Sheng wu gong cheng xue bao = Chinese journal of biotechnology最新文献

The homologous regions (hrs) of Bombyx mori nucleopolyhedrovirus (BmNPV) have been demonstrated to function as transcriptional enhancers and initiation sites of DNA replication. This study aimed to elucidate the protein-binding characteristics of hrs in the BmNPV genome and their regulatory mechanisms in viral infection. Using DNA pull-down coupled with LC-MS/MS, we systematically analyzed four highly interactive hrs (hr1, hr2L, hr3, and hr5), successfully identifying 215‒612 specific binding proteins for each region. Our findings revealed that these hrs not only bind to with numerous host proteins but also with multiple viral proteins. Notably, 15 proteins exhibited binding affinity to all four hrs, which suggested that these core interacting proteins may play pivotal roles in hrs-mediated regulation. Further analysis demonstrated that 67.3% of the binding proteins possessed multivalent binding properties, indicating that hrs may coordinate viral genome regulation through shared protein interaction networks. These results provide significant insights into the crucial regulatory functions of hrs in BmNPV infection, offer potential targets for developing antiviral strategies in silkworms, and contribute to a deeper understanding of baculovirus-host interactions at the molecular level.

Abscisic acid, stress and ripening-induced (ASR) proteins play crucial roles in plant ripening induction and stress responses. Although ASR proteins have been identified in various plant species, systematic studies in sugarcane remain limited, and their structural and functional characteristics are poorly understood. To this end, this study aimed to systematically identify members of the sugarcane ASR gene family at the genome-wide level, elucidate their molecular characteristics and evolutionary relationships, and investigate their expression patterns during growth, development, and stress responses. The results showed that a total of 40 ShASR genes from the Saccharum spp. cultivar 'R570' and 15 SsASR genes from the wild species Saccharum spontaneum were identified. All the predicted ASR proteins contained the characteristic ABA/WDS domain. Phylogenetic analysis, using rice ASR proteins as a reference, classified the sugarcane ASRs into three distinct subfamilies. Physicochemical property predictions indicated that sugarcane ASR proteins were stable and hydrophilic, mainly localized to the nucleus. Further analyses of gene structures, duplication patterns, and chromosomal distribution revealed that ASR genes mainly expanded through whole-genome or segmental duplication events and exhibited subfamily-specific clustering on chromosomes. Promoter analysis showed enrichment of cis-acting elements related to stress and plant hormone responses, as well as growth and development. Transcriptomic data revealed that most ASR genes were expressed in sugarcane leaf, leaf sheath, pith, skin, and bud samples and were responsive to smut pathogen infection and drought stress. RT-qPCR results confirmed that six ShASR genes (ShASR1, ShASR6, ShASR14, ShASR20, ShASR25, and ShASR40) showed differential expression patterns in response to exogenous plant hormone treatments and smut pathogen challenge. These findings suggested that ShASR genes might play important roles in regulating stress responses, disease resistance, and development in sugarcane. This study provides a comprehensive understanding of the sugarcane ASR gene family and offers valuable genetic resources for molecular breeding of stress-tolerant sugarcane cultivars.

Peanut (Arachis hypogaea L.) is an important oilseed crop widely cultivated in tropical and subtropical regions. The growth-regulating factors (GRFs) are key transcription factors that regulate plant growth and responses to stress. To improve the peanut yield and stress tolerance, it is crucial to investigate the roles of GRFs in growth, development, and stress responses. In this study, we analyzed the physicochemical properties, evolutionary relationships, chromosomal localization, and sequence variations of the AhGRF gene family by bioinformatics methods. Using qRT-PCR, we revealed the expression patterns of AhGRF genes under drought and cold stress conditions. Subcellular localization expression vectors were constructed to determine the cellular distribution of AhGRF2b and AhGRF3b. Finally, yeast two-hybrid (Y2H) assays were performed to identify interacting proteins of AhGRF3b. The results revealed that twenty-four AhGRF genes were identified in peanut, which were unevenly distributed across 16 chromosomes. The deduced proteins ranged from 268 to 630 aa in length, with molecular weights spanning 29 842.27 to 67 980.83 Da. Most AhGRFs were acidic and predicted to be localized in the nucleus. Phylogenetic analysis classified the AhGRF family members into six distinct clades. Multiple sequence alignment demonstrated that the majority of AhGRF genes contained conserved QLQ and WRC domains. Under drought and cold stress conditions, several AhGRF genes, particularly AhGRF2b and AhGRF3b, exhibited significantly upregulated expression, which indicated their responsiveness to abiotic stresses. Transient expression in tobacco showed that AhGRF2b was localized in both the nucleus and cytoplasm, while AhGRF3b was localized in the nucleus. Furthermore, Y2H assays revealed that AhGRF3b may interact with AhCAT3 (catalase), suggesting that AhGRF genes may enhance stress tolerance by regulating reactive oxygen species scavenging. These findings provide a theoretical basis for improving the stress tolerance in peanut breeding programs.

This study aimed to explore novel β-glucosidases with unique environmental adaptability and investigate their potential application in hydrolyzing ginsenoside Rb1. A GH3 family β-glucosidase gene TsBgl3 was successfully cloned from the marine-derived intestinal bacterium Tamlana sp. I1, and a recombinant enzyme with good solubility was obtained through an optimized Escherichia coli heterologous expression system. It was identified that the molecular weight of the recombinant enzyme TsBgl3 was 80.8 kDa, and the optimal reaction conditions were pH 6.0 and 37 ℃. This enzyme exhibited remarkable low-temperature catalytic properties and maintained a relative activity of 16.56% at 0 ℃. Kinetic analysis indicated that TsBgl3 exhibited high substrate affinity and catalytic efficiency for the substrate 4-nitrophenyl-beta-D-glucopyranoside (pNPG), with the K_m, V_max, and k_cat/K_m values of 3.65 mmol/L, 578.04 μmol/(mg·min), and 213.01 L/(mmol·s), respectively. It is worth noting that TsBgl3 exhibited excellent salt tolerance, with its enzymatic activity increasing by 57.47% in a 2 mol/L NaCl solution. In addition, the saponin hydrolysis experiment demonstrated that TsBgl3 could specifically hydrolyze the β-(1, 6)-glucosidic bond at the C-20 position in the ginsenoside Rb1 molecule, showing high specificity. Moreover, the substrate could be completely converted to ginsenoside Rd within 11 h (HPLC detected conversion rate > 99%). In conclusion, we successfully obtained a novel β-glucosidase, TsBgl3, which possessed both cold adaptability and high salt tolerance. This enzyme not only provides an efficient biocatalyst for the green preparation of rare ginsenosides but also offers a new path for the development and utilization of marine microbial resources.

Electrogenetics is a new field of synthetic biology, combining electronic devices and genetic methods to control gene expression and related cell functions. It covers a variety of fields such as synthetic biology, genetics, and electrochemistry and has been widely concerned by scientific research and academic circles at home and abroad, demonstrating great application prospects and potential in cell-cell communication, cell physiology and metabolism regulation, digital information storage, and disease treatment. In this paper, we summarized the components, advantages, and development process of electrogenetic systems and introduced the key response regulatory elements (such as metabolite transcription factors, oxidative stress transcription factors, and mammalian nuclear factors) of these systems. Then, we described electrogenetic regulatory systems based on these regulatory elements and discussed their applications in detail. Finally, we summed up the development and looked into the prospects of electrogenetic technology in synthetic biology. In the meanwhile, our paper pointed out the deficiencies of electrogenetics at present and proposed its future research directions and possible development trends, aiming at providing references and ideas for relevant researchers to promote the progress of electrogenetics research in synthetic biology.

Phthalate esters (PAEs) are widely used as plasticizers to improve the flexibility and durability of plastics, while they have emerged as persistent environmental contaminants due to their widespread presence in environmental media and endocrine-disrupting effects. Microbial degradation is an effective remediation strategy for removing PAEs in the environment, among which bacteria have become the main research objects due to their excellent PAE tolerance and degradation ability. It is worth noting that some PAE-degrading bacteria have substrate preference, which may directly affect their repair efficiency in actual environmental pollution sites. We detail the degradation bacteria with PAE substrate preference reported in recent years and review the research progress in the metabolic pathways of PAE-degrading bacteria, the action mechanisms of esterases, the transport mechanisms of transporters, and the applications of the bacteria in the bioremediation of PAE pollution, aiming to provide more solutions for the governance of environmental pollution problems caused by PAEs.

Receptor for activated C kinase 1 (RACK1), a scaffold protein, functions in different biological processes in plants through interacting with various receptor kinases/proteins and heterotrimeric G proteins. The functions of RACK1 have been investigated extensively in the model plant Arabidopsis. However, little is known about the roles of RACK1 homologs in soybean. Soybean is a paleotetraploidy plant and each gene has two copies in its genome. As a result, the forward genetic approaches are not suitable for studying the gene functions in soybean. To resolve the gene redundancy, we used Bean pod mottle virus-induced gene silencing approach to interrogate gene functions in soybean. Using this approach, we successfully silenced two homologous genes of GmRACK1 (GmRACK1A/1B) in soybean. The GmRACK1A/1B-silenced plants exhibited significantly compromised resistance to Soybean mosaic virus, Pseudomonas syringae pv. glycinea (Psg), and Xanthomonas campestris pv. glycinea (Xag). The compromised disease resistance was correlated with the reduced activation of GmMPK3/6 in response to Psg infection. Taken together, our results indicate that GmRACK1A/1B play positive roles in soybean immunity possibly through activating GmMPK3/6, demonstrate that GmRACK1 could serve as a potential target for molecular breeding, laying the foundation for enhancing broad-spectrum resistance in soybean through genetic engineering approaches.

Cytochrome P450 enzymes constitute the largest superfamily of oxidoreductases in nature, playing pivotal roles in drug metabolism, plant secondary metabolism, and biotransformation of environmental pollutants. To generate artificial P450 enzyme sequences with high fidelity and diversity, we propose P450Diff2, a novel diffusion model-based approach for generating P450 enzyme sequences. Built upon the EvoDiff-Seq framework comprising 640 million parameters, P450Diff2 was trained on a comprehensive dataset of 1 041 254 non-redundant P450 protein sequences collected from NCBI, GMind annotations, RNA-Seq assemblies, and metagenomic databases. Evaluation of the generated sequences revealed that P450Diff2 outperformed the previously proposed P450Diffusion model across multiple metrics, including amino acid composition distribution, sequence feature space coverage, sequence similarity profiles, and structural plausibility. Notably, the generated sequences achieved an average pLDDT score of 72.29. Experimental results further demonstrate that 60% of the generated sequences can correctly fold into biologically active P450 enzymes, indicating that the proposed method not only effectively preserves the structural features of natural sequences but also exhibits strong potential for functional sequence generation. By integrating large-scale sequence generation and screening workflows, this approach holds promise for the rapid design of efficient novel enzymes while significantly reducing the time and cost of experimental validation, offering a valuable and scalable paradigm for de novo enzyme engineering.

To explore the reduction mechanism and stabilization mechanism in the biosynthesis of selenium nanoparticles (SeNPs) by the cell supernatant of Streptomyces avermitilis, we used the cell supernatant as a reduction system to prepare SeNPs. Inductively coupled plasma-optical emission spectrometry (ICP-OES) was employed to carry out qualitative and quantitative analyses of the selenium element in SeNPs. Meanwhile, scanning electron microscopy (SEM), Fourier transform-infrared spectroscopy (FT-IR), and a potentiometer were utilized to characterize SeNPs. In addition, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify and analyze the components in the supernatant and the proteins on the surface of SeNPs. The results showed that when the concentration of Na₂SeO₃ in the cell supernatant of S. avermitilis was lower than 200 mmol/L, the supernatant had the ability to synthesize SeNPs in this particular experimental system. The selenium and protein content in the synthesized SeNPs reached 64.39% and 2.49%, respectively. The activity of proteins and pH in the supernatant significantly affected the synthesis of SeNPs, and SeNPs existed in two forms: a protein-binding form and a non-protein-binding form. FT-IR results revealed that the characteristic peaks of SeNPs synthesized by the supernatant showed no significant differences from those of SeNPs formed by S. avermitilis through other known methods. The Zeta potential was -22.9 mV. LC-MS/MS results showed that L-cysteine in the cell supernatant changed significantly before and after treatment with Na₂SeO₃. The results of protein identification on the surface of SeNPs indicated that a total of 119 proteins were involved in the formation of SeNPs, with the lengths ranging from 76 to 1 299 aa, molecular weights between 8 145.08 and 145 036.30 Da, and pI values in the range of 4.39 to 11.50. Among these proteins, 100 contained cysteine residues. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) had the highest content, and AhpE, a thiol-specific antioxidant protein containing a thioredoxin domain, was also detected. After iodoacetic acid was added to the supernatant, SeNPs were not detected in any of the samples. In conclusion, the cell supernatant of S. avermitilis has the ability to synthesize SeNPs. L-cysteine and the thiol groups of proteins containing cysteine residues in the cell supernatant reduce Na₂SeO₃ to produce red elementary selenium, which is then wrapped by biological macromolecules to form SeNPs. This study can provide new options for the development and application of SeNPs and offer a reference for deciphering the biosynthesis mechanism of SeNPs by S. avermitilis.

5-aminolevulinic acid (5-ALA) is an important non-proteinogenic amino acid that is widely used in biomedicine, agriculture, and the food industry. In recent years, with the development of synthetic biotechnology, building microbial cell factories for efficient production of 5-ALA has become a research hotspot. This article reviews the latest advances in producing 5-ALA via synthetic biotechnology strategies, including metabolic pathway optimization, key enzyme engineering, and fermentation process optimization. By reconstructing natural C4 and C5 pathways and developing non-natural synthetic routes, precise regulation of the precursor metabolic flux for 5-ALA has been achieved. In addition, the directed evolution and rational design of key enzymes such as 5-aminolevulinic acid synthase (ALAS), glutamyl tRNA reductase (HemA), and glutamate-1-semialdehyde aminotransferase (HemL) significantly improved catalytic efficiency. Regarding chassis cells, microorganisms such as Escherichia coli and Corynebacterium glutamicum have been widely used to construct efficient production platforms. The precise regulation of metabolic pathways enables effective balancing of metabolic burden and toxicity, thereby increasing the 5-ALA yield. Although significant progress has been made in the biosynthesis research and large-scale production of 5-ALA, the weak lipophilicity, low stability, and poor bioavailability of 5-ALA have reduced its application efficiency. How to improve its stability and lipophilicity is a key issue to be addressed in the future. Integrating artificial intelligence-assisted design with synthetic biology-driven optimization and novel chassis development is expected to further advance green and efficient industrial-scale production of 5-ALA. By comprehensively outlining synthetic biology strategies to boost 5-ALA yield and identifying key market challenges, this review provides a roadmap for industrial-scale production via multidisciplinary integration, thereby informing and guiding future research and industrial efforts in this field.