The two-step strategy for enhancing the specific activity and thermostability of alginate lyase AlyG2 with mechanism for improved thermostability.

To overcome the trade-off challenge encountered in the engineering of alginate lyase AlyG2 from Seonamhaeicola algicola Gy8T and to expand its potential industrial applications, we devised a two-step strategy encompassing activity enhancement followed by thermal stability engineering. To enhance the specific activity of efficient AlyG2, we strategically substituted residues with bulky steric hindrance proximal to the active pocket with glycine or alanine. This led to the generation of three promising positive mutants, with particular emphasis on the T91S mutant, exhibiting a 1.91-fold specific activity compared to the wild type. To mitigate the poor thermal stability of T91S, mutants with negative ΔΔG values in the thermal flexibility region were screened out. Notably, the S72Y⁎ mutant not only displayed 17.96 % further increase in specific activity but also exhibited improved stability compared to T91S, manifesting as a remarkable 30.97 % increase in relative activity following a 1-hour incubation at 42 °C. Furthermore, enhanced kinetic stability was observed. To gain deeper insights into the mechanism underlying the enhanced thermostability of the S72Y⁎ mutant, we conducted molecular dynamics simulations, principal component analysis (PCA), dynamic cross-correlation map (DCCM), and free energy landscape (FEL) analysis. The results unveiled a reduction in the flexibility of the surface loop, a stronger correlation dynamic and a narrower motion subspace in S72Y⁎ system, along with the formation of more stable hydrogen bonds. Collectively, our findings suggest amino acids substitutions resulting in smaller side chains proximate to the active site can positively impact enzyme activity, while reducing the flexibility of surface loops emerges as a pivotal factor in conferring thermal stability. These insights offer valuable guidance and a framework for the engineering of other enzyme types.

Lauinoids A-X: Labdane-type diterpenoids with anti-inflammatory activity from Croton laui.

Croton laui (Euphorbiaceae) is a traditional medicinal plant used by the Li ethnic group in China to treat headaches, stomachaches, and diphtheria. To understand the pharmacological basis of its medicinal use, an extensive investigation of the ethanolic extract of the bark of C. laui was performed. After repeated chromatography, twenty-four undescribed labdane-type diterpenoids, lauinoids A-X (1-24), and five known analogs (25-29) were isolated. Their structures and absolute configurations were established using a combination of spectroscopic analyses, electronic circular dichroism, nuclear magnetic resonance calculations, and single-crystal X-ray diffraction. Among them, compounds 1-3 exhibited an 11(12 â†’ 13)-abeo-16-nor-labdane skeleton, which originated putatively from 9 through a plausible pathway that involves a semipinacol rearrangement process. Compounds 11 and 12 belong to the rare class of 14,15-dinor-labdane diterpenoids. Compounds 18 and 28 exhibited substantial inhibitory effects by suppressing lipopolysaccharide-induced NO production in RAW 264.7 macrophages, with IC50 values of 3.37 ± 0.23 and 5.82 ± 0.28 µM, respectively. This study has greatly expanded the chemical diversity of labdane diterpenoids from C. laui and will guide future research on this ethnomedicinal plant.

Crotonoids A-H, Labdane-Type Diterpenoids with Anti-Inflammatory Activity from Croton sublyratus.

Croton sublyratus (Euphorbiaceae) is a traditional medicinal plant used by the Thai populace to treat helminthic infections and dermatologic conditions. In present study, eight new labdane-type diterpenoids, crotonoids A-H (1-8) and one known analogue (9) were isolated from the aerial parts of C. sublyratus. Compounds 6 and 7 belong to the rare class of 14,15-dinor-labdane diterpenoids. Compound 8 exhibited a rare 14,15,17-trinor-labdane skeleton. The structures of all these diterpenoids were elucidated by spectroscopic data analysis, electronic circular dichroism calculations, and single-crystal X-ray diffraction analysis. Compound 9 exhibited moderate anti-inflammatory activity via the inhibition of NO production in lipopolysaccharide-induced RAW 264.7 cells.

Cephalotane diterpenoids: structural diversity, biological activity, biosynthetic proposal, and chemical synthesis.

Covering: up to the end of 2023Cephalotane diterpenoids are a unique class of natural products exclusive to the genus Cephalotaxus, featuring a rigid 7,6,5,6-fused tetracyclic architecture. The study of cephalotanes dates back to the 1970s, when harringtonolide (1), a Cephalotaxus troponoid with a peculiar norditerpenoid carbon skeleton, was first discovered. In recent years, prototype C20 diterpenoids proposed as cephalotane were disclosed, which triggered intense studies on this diterpenoid family. To date, a cumulative total of 105 cephalotane diterpenoids with great structural diversity and biological importance have been isolated. In addition, significant advances have been made in the field of total synthesis and biosynthesis of cephalotanes in recent years. This review provides a complete overview of the chemical structures, bioactivities, biosynthetic aspects, and completed total synthesis of all the isolated cephalotane diterpenoids, which will help guide future research on this class of compounds.

Highly modified cephalotane-type diterpenoids from Cephalotaxus fortunei var. alpina and C. sinensis.

Cephalotanes are a rare class of diterpenoids occurring exclusively in Cephalotaxus plants. The intriguing structures and promising biological activities for this unique compound class prompt us to investigate C. fortunei var. alpina and C. sinensis, leading to the isolation of six undescribed cephalotane-type diterpenoids and/or norditerpenoids, ceforloids A-F (1-6). Their structures were elucidated by comprehensive analysis of spectroscopic data, including ECD and single-crystal X-ray diffraction studies, as well as quantum chemical calculations. Compound 1 possesses an unprecedented norditerpenoid skeleton featuring an unusual acetophenone moiety, and originated putatively from a disparate biogenetic pathway. Compounds 4 and 5 incorporate a unique 12,13-p-hydroxybenzylidene acetal motif. Compound 6 is a rare cephalotane-type diterpenoid glycoside. Immunosuppressive assays showed that compounds 2 and 6 exhibited mild suppressive activity against the activated T and B lymphocytes proliferation. These findings not only expanded the structural diversity of this small group of diterpenoids, but also explored their potential as novel structures for the development of immunosuppressive agents.

Unprecedented sesterterpenoids, orientanoids A-C: discovery, bioinspired total synthesis and antitumor immunity.

Sesterterpenoids are a very rare class of important natural products. Three new skeletal spiro sesterterpenoids, named orientanoids A-C (1-3), were isolated from Hedyosmum orientale. Their structures were determined by a combination of spectroscopic data, X-ray crystallography, and total synthesis. To obtain adequate materials for biological research, the bioinspired total syntheses of 1-3 were effectively achieved in 7-8 steps in overall yields of 2.3-6.4% from the commercially available santonin without using any protecting groups. In addition, this work also revised the stereochemistry of hedyosumins B (6) and C (10) as 11R-configuration. Tumor-associated macrophages (TAMs) have emerged as important therapeutic targets in cancer therapy. The in-depth biological evaluation revealed that these sesterterpenoids antagonized the protumoral and immunosuppressive functional phenotype of macrophages in vitro. Among them, the most potent and major compound 1 inhibited protumoral M2-like macrophages and activated cytotoxic CD8+ T cells, and consequently inhibited tumor growth in vivo.

Highly Modified Sesquiterpene Lactones with Cytotoxic Activities from Strobocalyx chunii.

Three new germacranolide sesquiterpene lactones (SLs), strochunolides A-C (1-3, respectively), and a new guaianolide SL, strochunolide D (4), were isolated from Strobocalyx chunii and structurally characterized. Compound 1 is the first example of a dihomo-germacranolide SL, characterized by an unprecedented 6/10/5 tricyclic scaffold incorporating an additional fused δ-lactone C-ring. The structure of a known germacranolide SL, spicatolide C (5), was revised as its 8-epimer. Compound 3 exhibited potent in vitro cytotoxic activity against the HL-60 cell line, with an IC50 value of 0.18 ± 0.01 µM.

Sumatranins A-J: Lignans with Immunosuppressive Activity from Cleistanthus sumatranus.

Chemical investigation of the twigs of Cleistanthus sumatranus (Phyllanthaceae) led to the isolation of 10 undescribed lignans, sumatranins A-J (1-10). Compounds 1-4 are unprecedented furopyran lignans characterized by a unique 2,3,3a,9a-tetrahydro-4H-furo[2,3-b]chromene heterotricyclic framework. Compounds 9 and 10 are rare 9'-nor-dibenzylbutane lignans. Structures were established based on analyses of spectroscopic data, X-ray crystallographic data, and experimental ECD spectra. Immunosuppressive assays revealed compounds 3 and 9 displayed moderate inhibitory effects with good selectivity indexes against LPS-induced B lymphocyte proliferation.

Cephalotane-type C20 diterpenoids from Cephalotaxus fortunei var. alpina.

Seventeen new cephalotane-type diterpenoids, fortalides A-Q (1-17), along with five known analogues, were isolated from the seeds of Cephalotaxus fortunei var. alpina. Their structures were determined by extensive spectroscopic methods, as well as electronic circular dichroism (ECD) and X-ray crystallographic data analyses. Some isolates exhibited unusual structural features that were first found in cephalotane-type diterpenoids, such as the occurrence of the 7-oxabicyclo[4.1.1]octane moiety in 14 and 15 and the cis-arrangement of 3-OH and Me-19 in 9. Besides, the antiplasmodial activity of these compounds was evaluated in this study.

Genomic Analysis and Characterization of Pseudotabrizicola formosa sp. nov., a Novel Aerobic Anoxygenic Phototrophic Bacterium, Isolated from Sayram Lake Water.

Aerobic anoxygenic photosynthetic bacteria (AAPB) are a kind of heterotrophic prokaryote that can use bacteriochlorophyll (BChl) for photosynthesis without oxygen production and they are widely distributed in aquatic environments, including oceans, lakes, and rivers. A novel aerobic anoxygenic photosynthetic bacterium strain XJSPT was isolated during a study of water microbial diversity in Sayram Lake, Xinjiang Province, China. Strain XJSPT was found to grow optimally at 33 °C, pH 7.5 with 1.0% (w/v) NaCl, and to produce bacteriochlorophyll a and carotenoids. Phylogenetic analysis based on 16S rRNA gene sequence and concatenated alignment sequences of 120 ubiquitous single-copy proteins both supported that strain XJSPT belonged to the genus Pseudotabrizicola. Both average nucleotide identity (ANI) and DNA-DNA hybridization (DDH) values were below the species delineation threshold. The primary polar lipids were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, one unknown lipid, and one unidentified phospholipid. Based on the results of polyphasic analyses performed in this study, strain XJSPT represents a new member of the genus Pseudotabrizicola, for which the name Pseudotabrizicola formosa sp. nov. is proposed. The type strain is XJSPT (=KCTC 52636T = MCCC 1H00184T = SDUM 107003T). Comparative genomic analysis showed that four species of the genus Pseudotabrizicola shared 2570 core genes and possessed a complete anoxygenic photosystem II.

Digestive Tract Morphology and Gut Microbiota Jointly Determine an Efficient Digestive Strategy in Subterranean Rodents: Plateau Zokor.

Rodents' lifestyles vary in different environments, and to adapt to various lifestyles specific digestion strategies have been developed. Among these strategies, the morphology of the digestive tracts and the gut microbiota are considered to play the most important roles in such adaptations. However, how subterranean rodents adapt to extreme environments through regulating gut microbial diversity and morphology of the digestive tract has yet to be fully studied. Here, we conducted the comparisons of the gastrointestinal morphology, food intake, food assimilation, food digestibility and gut microbiota of plateau zokor Eospalax baileyi in Qinghai-Tibet Plateau and laboratory rats Rattus norvegicus to further understand the survival strategy in a typical subterranean rodent species endemic to the Qinghai-Tibet Plateau. Our results revealed that plateau zokor evolved an efficient foraging strategy with low food intake, high food digestibility, and ultimately achieved a similar amount of food assimilation to laboratory rats. The length and weight of the digestive tract of the plateau zokor was significantly higher than the laboratory rat. Particularly, the weight and length of the large intestine and cecum in plateau zokor is three times greater than that of the laboratory rat. Microbiome analysis showed that genus (i.e., Prevotella, Oscillospira, CF231, Ruminococcus and Bacteroides), which are usually associated with cellulose degradation, were significantly enriched in laboratory rats, compared to plateau zokor. However, prediction of metagenomic function revealed that both plateau zokor and laboratory rats shared the same functions in carbohydrate metabolism and energy metabolism. The higher digestibility of crude fiber in plateau zokor was mainly driven by the sizes of cecum and cecum tract, as well as those gut microbiota which associated with cellulose degradation. Altogether, our results highlight that both gut microbiota and the morphology of the digestive tract are vital to the digestion in wild rodents.

Multispecies comparative analysis reveals transcriptional specificity during Mongolian horse testicular development.

Mongolian horses have been bred and used for labor and transport for centuries. Nevertheless, traits of testicular development in Mongolian horses have rarely been studied; particularly, studies regarding the transcriptional regulation characteristics of testicular development are lacking. In this paper, transcription specificity during testicular development in Mongolian horses is highlighted via a multispecies comparative analysis and weighted gene co-expression network analysis (WGCNA). Interestingly, the results showed that most genes were up-regulated in the testes after sexual maturity, which is a phenomenon conserved across species. Moreover, we observed nine key genes involved in regulating Mongolian horse testicular development. Notably, unique transcription signatures of testicular development in Mongolian horses are emphasized, which provides a novel insight into the mechanistic study of their testicular development.

Phorneroids A-M, diverse types of diterpenoids from Euphorbia neriifolia.

A chemical investigation on the aerial parts of Euphorbia neriifolia led to the identification of thirteen undescribed diterpenoids, phorneroids A-M, including ent-abietane (A-D), ent-kaurane (E-G), ent-atisane (H-K), and ent-isopimarane (L and M) types, together with three known compounds. Phorneroid A represents the first example of 8-spiro-fused 9,10-seco-ent-abietane diterpenoid lactone featuring a unique 6/5/6/5 spirocyclic framework. Biological assays showed that some of the compounds displayed moderate cytotoxicity against two human tumor cell lines, A549 and HL-60.

Sublyratins A-O, Labdane-Type Diterpenoids from Croton sublyratus.

Fifteen new labdane-type diterpenoids, sublyratins A-O (1-15), along with four known analogues (16-19) were isolated from the aerial parts of Croton sublyratus. Their structural assignments were challenging due to the stereoisomeric features evident and were achieved by analyzing comprehensively the spectroscopic data and electronic circular dichroism spectra and using X-ray crystallographic analysis. Compounds 9 and 16-18 displayed cytotoxic activity against the HL-60 cell line with IC50 values of 1.5-2.8 µM.

1,2-Difunctionalized bicyclo[1.1.1]pentanes: Long-sought-after mimetics for ortho/meta-substituted arenes.

The development of a versatile platform for the synthesis of 1,2-difunctionalized bicyclo[1.1.1]pentanes to potentially mimic ortho/meta-substituted arenes is described. The syntheses of useful building blocks bearing alcohol, amine, and carboxylic acid functional handles have been achieved from a simple common intermediate. Several ortho- and meta-substituted benzene analogs, as well as simple molecular matched pairs, have also been prepared using this platform. The results of in-depth ADME (absorption, distribution, metabolism, and excretion) investigations of these systems are presented, as well as computational studies which validate the ortho- or meta-character of these bioisosteres.

Marinobacter vulgaris sp. nov., a moderately halophilic bacterium isolated from a marine solar saltern.

A facultatively anaerobic, Gram-stain-negative and non-gliding bacterium, designated F01T, was isolated from marine solar saltern in Weihai, PR China. Cells of F01T were 0.2-0.4 µm wide and 1.4-4.1 µm long, weakly catalase-positive and oxidase-negative. Growth of F01T was determined to occur at 4-40 °C (optimum, 33-37 °C), pH 6.5-8.5 (optimum, 7.0-8.0), and with 0.5-18.0 % (w/v) NaCl (optimum, 3.0-6.0 %). The 16S rRNA gene sequence analysis indicated that F01T represented a member of the genus Marinobacter within the family Alteromonadaceae. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate was most closely related to Marinobacter algicola DSM 16394T, with a sequence similarity of 97.5 %. The DNA G+C content of the isolate was 57.6 mol%. The major respiratory quinone of F01T was ubiquinone-9 (Q-9) and the major fatty acids were anteiso-C15 : 0, C16 : 0 and C18 : 1ω9c. The major polar lipids were phosphoaminolipid, phosphatidylglycerol and phosphatidylethanolamine. On the basis of the results of the phylogenetic analysis and phenotypic properties, it is concluded that F01T can be considered to represent a novel species in the genus Marinobacter, for which the name Marinobacter vulgaris sp. nov. is proposed. The type strain is F01T (=MCCC 1H00290T=KCTC 52700T).

Cytotoxic Tigliane Diterpenoids from Croton damayeshu.

Chemical investigation of an EtOH extract of the twigs and leaves of Croton damayeshu afforded 10 new tigliane diterpenoids, crodamoids A-J (1-10), along with five known compounds. Their structures were elucidated by physical data analysis. Compounds 8, 9, and 15 displayed cytotoxic effects against two human tumor cell lines, A549 and HL-60 (IC50: 0.9-2.4 µM).

Dysoxylactam A: A Macrocyclolipopeptide Reverses P-Glycoprotein-Mediated Multidrug Resistance in Cancer Cells.

A 17-membered macrocyclolipopeptide, named dysoxylactam A (1) comprising an unprecedented branched C19 fatty acid and an l-valine, was isolated from the plants of Dysoxylum hongkongense. The challenging relative configuration of 1 was established by means of residual dipolar coupling-based NMR analysis. The absolute configuration of 1 was determined by single-crystal X-ray diffraction on its p-bromobenzoate derivative (2). Compound 1 dramatically reversed multidrug resistance in cancer cells with the fold-reversals ranging from 28.4 to 1039.7 at the noncytotoxic concentration of 10 µM. The mode-of-action study of 1 revealed that it inhibited the function of P-glycoprotein (P-gp), a key mediator in multidrug resistance.

Motilimonas pumila sp. nov., isolated from the gut of sea cucumber Apostichopus japonicus.

A facultatively anaerobic and Gram-negative bacterium, designated strain PLHSC7-2T, was isolated from the gut of sea cucumber Apostichopusjaponicus that had been collected from the coastal area of Yantai, China. The cells were rod-shaped and motile by means of polar flagella. The novel isolate grew optimally at 28-30 °C, in the presence of 2.0-3.0 % (w/v) NaCl and at pH 7.0-7.5. The sole respiratory quinone was Q-8 and the major fatty acids were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and C17 : 0. The predominant polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain PLHSC7-2T was phylogenetically affiliated with the genus Motilimonas, and exhibited sequence similarity of 96.2 % toMotilimonas eburnea YH6T. The DNA G+C content was 45.5 mol%. On the basis of phenotypic , phylogenetic and genetic distinctiveness, strain PLHSC7-2T (=MCCC 1K03522T=KCTC 62589T) was classified as a novel species within the genus Motilimonas, for which the name Motilimonas pumila sp. nov. is proposed.

Hanstruepera crassostreae sp. nov., a novel marine bacterium of the family Flavobacteriaceae isolated from an oyster.

A Gram-stain-negative, rod-shaped, non-motile, aerobic and orange-pigmented marine bacterium, designated strain L53T, was isolated from an oyster sample collected from the coast of Weihai, China (122.0° E 37.5° N). Growth of strain L53T occurred at 4-40 °C (optimum, 33 °C), at pH 6.5-8.0 (optimum, pH 7.5) and with 1.0-7.0 % (w/v) NaCl (optimum, 3.0 %). Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain L53T was closely related to Bizionia echini KCTC 22015T (96.9 %) and Hanstruepera neustonica JCM 19743T (96.1 %). Strain L53T was located in a distinct phyletic lineage in a discrete clade associated with H. neustonica JCM 19743T. The DNA G+C content of strain L53T was 33.5 mol%. The sole menaquinone was MK-6. The polar lipids comprised one phosphatidylethanolamine, four unidentified aminolipids and four unidentified lipids. The major fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH and iso-C15 : 0 G. Based on morphological, physiological and molecular properties as well as on phylogenetic distinctiveness, strain L53T should be placed in the genus Hanstruepera as representing a novel species, for which the name Hanstruepera crassostreae sp. nov. is proposed. The type strain is L53T (=KCTC 62247T=MCCC 1H00246T).