the results from ultra-high-performance liquid chromatography-quadruplex time-of-flight-double mass spectrometry (UHPLC-QTOF-MS/MS) analysis. The results showed that deletion of PcSE inhibited triterpene synthesis. In vivo verification of PcSE function was performed using a PEG-mediated protoplast transformation approach. CONCLUSION: The findings from this study provide a foundation for further studies on heterologous biosynthesis of P. cocos secondary metabolites.

Characterization of the chloroplast genome of medicinal herb Polygonatum cyrtonema and identification of molecular markers by comparative analysis.

Polygonatum cyrtonema Hua is a traditional Chinese herb medicine, and it is widely distributed in China. The intrageneric taxonomy and phylogenetic relationships within Polygonatum have long been controversial due to their morphological similarity and lacking special DNA barcodes. In this paper, the complete chloroplast genome is a relatively conserved quadripartite structure including a large single copy region of 84 711 bp, a small single copy region of 18 210 bp, and a pair of inverted repeats region of 26 142 bp. A total of 342 simple sequence repeats were identified, and most of them were found to be composed of A/T, including 126 mono-nucleotides and 179 di-nucleotides. Nucleotide diversity was analyzed and eight highly variable regions (psbl∼trnT-CGU, atpF∼atpH, trnT-GGU∼psbD, psaJ∼rps20, trnL-UAG∼ndhD, ndhG∼ndhl, ndhA, and rpl32∼ccsA) were identified as potential molecular markers. Phylogenetic analysis based on the whole chloroplast genome showed that P. cyrtonema, within the family Asparagaceae, is closely related to Polygonatum sibiricum and Polygonatum kingianum. The sequence matK, trnT-GGU & ccsA, and ndhG∼ndhA were identified as three DNA barcodes. The assembly and comparative analysis of P. cyrtonema complete chloroplast genome will provide essential molecular information about the evolution and molecular biology for further study.

Supramolecular Host-Guest Inclusion for Distinguishing Cucurbit[7]uril-Based Pseudorotaxanes from Small-Molecule Ligands in Coordination Assembly with a Uranyl Center.

Although the capability of supramolecular pseudorotaxane/rotaxane systems as ligands for coordination with actinides has been identified by the on-going emerging of uranyl-organic polyrotaxane compounds, it is, however, still unknown how supramolecular inclusion affects the coordination assembly of the simple "axle" ligand with uranyl species. Herein, a semi-rigid organic dicarboxylate compound [BzBPCEt]Br2 (L1 ) is selected as a small-molecule "axle" ligand and the corresponding cucurbit[7]uril (CB7)-based [2]pseudorotaxane ligand, [BzBPCEt]Br2 @CB7 (L1 @CB7) has been also synthesized through CB7-based inclusion in this work. A detailed comparison between uranyl complexes from the "axle" ligand L1 and those from pseudorotaxane L1 @CB7 has been conducted, demonstrating the significant role of CB7-based inclusion in distinguishing supramolecular pseudorotaxane ligands from small-molecule dicarboxylates in uranyl coordination assembly. Notably, the impact of supramolecular inclusion on the "axle" linker in the system with cucurbituril macrocycles involved is established for the first time. Detailed structure decipherment suggests that the significant effect of CB7 is attributed to hydrothermal stabilization of the "axle" ligand or increased steric hindrance to the groups nearby originated from the bulky size of macrocyclic CB7.

Supramolecular Isomers of Coordination-Directed Side-Chain Polypseudorotaxanes Based on Trimeric Uranyl Oxalate Nodes.

Although the prosperity of rotaxane coordination polymers with rotaxane molecules serving as main-chain linkers is known, side-chain metal-organic polypseudorotaxanes incorporating macrocyclic host molecules have not been reported to date. Herein a new type of coordination-driven cucurbit[6]uril-bearing side-chain polypseudorotaxane, with two-dimensional trimeric uranyl-oxalate as main chains, has been synthesized. This was carried out through hydrothermal reactions of uranyl components with an in situ-formed carboxylated pseudorotaxane ligand in the presence of oxalate co-ligands. Varying the substitution site of coordination groups led to two different supramolecular isomers. Further mechanistic analysis indicated that condition-dependent hydrolysis of the cyano groups of the pseudorotaxane ligand, as well as the participation of oxalate groups into the coordination sphere of uranyl moieties, contributes to the formation of this new type of side-chain polypseudorotaxane.

Temperature-induced reversible single-crystal-to-single-crystal isomerisation of uranyl polyrotaxanes: an exquisite case of coordination variability of the uranyl center.

The first reversible solid-state single-crystal-to-single-crystal isomerisation mediated by the change of uranyl-ligand coordination modes, that is from seven-coordinated uranium(vi) of α-UP to six-coordinated uranium(vi) of the supramolecular isomer, ß-UP, has been achieved in the uranyl polyrotaxane system by a temperature-induced strategy.

Mixed-Ligand Uranyl Polyrotaxanes Incorporating a Sulfate/Oxalate Coligand: Achieving Structural Diversity via pH-Dependent Competitive Effect.

A mixed-ligand system provides an alternative route to tune the structures and properties of metal-organic compounds by introducing functional organic or inorganic coligands. In this work, five new uranyl-based polyrotaxane compounds incorporating a sulfate or oxalate coligand have been hydrothermally synthesized via a mixed-ligand method. Based on C6BPCA@CB6 (C6BPCA = 1,1'-(hexane-1,6-diyl)bis(4-(carbonyl)pyridin-1-ium), CB6 = cucurbit[6]uril) ligand, UPS1 (UO2(L)0.5(SO4)(H2O)·2H2O, L = C6BPCA@CB6) is formed by the alteration of initial aqueous solution pH to a higher acidity. The resulting 2D uranyl polyrotaxane sheet structure of UPS1 is based on uranyl-sulfate ribbons connected by the C6BPCA@CB6 pseudorotaxane linkers. By using oxalate ligand instead of sulfate, four oxalate-containing uranyl polyrotaxane compounds, UPO1-UPO4, have been acquired by tuning reaction pH and ligand concentration: UPO1 (UO2(L)0.5(C2O4)0.5(NO3)·3H2O) in one-dimensional chain was obtained at a low pH value range (1.47-1.89) and UPO2 (UO2(L)(C2O4)(H2O)·7H2O)obtained at a higher pH value range (4.31-7.21). By lowering the amount of oxalate, another two uranyl polyrotaxane network UPO3 ((UO2)2(L)0.5(C2O4)2(H2O)) and UPO4 ((UO2)2O(OH)(L)0.5(C2O4)0.5(H2O)) could be acquired at a low pH value of 1.98 and a higher pH value over 6, respectively. The UPO1-UPO4 compounds, which display structural diversity via pH-dependent competitive effect of oxalate, represent the first series of mixed-ligand uranyl polyrotaxanes with organic ligand as the coligand. Moreover, the self-assembly process and its internal mechanism concerning pH-dependent competitive effect and other related factors such as concentration of the reagents and coordination behaviors of the coligands were discussed in detail.

Solvent-Dependent Synthesis of Porous Anionic Uranyl-Organic Frameworks Featuring a Highly Symmetrical (3,4)-Connected ctn or bor Topology for Selective Dye Adsorption.

Two highly symmetrical (3,4)-connected uranyl-organic frameworks (UOFs) were synthesized by a judicious combination of D3h -symmetrical triangular [UO2 (COO)3 ]- and Td symmetrical tetrahedral tetrakis(4-carboxyphenyl)methane (H4 MTB). These two as-synthesized UOFs possess similar structural units and coordination modes but totally different topological structures, namely ctn net and bor net. Solvent-induced interpenetration and a morphology change are observed. The two compounds exhibit crystal transformation via a dissolution-crystallization process. Adsorption experiments in CH3 OH solution indicate that both of them can selectively remove positively charged dyes over negatively charged and neutral dyes. Moreover, the electronic structural and bonding properties of the two compounds were systematically explored by density functional theory (DFT) calculations.

First three-dimensional actinide polyrotaxane framework mediated by windmill-like six-connected oligomeric uranyl: dual roles of the pseudorotaxane precursor.

The first 3D actinide polyrotaxane framework (named IHEP-URCP-2) has been obtained based on windmill-like six-connected high-nuclear oligomeric uranyl nodes under hydrothermal conditions. Notably, the in situ formed pseudorotaxane ligand simultaneously plays dual roles of both a bulky pseudorotaxane linker and a supramolecular guest.

An Unprecedented Two-Fold Nested Super-Polyrotaxane: Sulfate-Directed Hierarchical Polythreading Assembly of Uranyl Polyrotaxane Moieties.

The hierarchical assembly of well-organized submoieties could lead to more complicated superstructures with intriguing properties. We describe herein an unprecedented polyrotaxane polythreading framework containing a two-fold nested super-polyrotaxane substructure, which was synthesized through a uranyl-directed hierarchical polythreading assembly of one-dimensional polyrotaxane chains and two-dimensional polyrotaxane networks. This special assembly mode actually affords a new way of supramolecular chemistry instead of covalently linked bulky stoppers to construct stable interlocked rotaxane moieties. An investigation of the synthesis condition shows that sulfate can assume a vital role in mediating the formation of different uranyl species, especially the unique trinuclear uranyl moiety [(UO2 )3 O(OH)2 ](2+) , involving a notable bent [O=U=O] bond with a bond angle of 172.0(9)°. Detailed analysis of the coordination features, the thermal stability as well as a fluorescence, and electrochemical characterization demonstrate that the uniqueness of this super-polyrotaxane structure is mainly closely related to the trinuclear uranyl moiety, which is confirmed by quantum chemical calculations.