Search Results (11)

Endophytic bacteria from pea (Pisum sativum L.) plants play important roles in regulating plant growth, health, and nutrition. To enhance the understanding of endophytic bacteria in peas, twenty pea cultivars, two chickpeas, and two broad bean cultivars were planted into artificial soils for 4 weeks. Leaves and roots were collected from plants and sterilized. Endophytic bacterial DNAs were isolated from sterilized materials (leaves, roots, and seeds) and used as templates to detect the bacterial diversity by amplifying the 16S V3–V4 region. The Remel Tryptose Soya Agar (TSA) medium, the aluminum sec-butoxide (ASb) medium, and the yeast extract mannitol agar (YMA) medium were used to isolate bacteria from sterilized leaves and roots, respectively. The plant growth-promoting (PGP) properties of these isolated bacteria, such as the solubilization of phosphorus and potassium and the production of Indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, nitrogenase, pectinase, and cellulose, were studied in vitro. Bacterial isolates were processed for 16S rDNA gene sequencing and performed molecular identification by reconstruction of the phylogenetic tree using the neighborhood association approach in the software MEGA X. Results indicated that the majority of the bacterial communities were shared among leaves, roots, and seeds of pea plants. In both the leaves and roots of pea plants, the prominent phyla identified were Pseudomonadota, Bacteroidota, and Bacillota, with dominant genera such as Rhizobium, Bacteroides, Blautia, and Prevotella prevailing at the genus level. The samples from leaves and roots had unique dominant bacterial genera. In total, 48 endophytic bacteria strains were isolated from leaves and roots, of which 16 strains were from roots and 32 strains were from leaves. The majority of the isolates from leaves (78.13%) and roots (75%) had the ability to produce indole-3-acetic acid (IAA). Moreover, isolates from roots also had greater ability to produce 1-amino-cyclopropane-1-carboxylic acid (ACC) deaminase (81.25%) than those from leaves (62.5%). This study demonstrated the unique distribution of endophytes in leaves and roots of pea, which can have great potential in pea production. Full article

Hybrid breeding is an effective approach to generate better varieties and prevent variety degradation. The present study investigated the metabolic changes underlying growth superiority in the novel Gymnocypris hybrid (GH), Gymnocypris przewalskii ♀ (GP) × G. eckloni ♂ (GE). The ranking of survival rate was GH > GE > GP, whereas the ranking of growth rate was GE > GH > GP. A proteomic analysis of G. hybrid and its parents was conducted to elucidate the metabolic changes underlying growth superiority. Identified pathways were primarily associated with amino acid, carbohydrate, energy, lipid, and nucleotide metabolism. These metabolic pathways, which are closely associated with growth, are controlled through regulation of the expression of numerous proteins, including adenosylhomocysteinase, hydroxypyruvate reductase, glutamate-cysteine ligase, L-lactate dehydrogenase, creatine kinase, GDP-L-fucose synthase, pyruvate kinase, fructose-bisphosphate aldolase, carbonic anhydrase, phosphopyruvate hydratase, phosphoglycerate kinase, S-(hydroxymethyl) glutathione dehydrogenase, and AMP deaminase. Real-time PCR assays showed that the level of mRNA expression of differentially expressed genes was positively correlated with growth. Proteins that were differentially expressed in GH exhibited fewer differences from GP and more differences from GE. These data are the first to reveal the molecular mechanism whereby growth is regulated in G. hybrid and its parents at the protein level. The study thus provides important information for genetic breeding and improvement of G. hybrid for aquaculture production. Full article

The flavor of chicken meat is influenced by muscle metabolites and regulatory genes and varies with age. In this study, the metabolomic and transcriptomic data of breast muscle at four developmental stages (days 1, 56, 98, and 120) of Beijing-You chickens (BJYs) were integrated and 310 significantly changed metabolites (SCMs) and 7,225 differentially expressed genes (DEGs) were identified. A Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that SCMs and DEGs were enriched in amino acid, lipid, and inosine monophosphate (IMP) metabolism pathways. Furthermore, genes highly associated with flavor amino acids, lipids, and IMP were identified by a weighted gene co-expression network analysis (WGCNA), including cystathionine β-synthase (CBS), glycine amidinotransferase (GATM), glutamate decarboxylase 2 (GAD2), patatin-like phospholipasedomain containing 6 (PNPLA6), low-specificity L-threonine aldolase (ItaE), and adenylate monophosphate deaminase 1 (AMPD1) genes. A regulatory network related to the accumulation of key flavor components was constructed. In conclusion, this study provides new perspectives regarding the regulatory mechanisms of flavor metabolites in chicken meat during development. Full article

The formation and development of chloroplasts play a vital role in the breeding of high-yield rice (Oryza sativa L.). Porphobilinogen deaminases (PBGDs) act in the early stage of chlorophyll and heme biosynthesis. However, the role of PBGDs in chloroplast development and chlorophyll production remains elusive in rice. Here, we identified the spotted leaf 42 (spl42) mutant, which exhibited a reddish-brown spotted leaf phenotype. The mutant showed a significantly lower chlorophyll content, abnormal thylakoid morphology, and elevated activities of reactive oxygen species (ROS)-scavenging enzymes. Consistently, multiple genes related to chloroplast development and chlorophyll biosynthesis were significantly down-regulated, whereas many genes involved in leaf senescence, ROS production, and defense responses were upregulated in the spl42 mutant. Map-based cloning revealed that SPL42 encodes a PBGD. A C-to-T base substitution occurred in spl42, resulting in an amino acid change and significantly reduced PBGD enzyme activity. SPL42 targets to the chloroplast and interacts with the multiple organelle RNA editing factors (MORFs) OsMORF8-1 and OsMORF8-2 to affect RNA editing. The identification and characterization of spl42 helps in elucidating the molecular mechanisms associated with chlorophyll synthesis and RNA editing in rice. Full article

Salvianic acid A (SAA), as the main bioactive component of the traditional Chinese herb Salvia miltiorrhiza, has important application value in the treatment of cardiovascular diseases. In this study, a two-step bioprocess for the preparation of SAA from l-DOPA was developed. In the first step, l-DOPA was transformed to 3,4-dihydroxyphenylalanine (DHPPA) using engineered Escherichia coli cells expressing membrane-bound L-amino acid deaminase from Proteus vulgaris. After that, the unpurified DHPPA was directly converted into SAA by permeabilized recombinant E. coli cells co-expressing d-lactate dehydrogenase from Pediococcus acidilactici and formate dehydrogenase from Mycobacterium vaccae N10. Under optimized conditions, 48.3 mM of SAA could be prepared from 50 mM of l-DOPA, with a yield of 96.6%. Therefore, the bioprocess developed here was not only environmentally friendly, but also exhibited excellent production efficiency and, thus, is promising for industrial SAA production. Full article

High soil acidity is one of the main unfavorable soil factors that inhibit the growth and mineral nutrition of plants. This is largely due to the toxicity of aluminum (Al), the mobility of which increases significantly in acidic soils. Symbiotic microorganisms have a wide range of beneficial properties for plants, protecting them against abiotic stress factors. This report describes the mechanisms of positive effects of plant growth-promoting rhizobacteria Pseudomonas fluorescens SPB2137 on four pea (Pisum sativum L.) genotypes grown in hydroponics and treated with 80 µM AlCl3. In batch culture, the bacteria produced auxins, possessed 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, alkalized the medium and immobilized Al, forming biofilm-like structures and insoluble phosphates. Inoculation with Ps. fluorescens SPB2137 increased root and/or shoot biomass of Al-treated plants. The bacteria alkalized the nutrient solution and transferred Al from the solution to the residue, which contained phosphorus that was exuded by roots. As a result, the Al concentration in roots decreased, while the amount of precipitated Al correlated negatively with its concentration in the solution, positively with the solution pH and negatively with Al concentration in roots and shoots. Treatment with Al induced root exudation of organic acids, amino acids and sugars. The bacteria modulated root exudation via utilization and/or stimulation processes. The effects of Al and bacteria on plants varied depending on pea genotype, but all the effects had a positive direction and the variability was mostly quantitative. Thus, Ps. fluorescens SPB2137 improved the Al tolerance of pea due to immobilization and exclusion of toxicants from the root zone. Full article

Honey peach (Prunus persica L.) is highly nutritious; it is an excellent source of sugars, proteins, amino acids, vitamins, and mineral elements. However, it is a perishable climacteric fruit that is difficult to preserve. In this study, “Feicheng” honey peach fruit was used as a test material to investigate the synergistic preservation effect of 1-methylcyclopropene (1-MCP) and laser microporous film (LMF). The peach fruits were fumigated for 24 h with 2 μL L⁻¹ 1-MCP, then packed in LMF. In comparison with the control treatment, 1-MCP + LMF treatment markedly decreased the respiration rate, weight loss, and rot rate of peach fruits. Moreover, the combination of 1-MCP and LMF suppressed the increase in soluble solids (SS) and reducing sugars (RS), as well as the decrease in titratable acid (TA) and ascorbic acid (AsA). The combined application also maintained a high protopectin content and low soluble pectin content; it reduced the accumulation of superoxide anions (O₂⁻) and hydrogen peroxide (H₂O₂). Except in a few samples, the catalase (CAT) and ascorbate peroxidase (APX) activities were higher when treated by 1-MCP + LMF. Conversely, the phenylalanine deaminase (PAL), peroxidase (POD), lipase, lipoxygenase (LOX), polygalacturonase (PG), β-glucosidase, and cellulase (Cx) activities were lower than in the control. Furthermore, 1-MCP + LMF treatment reduced the relative abundances of dominant pathogenic fungi (e.g., Streptomyces, Stachybotrys, and Issa sp.). The combined treatment improved the relative abundances of antagonistic fungi (e.g., Aureobasidium and Holtermanniella). The results indicated that the co-application of 1-MCP and LMF markedly reduced weight loss and spoilage, delayed the decline of nutritional quality, and inhibited the physiological and biochemical metabolic activities of peach during storage. These changes extended its shelf-life to 28 days at 5 °C. The results provide a reference for the commercial application of this technology. Full article

l-amino acid deaminases (LAADs) are flavoenzymes that catalyze the stereospecific oxidative deamination of l-amino acids into α-keto acids, which are widely used in the pharmaceutical, food, chemical, and cosmetic industries. However, the substrate specificity of available LAADs is limited, and most substrates are concentrated on several bulky or basic l-amino acids. In this study, we employed a LAAD from Proteus mirabilis (PmiLAAD) and broadened its substrate specificity using a semi-rational design strategy. Molecular docking and alanine scanning identified F96, Q278, and E417 as key residues around the substrate-binding pocket of PmiLAAD. Site-directed saturation mutagenesis identified E417 as the key site for substrate specificity expansion. Expansion of the substrate channel with mutations of E417 (E417L, E417A) improved activity toward the bulky substrate l-Trp, and mutation of E417 to basic amino acids (E417K, E417H, E417R) enhanced the universal activity toward various l-amino acid substrates. The variant PmiLAAD^E417K showed remarkable catalytic activity improvement on seven substrates (l-Ala, l-Asp, l-Ile, l-Leu, l-Phe, l-Trp, and l-Val). The catalytic efficiency improvement obtained by E417 mutation may be attributed to the expansion of the entrance channel and its electrostatic interactions. These PmiLAAD variants with a broadened substrate spectrum can extend the application potential of LAADs. Full article

L-amino acid deaminases (LAADs) are membrane flavoenzymes that catalyze the deamination of neutral and aromatic L-amino acids to α-keto acids and ammonia. LAADs can be used to develop many important biotechnological applications. However, the transmembrane α-helix of LAADs restricts its soluble active expression and purification from a heterologous host, such as Escherichia coli. Herein, through fusion with the maltose-binding protein (MBP) tag, the recombinant E. coli BL21 (DE3)/pET-21b-MBP-PmLAAD was constructed and the LAAD from Proteus mirabilis (PmLAAD) was actively expressed as a soluble protein. After purification, the purified MBP-PmLAAD was obtained. Then, the catalytic activity of the MBP-PmLAAD fusion protein was determined and compared with the non-fused PmLAAD. After fusion with the MBP-tag, the catalytic efficiency of the MBP-PmLAAD cell lysate was much higher than that of the membrane-bound PmLAAD whole cells. The soluble MBP-PmLAAD cell lysate catalyzed the conversion of 100 mM L-phenylalanine (L-Phe) to phenylpyruvic acid (PPA) with a 100% yield in 6 h. Therefore, the fusion of the MBP-tag not only improved the soluble expression of the PmLAAD membrane-bound protein, but also increased its catalytic performance. Full article

Reactive intermediate deaminase (Rid) protein family is a recently discovered group of enzymes that is conserved in all domains of life and is proposed to play a role in the detoxification of reactive enamines/imines. UK114, the mammalian member of RidA subfamily, was identified in the early 90s as a component of perchloric acid-soluble extracts from goat liver and exhibited immunomodulatory properties. Multiple activities were attributed to this protein, but its function is still unclear. This work addressed the question of whether UK114 is a Rid enzyme. Biochemical analyses demonstrated that UK114 hydrolyzes α-imino acids generated by l- or d-amino acid oxidases with a preference for those deriving from Ala > Leu = l-Met > l-Gln, whereas it was poorly active on l-Phe and l-His. Circular Dichroism (CD) analyses of UK114 conformational stability highlighted its remarkable resistance to thermal unfolding, even at high urea concentrations. The half-life of heat inactivation at 95 °C, measured from CD and activity data, was about 3.5 h. The unusual conformational stability of UK114 could be relevant in the frame of a future evaluation of its immunogenic properties. In conclusion, mammalian UK114 proteins are RidA enzymes that may play an important role in metabolism homeostasis also in these organisms. Full article

Ornithine cyclodeaminase (OCD) is an NAD⁺-dependent deaminase that is found in bacterial species such as Pseudomonas putida. Importantly, it catalyzes the direct conversion of the amino acid L-ornithine to L-proline. Using molecular dynamics (MD) and a hybrid quantum mechanics/molecular mechanics (QM/MM) method in the ONIOM formalism, the catalytic mechanism of OCD has been examined. The rate limiting step is calculated to be the initial step in the overall mechanism: hydride transfer from the L-ornithine’s C_α–H group to the NAD⁺ cofactor with concomitant formation of a C_α=NH₂⁺ Schiff base with a barrier of 90.6 kJ mol⁻¹. Importantly, no water is observed within the active site during the MD simulations suitably positioned to hydrolyze the C_α=NH₂⁺ intermediate to form the corresponding carbonyl. Instead, the reaction proceeds via a non-hydrolytic mechanism involving direct nucleophilic attack of the δ-amine at the C_α-position. This is then followed by cleavage and loss of the α-NH₂ group to give the Δ¹-pyrroline-2-carboxylate that is subsequently reduced to L-proline. Full article