The eukaryotic translation inhibitor cycloheximide (CHX) and its analogue actiphenol (APN) feature a glutarimide moiety and a six-membered carbocyclic ring system. The biosynthesis of CHX and APN is not yet fully understood, particularly with respect to the mechanism of formation of the fully reduced cyclohexanone ring in CHX and the aromatic phenol ring in APN. In this work, a combination of gene inactivation, chemical synthesis, and in vitro biochemical experiments highlighted an ensemble of three tailoring redox enzymes as being responsible for the biosynthesis of the six-membered carbocyclic ring systems. Specifically, two redox enzymes (ChxJ and ChxI) alone can generate an active intermediate that undergoes a cascade of non-enzymatic transformations to create APN, while a reductive enzyme (ChxG) acts as a gatekeeper, directing the same intermediate down a different pathway toward CHX. Finally, the full nature of each biosynthetic pathway was established in detail, including the formation mechanisms of six-membered carbocyclic rings.

Complex phenylethanoid glycosides (PhGs), such as verbascoside and echinacoside, comprise a vital family of natural products with renowned nutraceutical and pharmaceutical significance. Despite the high demand for these compounds across various industries, traditional plant extraction methods yield insufficient quantities, highlighting the need for alternative production methods. Therefore, this paper reports the successful engineering of Saccharomyces cerevisiae cell factories for the efficient production of complex PhGs from glucose. First, key pathway enzymes with enhanced catalytic activities in yeast were primarily screened from various verbascoside-producing plants. Second, intermediate osmanthuside B was produced with a titer of 21.5 ± 1.5 mg/L from glucose by overexpressing several enzymes, including glucosyltransferase RrUGT33 from Rhdiola rosea, acyltransferase SiAT, and 1,3-rhamnosyltransferase SiRT from Sesamum indicum, UDP-L-rhamnose synthase AtRHM2, and 4-coumarate: coenzyme A ligase At4CL1 from Arabidopsis thaliana in a p-coumaric acid-overproducing S. cerevisiae strain. Third, the production of osmanthuside B was further enhanced by increasing the copy number of SiAT and AtRHM2 in genome and diverting L-tyrosine into tyrosol biosynthesis by introducing an aromatic aldehyde synthase PcAAS from Petroselinum crispum with a titer of 320.6 ± 59.3 mg/L. Fourth, the biosynthesis of verbascoside was accomplished by integrating genes CYP98A20 and AtCPR1 into the chromosomes of the osmanthuside B-producing strain, the titer reached 184.7 ± 5.7 mg/L. Furthermore, the overexpression of the glucose-6-phosphate dehydrogenase (ZWF1) led to significantly enhanced verbascoside production to 230.6 ± 11.8 mg/L. The strains were further engineered to produce echinacoside with a titer of 184.2 ± 11.2 mg/L. Finally, the fed-batch fermentation in a 5-L bioreactor yielded 4497.9 ± 285.2 mg/L of verbascoside or 3617.4 ± 117.4 mg/L of echinacoside. This work provides a crucial foundation for the green, industrial, and sustainable production of verbascoside and echinacoside and sets an initial point for the microbial production of other complex PhG derivatives.

During a screening for antifungal secondary metabolites, six new mono-/bis-alkenoic acid derivatives ( 2 – 7 ) and one known alkenoic acid derivative ( 1 ) were isolated from an endophytic fungi Scopulariopsis candelabrum . Their chemical structures were identified by 1 H-NMR, 13 C-NMR, 2D NMR, and high-resolution mass spectrometry, as well as comparisons with previously reported literatures. Among them, fusariumesters C‒F ( 2 – 5 ) are bis-alkenoic acid derivatives dimerized by an ester bond, while acetylfusaridioic acid A ( 6 ) and fusaridioic acid D ( 7 ) are alkenoic acid monomers. All the isolates were submitted to an antifungal assay against Candida albicans and the corn pathogen Exserohilum turcicum using the filter paper agar diffusion method. As a result, only compound 1 decorating with β -lactone ring turned out to be active against these two tested fungi. The broth microdilution assay against Candida albicans showed the minimum inhibitory concentration (MIC) value of 1 to be 20 μ g/ml, while the minimum inhibitory concentration value of the positive control (naystatin) was 10 μ g/ml. And the half maximal inhibitory concentration (IC 50 ) value (21.23 μ g/ml) of 1 against Exserohilum turcicum was determined by analyzing its inhibition effect on the mycelial growth, using cycloheximide (IC 50 = 46.70 μ g/ml) as the positive control.

Inthomycins are polyketide antibiotics which contain a terminal carboxamide group and a triene chain. Inthomycin B (1) and its two new analogues 2 and 3 were isolated from the crude extract of Streptomyces pactum L8. Identification of the gene cluster for inthomycin biosynthesis as well as the 15N-labeled glycine incorporation into inthomycins are described. Combined with the gene deletion of the rare P450 domain in the NRPS module, a formation mechanism of carboxamide moiety in inthomycins was proposed via an oxidative release of the assembly chain assisted by the P450 domain.

Seven new trialkyl-substituted benzene derivatives named benwamycins A-G (1-7), together with three known congeners, 8-10, were isolated from culture broth of the soil-derived Streptomyces sp. KIB-H1471. Their structures were elucidated by using 1D and 2D NMR analyses in combination with HRESIMS data. The absolute configurations of 1-9 were determined by chemical conversion and comparison of circular dichroism spectra and confirmed for 1 by single-crystal X-ray crystallography. Compounds 6 and 7 have a unique γ-pyrone-like ring on one side chain. Compounds 2 and 6 inhibited human T cell proliferation with IC50 values of 14.3 and 12.5 μM, respectively, without obvious cytotoxicity for naïve human T cells. Compounds 3 and 6 could weakly enhance insulin-stimulated glucose uptake.

Nine new pentacyclic polyketides, fasamycins G-K (1-5) and formicamycins N-Q (6-9), along with 10 known analogues (10-19), were isolated from a rhizospheric soil-derived Streptomyces sp. KIB-1414. Their structures and absolute configurations were elucidated by interpretation of NMR and HRMS data and comparisons of CD data. The compounds were active against methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus, Bacillus subtilis, and Escherichia coli strains, with MIC values ranging from 0.20 to 50.00 μg/mL.

Two new peptides, MCh-1 and MCh-2, along with three known trypsin inhibitors (MCTI-I, MCTI-II and MCTI-III), were isolated from the seeds of the tropical vine Momordica charantia. The sequences of the peptides were determined using mass spectrometry and NMR spectroscopy. Using a strategy involving partial reduction and stepwise alkylation of the peptides, followed by enzymatic digestion and tandem mass spectrometry sequencing, the disulfide connectivity of MCh-1 was elucidated to be CysI-CysIV, CysII-CysV and CysIII-CysVI. The three-dimensional structures of MCh-1 and MCh-2 were determined using NMR spectroscopy and found to contain the inhibitor cystine knot (ICK) motif. The sequences of the novel peptides differ significantly from peptides previously isolated from this plant. Therefore, this study expands the known peptide diversity in M. charantia and the range of sequences that can be accommodated by the ICK motif. Furthermore, we show that a stable two-disulfide intermediate is involved in the oxidative folding of MCh-1. This disulfide intermediate is structurally homologous to the proposed ancestral fold of ICK peptides, and provides a possible pathway for the evolution of this structural motif, which is highly prevalent in nature.

Plant cyclopeptides are a large group of small molecule metabolites found in a wide variety of plants, including traditional Chinese medicinal plants. Many of the cyclopeptides have highly unusual structures and potent biological activities. However, the majority of the cyclopeptides have not been studied for their biosynthetic mechanisms. In this study, we have established a culture system for the biosynthetic study of heterophyllin B (HB), a cyclopeptide produced by the medicinal plant Pseudostellaria heterophylla. We first developed a shoot culture of P. heterophylla that produced HB consistently under laboratory conditions. Using 14 C-labeled proline as tracer, we showed that labeled HB was produced by the cultured shoots, indicating that this system has de novo biosynthetic activity. Next, we chemically synthesized HB's linear peptide precursor (LHB) and the N-acetyl cysteamine thioester of LHB (LHB-SNAC). When LHB-SNAC was incubated with total cell free extracts of the cultured shoots, a small amount of cyclized product (HB), in addition to the hydrolyzed product (LHB), was produced. The in vivo and in vitro results demonstrate the presence of an HB biosynthetic system, which provides insight into the molecular mechanism for plant cyclopeptide biosynthesis.

Many Violaceae plants contain cyclotides, which are plant cyclopeptides distinguished by a cyclic cystine knot motif with 28-37 amino acid residues. In the current study, four new cyclotides, vila A - D (1-4 resp), together with a known cyclotide, vary D (5), were isolated from Viola labridorica (Violaceae) A chromatography-based method was used to isolate the cyclotides, which were characterized using tandem mass spectrometry and 2D-NMR spectroscopy Several of the cyclotides showed cytotoxic activities against five cancer cell lines, i e, U251 MDA-MB-231, A549 DU145, and BEL-7402, with vila A and B (1 and 2, resp) being the most cytotoxic The isolated cyclotides showed no antibacterial activity against Staphyloccocus aureus and Candida albicans Homology modeling of the cyclotide structures was used to analyze structure-activity relationships.

Many plants of the Violaceae plant family have been used in traditional remedies, and these plants often contain cyclotides, a particular type of plant cyclopeptide that is distinguished by a cyclic cystine knot motif. In general, bioactive plant cyclopeptides are interesting candidates for drug development. In the current study, a suite of 14 cyclotides, which includes seven novel cyclotides [vitri B, C, D, E, F, varv Hm, and He], together with seven known cyclotides [varv A, D, E, F, H, vitri A, and cycloviolacin O2], was isolated from Viola tricolor, a common flower. A chromatography-based method was used to isolate the cyclotides, which were characterized using tandem mass spectrometry and NMR spectroscopy. Several of the cyclotides showed cytotoxic activities against five cancer cell lines, U251, MDA-MB-231, A549, DU145, and BEL-7402. Three cyclotides, vitri A, vitri F, and cycloviolacin O2, were the most cytotoxic. The cytotoxic activity of the cyclotides did not correlate well with their hemolytic activity, indicating that different interactions, most likely with membranes, are involved for cytotoxic and hemolytic activities. Homology modeling of the structures was used in deriving structure–activity relationships.

Cyclotides are a family of plant-derived macrocyclic peptides, which are formed by 28-37 amino acid residues and contain three disulfide bonds and the unique protein structural motif termed cyclic cystine knot. For their unique structures and various bioactivities, such as uterotonic, hemolytic, cytotoxic and anti-bacterial activities, and stability to resist thermal, acidic and proteolytic degradation, cyclotides can be used as a peptide-based combinational template and carrier for drug design, and attract more attentions from scientists. Now over 100 cyclotides have been isolated from about 30 species of Violaceae, Rubiaceae and Cucurbitaceae. Most contributions on cyclotides have been achieved by the research groups in Australia, Sweden and USA. Our lab is also investigating on cyclotides. This paper introduces cyclotides' research history, methods for extraction, isolation and detection, structural determination and classification, homological analysis of sequences, synthesis and biosynthesis, bioactivity and so on.

Cyclotides are a family of plant defense proteins that are highly resistant to adverse chemical, thermal, and enzymatic treatment. Here, we present the first crystal structure of a cyclotide, varv F, from the European field pansy, Viola arvensis, determined at a resolution of 1.8 angstrom. The solution state NMR structure was also determined and, combined with measurements of biophysical parameters for several cyclotides, provided an insight into the structural features that account for the remarkable stability of the cyclotide family. The x-ray data confirm the cystine knot topology and the circular backbone, and delineate a conserved network of hydrogen bonds that contribute to the stability of the cyclotide fold. The structural role of a highly conserved Glu residue that has been shown to regulate cyclotide function was also determined, verifying its involvement in a stabilizing hydrogen bond network. We also demonstrate that varv F binds to dodecylphosphocholine micelles, defining the binding orientation and showing that its structure remains unchanged upon binding, further demonstrating that the cyclotide fold is rigid. This study provides a biological insight into the mechanism by which cyclotides maintain their native activity in the unfavorable environment of predator insect guts. It also provides a structural basis for explaining how a cluster of residues important for bioactivity may be involved in self-association interactions in membranes. As well as being important for their bioactivity, the structural rigidity of cyclotides makes them very suitable as a stable template for peptide-based drug design.

用碘、碘化铋钾、茚三酮和考马斯亮蓝G-250四种显色剂对包括植物环蛋白在内的3种寡肽以及氨基酸和蛋白质在薄层层析板上进行了5组显色反应研究.结果表明,可以综合考马斯亮蓝G-250和水解前后对茚三酮的显色来识别环蛋白.应用该显色方法开展了三色堇、紫花地丁、如意草、木鳖子和苦瓜等5种植物的环蛋白的检测和纯化工作,均检测出环蛋白,并分离获得十多个环蛋白,鉴定了其中的3个,为已知环蛋白cycloviolacin O2,kalataB1和vary peptide A.

Iodine, Dragendorff's reagent, ninhydrin and Coomassie brilliant blue G-250 reagents were used to detect cyclotides from proteins, cyclopeptides, linear-peptides and amino acids in the thin layer chromatography (TLC). Cyclotides could be distinguished from the others by comparing the TLC coloring plots with G-250 and, ninhydrin reagents. With this method, cyclotides were discovered in Viola labridorica, V. tricolor, V. hamiltoniana, Momordica charantia, and M. cochinchinensis and over 10 cyclotides were isolated, three of which were determined as known cyclotides cycloviolacin O2, kalata B1 and vary peptide A.

简要介绍了植物环蛋白的定义、结构特点、研究历史、分布、提取分离方法、化学合成与生物合成、生物活性与生物功能.并主要以从紫花蔓地丁(Viola labridorica)中分离得到的六个环蛋白之一,cycloviolacin 02为例介绍通过还原酶解-质谱与二维核磁共振谱结合鉴定环蛋白结构的研究方法.