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

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.

The global energy crisis and environmental pollution are becoming increasingly serious. The development of sustainable and clean renewable energy has become a key direction of scientific research. Microalgae are ideal raw materials for biodiesel production due to their efficient photosynthetic ability, fast growth rate, and rich lipid content. Chlamydomonas reinhardtii, as a model organism of unicellular eukaryotic green algae, has the advantages of a clear genetic background and convenient operation, which makes it an ideal target for the study of lipid metabolism in microalgae. Nitrogen stress can induce lipid accumulation in microalgae, while its molecular mechanism has not been fully elucidated. In this study, we used a CRISPR interference (CRISPRi) system to regulate key genes of nitrogen metabolism in a targeted manner and thus simulated the nitrogen stress environment to investigate its effect on lipid accumulation in C. reinhardtii, aiming to provide a new technological strategy for the efficient production of microalgal lipids. The CRISPRi system was constructed to inhibit the expression of the nitrate reductase gene (CrNIT1) and the nitrite reductase gene (CrNII1) in C. reinhardtii FACHB-2220. We evaluated the effects of nitrogen metabolism inhibition on lipid accumulation by measuring the cell growth, lipid content, and expression levels of key genes. The algal strain ΔNIT1-4 with inhibited CrNIT1 expression showed the CrNIT1 expression 10.27% that of the wild type (WT, and the strain ΔNII1-4 with inhibited CrNII1 expression showed the CrNII1 expression16.02% that of WT, indicating that the CRISPRi system effectively inhibited the transcription of the target genes. Under the condition of nitrogen abundance, the cell density of ΔNIT1-4 and ΔNII1-4 was only 33.7% and 40.2%, respectively, of that of WT. The total lipid content of ΔNIT1-4 and ΔNII1-4 was 34.41% and 33.45% of the dry weight, respectively, which was significantly higher than that of WT. In this study, we successfully simulated the nitrogen stress effect by suppressing the key genes of nitrogen metabolism through the CRISPRi system and significantly improved the lipid accumulation efficiency of C. reinhardtii. This study elucidates the regulatory relationship between nitrogen metabolism and lipid synthesis, providing a theoretical basis and technical support for the industrial application of microalgae in bioenergy production.

Rosin, a characteristic bioactive compound of the endangered medicinal plant Rhodiola rosea, exhibits diverse pharmacological properties. However, conventional approaches such as chemical synthesis and plant extraction fail to meet the requirements of sustainable development. In this study, we engineered Saccharomyces cerevisiae to construct a glucose-based microbial platform for rosin biosynthesis. First, feedback inhibition in the aromatic amino acid synthesis pathway was alleviated by overexpression of feedback-resistant mutant enzymes and introduction of exogenous isozymes. Concurrently, the phosphoketolase pathway was integrated to enhance erythrose-4-phosphate (E4P) supply, thereby reinforcing aromatic amino acid biosynthesis. The reported cinnamoyl-CoA reductase (CCR) and carboxylic acid reductase (CAR) pathways are both capable of synthesizing cinnamyl alcohol. This study systematically evaluated, these two pathways in S. cerevisiae, achieving de novo biosynthesis of cinnamyl alcohol with shake-flask titers of 0.35 mg/L and 35.51 mg/L, respectively. Subsequently, seven glycosyltransferases (GTs) were screened for cinnamyl alcohol glycosylation, with AtUGT73C5^syn from Arabidopsis thaliana demonstrating the highest catalytic efficiency. By integrating Atugt73c5^syn into the cinnamyl alcohol-producing strain, we achieved de novo biosynthesis of rosin in S. cerevisiae for the first time, which reached a titer of 14.91 mg/L in shake flasks. Further optimization by increasing the copy number of glycosyltransferase and the UDP-glucose supply increased the titer of rosin to 23.54 mg/L. This study establishes a foundational platform for developing S. cerevisiae as a microbial cell factory for high-titer phenylpropanoid glycoside production.