Efficient Expression and Application of a Modified Rhizomucor miehei Lipase for Simultaneous Production of Biodiesel and Eicosapentaenoic Acid Ethyl Ester from Nannochloropsis Oil.

Few reports exist on one-step enzymatic methods for the simultaneous production of biodiesel and eicosapentaenoic acid ethyl ester (EPA-EE), a high-value pharmaceutical compound. This study aimed to efficiently express Rhizomucor miehei lipase (pRML) in Pichia pastoris X-33 via propeptide mutation and high-copy strain screening. The mutated enzyme was then used to simultaneously catalyze the production of both biodiesel and EPA-EE. The P46N mutation in the propeptide (P46N-pRML) significantly boosted its production, with the four-copy strain increasing enzyme yield by 3.7-fold, reaching 3425 U/mL. Meanwhile, its optimal temperature increased to 45-50 °C, pH expanded to 7.0-8.0, specific activity doubled, Km reduced to one-third, and kcat/Km increased 7-fold. Notably, P46N-pRML efficiently converts Nannochloropsis gaditana oil's eicosapentaenoic acid (EPA). Under optimal conditions, it achieves up to 93% biodiesel and 92% EPA-EE yields in 9 h. Our study introduces a novel, efficient one-step green method to produce both biodiesel and EPA-EE using this advanced enzyme.

Characterization of a Novel Polysaccharide Derived from Rhizospheric Paecilomyces vaniformisi and Its Mechanism for Enhancing Salinity Resistance in Rice Seedlings.

Soil salinity is an important limiting factor in agricultural production. Rhizospheric fungi can potentially enhance crop salinity tolerance, but the precise role of signaling substances is still to be systematically elucidated. A rhizospheric fungus identified as Paecilomyces vaniformisi was found to enhance the salinity tolerance of rice seedlings. In this study, a novel polysaccharide (PPL2b) was isolated from P. vaniformisi and identified as consisting of Manp, Glcp, GalpA, and Galp. In a further study, PPL2b showed significant activity in alleviating salinity stress-induced growth inhibition in rice seedlings. The results indicated that under salinity stress, PPL2b enhances seed germination, plant growth (height and biomass), and biochemical parameters (soluble sugar and protein contents). Additionally, PPL2b regulates genes such as SOS1 and SKOR to decrease K+ efflux and increase Na+ efflux. PPL2b increased the expression and activity of genes related to antioxidant enzymes and nonenzyme substances in salinity-induced oxidative stress. Further study indicated that PPL2b plays a crucial role in regulating osmotic substances, such as proline and betaine, in maintaining the osmotic balance. It also modulates plant hormones to promote rice seedling growth and enhance their tolerance to soil salinity. The variables interacted and were divided into two groups (PC1 77.39% and PC2 18.77%) based on their relative values. Therefore, these findings indicate that PPL2b from P. vaniformisi can alleviate the inhibitory effects of salinity stress on root development, osmotic adjustment, ion balance, oxidative stress balance, and growth of rice seedlings. Furthermore, it suggests that polysaccharides produced by rhizospheric fungi could be utilized to enhance crop tolerance to salinity.

Evaluation of negative effect of Naphthenic acids (NAs) on physiological metabolism and polycyclic aromatic hydrocarbons adsorption of Phragmites australis.

Toxic substances in the environment disturb the adsorption of pollutants in plants but little is known about the underlying mechanisms of these processes. This study evaluated the PAH adsorption by Phragmites australis under NAs stress. Results showed that Naphthenic acids (NAs) significantly decreased the adsorption of PAHs and had higher selectivity for type and structure. P. australis root cell growth and mitosis were significantly affected by NAs, which was accompanied by serious disturbances in mitochondrial function. The physiological evaluation showed the NAs could increase Reactive Oxygen Species (ROS) accumulation by around 16-fold and cause damage to the root cell normal redox equilibrium. The levels of three key related antioxidants, PLA, CAT and POD, decreased significantly to 35-50% under NAs stress and were dependent upon NAs concentration. Furthermore, NAs could significantly change the concentration and species of root exudates of P. ausralis. Autotoxic substances, including alcohol and amines, increased by 28.63% and 23.96, respectively. Sixteen compounds were identified and assumed as potential biomarkers. Galactonic, glyceric, and octadecanoic acid had the general effect of activating PAH in soil. The global view of the metabolic pathway suggests that NAs influenced the citric acid cycle, fatty acid synthesis, amino acid metabolism and the phenylpropanoid pathway. Detection data results indicated that the energy products cause hypoxia and oxidative stress, which are the main processes under the NAs. Furthermore, verification of these processes was fulfilled through gene expression and biomarkers quantification. Our results provide novel metabolic insights into the mechanisms of PAHs adsorption by P. australis under NAs disturbance, suggesting that monitoring NAs in phytoremediation applications is necessary.

Perturbations in nitric oxide homeostasis promote Arabidopsis disease susceptibility towards Phytophthora parasitica.

Phytophthora species can infect hundreds of different plants, including many important crops, causing a number of agriculturally relevant diseases. A key feature of attempted pathogen infection is the rapid production of the redox active molecule nitric oxide (NO). However, the potential role(s) of NO in plant resistance against Phytophthora is relatively unexplored. Here we show that the level of NO accumulation is crucial for basal resistance in Arabidopsis against Phytophthora parasitica. Counterintuitively, both relatively low or relatively high NO accumulation leads to reduced resistance against P. parasitica. S-nitrosylation, the addition of a NO group to a protein cysteine thiol to form an S-nitrosothiol, is an important route for NO bioactivity and this process is regulated predominantly by S-nitrosoglutathione reductase 1 (GSNOR1). Loss-of-function mutations in GSNOR1 disable both salicylic acid accumulation and associated signalling, and also the production of reactive oxygen species, leading to susceptibility towards P. parasitica. Significantly, we also demonstrate that secreted proteins from P. parasitica can inhibit Arabidopsis GSNOR1 activity.

A new chitosan sub-micron and encapsulated Iturin A with enhanced antifungal activity against Ceratocystis fimbriata and Rhizopus strolonifer.

Iturin A is a natural antifungal agent that is widely used in the agriculture and food industries. In the present study, a new modified chitosan sub-micron, succinaldehydic acid (SAC) - chitosan (CS) system was synthesised by microwave irradiation and then as a carrier for capsulation of Iturin A (SAC-CS-IA). The structures of SAC-CS and SAC-CS-IA were characterised by SEM, NMR and FTIR. The size distribution suggested that the average size of SAC-CS and SAC-CS-IA was from 1.5 to 2.5 µm. An encapsulation efficiency of 92.02% under an adaptive pH (pH = 5) and time (5.5 h) was used. The study of release kinetics shows that about 80% of Iturin A was released in 25 days. An antifungal activity assay indicated that SAC-CS-IA exhibited higher antifungal activity against Ceratocystis fimbriata and Rhizopus strolonifer with 75.05 ± 3.24% and 80.54 ± 2.65%, respectively. The results indicate that the SAC-CS can improve the stability of IA on heat and pH with a wide range and tolerance most of enzymes. Actual tuber storage suggested that SAC-CS-IA can significantly inhibit pathogen fungal infection and reduce toxin product. Meanwhile, SAC-CS-IA could retain the water, starch, and soluble sugar contents. Low residue assay indicated that SAC-CS-IA could be used as an antifungal and anti-rotting agent in agriculture and food applications.

Fermentation conditions optimization, purification, and antioxidant activity of exopolysaccharides obtained from the plant growth-promoting endophytic actinobacterium Glutamicibacter halophytocola KLBMP 5180.

In this study, an endophytic actinobacterium Glutamicibacter halophytocola KLBMP 5180, was investigated for the production and antioxidant activity of exopolysaccharides (EPSs). First, the suitable fermentation time, temperature, inoculation volume, pH value, and the carbon and nitrogen sources for EPSs production were obtained using the one variable at a time method (OVAT). Then, a central composition design was used for fermentation conditions optimization to obtain the maximum EPS yield. The optimal medium and condition were as follows: 100 mL broth in 250 mL Erlenmeyer flasks, including 3.65 g/L maltose, 9.88 g/L malt extract, 3.40 g/L yeast extract, 1.41 g/L MnCl2, pH 7.5, culture temperature 28 °C, and 200 rpm for 7 days, which increased the yield of EPSs to 2.89 g/L. Two purified EPSs, 5180EPS-1 (MW 58.9 kDa) and 5180EPS-2 (10.5 kDa), comprising rhamnose, galacturonic acid, glucose, glucuronic acid, xylose, and arabinose, were obtained for chemical analysis and antioxidant evaluation. The scavenging ability and reducing power of the superoxide anion and hydroxyl radicals demonstrated the moderate in vitro antioxidant activities of the two EPSs, thus indicating their potential to be a new source of natural antioxidants. However, further structure elucidation and functional studies need to be continued.

Community Composition and Metabolic Potential of Endophytic Actinobacteria From Coastal Salt Marsh Plants in Jiangsu, China.

The diversity and functional roles of the plant associated endophytic actinobacteria in unique habitats remain poorly understood. In this paper, we examined the phylogenetic diversity and community composition of endophytic actinobacteria associated with native coastal salt marsh plants in Jiangsu, China using a combination of cultivation and 16S rRNA gene-based high-throughput sequencing (HTS) methods. Further, we evaluated the antifungal, fibrinolytic activities and the secondary metabolite biosynthesis potential of isolates via gene screening. A total of 278 actinobacterial isolates were isolated from 19 plant samples. 16S rRNA gene sequencing revealed that the isolates were highly diverse and belonged to 23 genera within the Actinomycetales order, with Streptomyces, Saccharopolyspora, and Pseudonocardia comprising the most abundant genera. In addition, more than 10 of the isolates were novel actinobacterial taxa distributed across eight genera. HTS analyses of seven representative plant root samples revealed that Actinobacteria phylum constituted 0.04-28.66% of root endophytic bacterial communities. A total of four actinobacterial classes, 14 orders, 35 families, and 63 known genera were detected via HTS, and these communities were found to be dominated by the members of the order Actinomycetales including the genera Streptomyces, Mycobacterium, Arthrobacter, Nocardioides, and Micromonospora. In addition, 30.4% of the representative isolates exhibited antifungal activities, 40.5% of them showed fibrinolytic activities, while 43.0% of the strains harbored secondary metabolite biosynthesis genes. These results demonstrated that coastal salt marsh plants in the Jiangsu Province represented an underexplored new reservoir of diverse and novel endophytic actinobacteria that may be of potential interest in the discovery of bioactive compounds with potential as biocontrol agents and for fibrinolytic enzyme production.

Synthesis and in vitro antifungal efficacy of oleoyl-chitosan nanoparticles against plant pathogenic fungi.

An antifungal dispersion system was prepared by oleoyl-chitosan (O-chitosan) nanoparticles, and the antifungal activity against several plant pathogenic fungi was investigated. Under scanning electron microscopy, the nanoparticles formulation appeared to be uniform with almost spherical shape. The particle size of nanoparticles was around 296.962 nm. Transmission electron microscopy observation showed that nanoparticles could be well distributed in potato dextrose agar medium. Mycelium growth experiment demonstrated that Nigrospora sphaerica, Botryosphaeria dothidea, Nigrospora oryzae and Alternaria tenuissima were chitosan-sensitive, while Gibberella zeae and Fusarium culmorum were chitosan-resistant. The antifungal index was increased as the concentration of nanoparticles increased for chitosan-sensitive fungi. Fatty acid analyses revealed that plasma membranes of chitosan-sensitive fungi were shown to have lower levels of unsaturated fatty acid than chitosan-resistant fungi. Phylogenetic analysis based on ITS gene sequences indicated that two chitosan-resistant fungi had a near phylogenetic relationship. Results showed that O-chitosan nanoparticles could be a useful alternative for controlling pathogenic fungi in agriculture.

Tamaricihabitans halophyticus gen. nov., sp. nov., an endophytic actinomycete of the family Pseudonocardiaceae.

A novel actinomycete strain, designated KLBMP 1356T, was isolated from the root of halophyte Tamarix chinensis Lour. collected from the coastal area of Jiangsu province, PR China. The isolate was characterized using a polyphasic approach. Comparative analysis of the 16S rRNA gene sequence indicated that strain KLBMP 1356T was phylogenetically related to members of the family Pseudonocardiaceae and formed a distinct monophyletic clade between the genera Amycolatopsis (93.1-94.7 % 16S rRNA gene sequence similarity), Prauserella (93.6-95.1 %) and Saccharomonospora (93.2-94.3 %). The isolate displayed long spore chains containing rod-shaped and smooth-surfaced spores. Strain KLBMP 1356T contained meso-diaminopimelic acid as the diagnostic diamino acid, and galactose, arabinose and glucose as the whole-cell sugars. The major menaquinone was MK-9(H4) and the fatty acid profile was characterized by the predominance of iso-C16 : 0, C17 : 1ω8c, C17 : 1ω6c and C17 : 0. The polar lipids comprised diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, unknown aminophospholipids and an unknown glycolipid. Mycolic acids were not present. The G+C content of the genomic DNA was 67.2 mol%. On the basis of the evidence from this polyphasic study, strain KLBMP 1356T is considered to represent a novel species of a new genus in the family Pseudonocardiaceae, for which the name Tamaricihabitans halophyticus gen. nov., sp. nov. is proposed. The type strain of the type species is KLBMP 1356T ( = DSM 45765T = NBRC 109361T).

Nocardia rhizosphaerae sp. nov., a novel actinomycete isolated from the coastal rhizosphere of Artemisia Linn., China.

A novel actinomycete, designated strain KLBMP S0043(T), was isolated from the rhizosphere soil of Artemisia Linn. collected from the coastal region of Lianyungang, Jiangsu Province, in east China and was studied in detail for its taxonomic position. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain KLBMP S0043(T) is a member of the genus Nocardia. The 16S rRNA gene sequence similarity indicated that strain KLBMP S0043(T) is closely related to Nocardia asteroides NBRC 15531(T) (97.61 %) and Nocardia neocaledoniensis SBHR OA6(T) (97.38 %); similarity to other type strains of the genus Nocardia was found to be less than 97.2 %. The organism has chemical and morphological features consistent with its classification in the genus Nocardia such as meso-diaminopimelic acid as the diagnostic diamino acid in the cell wall peptidoglycan and arabinose and galactose as the diagnostic sugars. The predominant menaquinone was identified as MK-8(H4ω-cycl). Mycolic acids were detected. The diagnostic phospholipids were found to be diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannosides. The predominant cellular fatty acids were identified as C16:0, C18:0, C18:1ω9c, 10-methyl C18:0 [tuberculostearic acid (TBSA)] and summed feature 3 (C16:1ω7c/C16:1ω6c). The G+C content of the genomic DNA was determined to be 71.4 mol%. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of the strain from its most closely related strains. Based on morphological, chemotaxonomic and phylogenetic data, strain KLBMP S0043(T) is considered to represent a novel species of the genus Nocardia, for which the name Nocardia rhizosphaerae sp. nov. is proposed. The type strain is KLBMP S0043(T) (=CGMCC 4.7204 (T) = KCTC 29678(T)).

Purification and characterization of a fibrinolytic enzyme from Streptomyces sp. XZNUM 00004.

A fibrinolytic enzyme (SFE1) from Streptomyces sp. XZNUM 00004 was purified to electrophoretic homogeneity with the methods including ammonium sulfate precipitation, polyacrylamide gel, DEAE-Sepharose Fast Flow anion exchange and gel-filtration chromatography. The molecular weight of SFE1 was estimated to be 20 kDa by SDS-PAGE, fibrin zymography, and gel filtration chromatography. The isoelectric point was 4.9. K (m) and V (max) values were 0.96 mg/ml and 181.8 unit/ml, respectively. It was very stable at pH 5.0-8.0 and below 65 °C. The optimum pH for enzyme activity was 7.8. The optimum temperature was 35 °C. The fibrinolytic activity of SFE1 was enhanced by Na(+), K(+), Mn(2+), Mg(2+), Zn(2+) and Co(2+). Conversely, Cu(2+) showed strong inhibition. Furthermore, the fibrinolytic activity was strongly inhibited by PMSF, and partly inhibited by EDTA and EGTA. SFE1 rapidly hydrolyzed the Aα-chain of fibrinogen, followed by the Bß-chain and finally the γ-chain. The first 15 amino acids of the N-terminal sequence were APITLSQGHVDVVDI. Additionally, SFE1 directly digested fibrin and not by plasminogen activators in vitro. SFE1 can be further developed as a potential candidate for thrombolytic therapy.

Streptomyces phytohabitans sp. nov., a novel endophytic actinomycete isolated from medicinal plant Curcuma phaeocaulis.

A novel actinomycete, designated strain KLBMP 4601(T), was isolated from the root of the medicinal plant Curcuma phaeocaulis collected from Sichuan Province, south-west China. The strain produced extensively branched substrate and aerial hyphae that carried straight to flexuous spore chains. Chemotaxonomic properties of this strain were consistent with those of members of the genus Streptomyces. The cell wall of strain KLBMP 4601(T) contained LL-diaminopimelic acid as the characteristic diamino acid. The major menaquinone was MK-9(H(4)), with minor amounts of MK-9(H(6)), MK-9(H(8)) and MK-10(H(2)). The major fatty acids were C(16:0), iso-C(16:0), C(18:1)ω9c and C(16:1), iso G. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain KLBMP 4601(T) belongs to the genus Streptomyces and is most closely related to Streptomyces armeniacus JCM 3070(T) (97.9 %), Streptomyces pharmamarensis PM267(T) (97.6 %) and Streptomyces artemisiae YIM 63135(T) (97.5 %). The 16S rRNA gene sequence similarity between strain KLBMP 4601(T) and other members of this genus were lower than 97.5 %. DNA-DNA hybridization studies of strain KLBMP 4601(T) with the three closest species showed relatedness values of 36.3 ± 4.2 %, 27.3 ± 0.6 %, and 30.9 ± 2.5 %, respectively. On the basis of chemotaxonomic, phenotypic and genotypic characteristics, it is evident that strain KLBMP 4601(T) represents a novel species of the genus Streptomyces, for which the name Streptomyces phytohabitans sp. nov. is proposed. The type strain is KLBMP 4601(T) (=KCTC 19892(T) = NBRC 108772(T)).

Molecular characterization and expression analysis of a gene encoding for farnesyl diphosphate synthase from Euphorbia pekinensis Rupr.

The root of Euphorbia pekinensis as a traditional herbal medicine has been recorded in Chinese pharmacopoeias for the treatment of oedema, gonorrhea, migraine and wart cures. In this work, we reported on the cDNA cloning and characterization of a novel farnesyl diphosphate synthase (FPS) from E. pekinensis. The full-length cDNA named EpFPS (Genbank Accession Number FJ755465) contained 1431 bp with an open reading frame of 1029 bp encoding a polypeptie of 342 amino acids. The deduced amino acid sequence of the EpFPS named EpFPS exhibited a high homology with other plant FPSs, and contained five conserved domains. Phylogenetic analysis showed that EpFPS belonged to the plant FPS group. Southern blot analysis revealed that there exists a small FPS gene family in E. pekinensis. Expression pattern analysis revealed that EpFPS expressed strongly in root, weak in leaf and stem. In callus, expression of EpFPS gene and biosynthesis of triterpenoids were strongly induced by Methyl jasmonate and slightly induced by Salicylic acid. Functional complementation of EpFPS in an ergosterol auxotrophic yeast strain indicated that the cloned cDNA encoded a functional farnesyl diphosphate synthase.

Rapid isolation and purification of inotodiol and trametenolic acid from Inonotus obliquus by high-speed counter-current chromatography with evaporative light scatting detection.

INTRODUCTION: In Eastern Europe, especially Russia, the fruiting body of Inonotus obliquus has been used as a folk medicine for cancer since the sixteenth or seventeenth century. Inotodiol and trametenolic acid are considered to be the main bioactive compounds of the fruiting body of the mushroom. These compounds show various biological activities, including anti-tumour, anti-viral, hypoglycaemic, anti-oxidant and cyto-protective. However, effective methods for isolating and purifying inotodiol and trametenolic acid from the fruiting body of Inonotus obliquus are not currently available. OBJECTIVE: To develop a suitable preparative method in order to isolate inotodiol and trametenolic acid from a complex Inonotus obliquus extract by preparative high-speed counter-current chromatography (HSCCC). METHODOLOGY: Inotodiol and trametenolic acid were rapidly isolated and purified from the chloroform extract of Inonotus obliquus (Fr.) by HSCCC with evaporative light scatting detection (ELSD). The purity of the obtained target compounds was analysed by high-performance liquid chromatography (HPLC) with ELSD. The structures of the two compounds were identified by ¹H NMR and ¹³C NMR. RESULT: The target compounds were finally isolated and purified with a solvent system of hexane:ethyl acetate:methanol:water (1:0.4:1:0.4, v/v/v/v). In a single operation, 100 mg of the I. obliquus extracts yielded 13.0 mg of inotodiol and 7.0 mg of trametenolic acid. The entire separation and purification process took less than 5 h. The purities of obtained inotodiol and trametenolic acid were 97.51 and 94.04%, respectively. CONCLUSION: HSCCC-ELSD was an efficient and rapid method for the separation and purification of inotodiol and trametenolic acid from I. obliquus.

Molecular cloning, characterization and function analysis of the gene encoding HMG-CoA reductase from Euphorbia Pekinensis Rupr.

A new full-length cDNA encoding 3-hydroxy-3-methylglutoryl-Coenzyme A reductase (HMGR; EC1.1.1.34), which catalyzes the first committed step of isoprenoids biosynthesis in MVA pathway, was isolated from young leaves of Euphorbia Pekinensis Rupr. by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of HMGR (designated as EpHMGR, GenBank Accession NO. EF062569) was 2,200 bp containing a 1,752 bp ORF encoding 583 amino acids. Bioinformatic analyzes revealed that the deduced EpHMGR had extensive homology with other plant HMGRs and contained two transmembrane domains and a catalytic domain. The predicted 3-D model of EpHMGR had a typical spatial structure of HMGRs. Southern blot analysis indicated that at most two copies of EpHMGR gene existed in E. Pekinensis genome. Tissue expression analysis revealed that EpHMGR expressed strongly in roots, weakly in stems and leaves. The functional colour complementation assay indicated that EpHMGR could accelerate the biosynthesis of carotenoids in the Escherichia coli transformant, demonstrating that EpHMGR plays an influential role in isoprenoid biosynthesis.

Two-Step Purification of Cordycepin from Cordyceps Millitaris by High-Speed Countercurrent Chromatography.

Cordycepin is successfully isolated and purified from Cordyceps millitaris in two-step purification by high-speed countercurrent chromatography. Two solvent systems, ethyl acetate-1-butanol-water (3:2:5, v/v/v) and trichloromethane-methanol-1-butanol-water (2:1:0.25:1, v/v/v/v), were used for the two-step purification. The purity of the prepared cordycepin was 98.1% according to the high-performance liquid chromatography analysis.