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

To investigate the roles of long non-coding RNAs (lncRNAs) and potential micropeptides encoded by these lncRNAs in influenza A virus (IAV) replication, we analyzed the data obtained from RNA-seq and Ribo-seq. We found that the lncRNA zinc finger antisense 1 (ZFAS1) was significantly up-regulated by IAV infection. This finding was confirmed by in vitro experiments, which showed that IAV infection caused a significant increase in the expression of ZFAS1, with the effect exhibiting a dose- and time-dependent relationship in response to the viral infection. This up-regulation was also observed in cells infected with other RNA and DNA viruses. Furthermore, we discovered that the type Ⅰ interferon signaling pathway positively regulated ZFAS1 expression. Functional assays revealed that silencing ZFAS1 significantly promoted IAV replication, while its overexpression significantly suppressed the virus replication. Additionally, LC-MS/MS analysis and Western blotting suggested that ZFAS1 encoded a 56-aa micropeptide, named ZFAS1-P56, which can also inhibit IAV replication. These results reveal that IAV-induced expression of ZFAS1 is regulated via the type Ⅰ interferon signaling pathway, and both ZFAS1 and ZFAS1-P56 suppress the IAV replication. This study unveils a new mechanism by which host establishes an innate immunity against the viral infection.

L-threonine, a member of the L-aspartic acid group, is one of the amino acids that cannot be synthesized by humans and livestock. It is widely used in feed, medicine, and food fields. The fermentation production of L-threonine faces the problems of a narrow substrate utilization spectrum, slow growth of strains, and low yields. In this study, the lab-stored Escherichia coli THRN1 was used as the chassis for metabolic engineering to construct an industrial strain capable of efficiently, stably, and continuously producing L-threonine. Firstly, comparative genomic analysis was performed on the mutated strains THRN1 and FMME1 to reveal the potential metabolic mechanism of excessive accumulation of L-threonine and identify the target of further metabolic modification. Subsequently, three unreported effective thrA mutants were obtained by the MutaT7 system of directed evolution in vivo, which increased the L-threonine synthesis flux, and strain THRN2 was obtained. Secondly, to improve the industrial applicability of the strain, we knocked out mlc and introduced the allogenic glvAC (Lentibacillus salicampi) to enhance the utilization of glucose and maltose from hydrolysis of industrial starch, and obtained strain THRN7. The strain was fermented in a 5 L bioreactor for 34 h, with the L-threonine titer of 121.26 g/L and the yield of 60.47%. Finally, through the optimization of fermentation process, strain THRN7 can produce 120.42 g/L L-threonine in a 50 L bioreactor within 32 h, with the yield and productivity reaching 60.88% and 3.76 g/(L·h), respectively. In this study, a high yield L-threonine strain with no resistance and no plasmid was constructed, which laid a solid foundation for the industrial production of L-threonine.

Trichodermin, a sesquiterpenoid compound with antifungal, plant growth-regulating, and antitumor activities, demonstrates broad application prospects in both pharmaceutical and agricultural fields. Trichoderma taxi is an efficient trichodermin-producing filamentous fungus isolated from Taxus mairei, while its unique aconidial phenotype poses challenges to genetic transformation. To overcome this barrier and enable targeted engineering, this study established an Agrobacterium tumefaciens-mediated transformation (ATMT) system suitable for aconidial T. taxi, building on proven expertise in genetic transformation of sporulating filamentous fungi. Through systematic optimization of key parameters including mycelium pretreatment (mechanical grinding method), infection time, and co-culture conditions, we successfully achieved efficient transformation of T. taxi by A. tumefaciens. Utilizing this technical platform, we targeted tri3 (a gene encoding acetyltransferase, a key enzyme in trichodermin biosynthesis) to construct tri3-overexpressing strains. The engineered strains exhibited trichodermin production reaching 1.17 g/L, representing a 33.3% increase compared with that of the parent strain. The established transformation system not only provides a reliable technical approach for metabolic pathway engineering and high-yielding strain development of T. taxi, but also opens new avenues for genetic manipulation of other aconidial filamentous fungi in industrial applications.

A CRISPR-Cas6-mediated lycopene synthase assembly regulation strategy was developed to optimize the metabolic pathway of lycopene biosynthesis in Escherichia coli and enhance production efficiency. Leveraging the orthologous properties of EcCas6e and Csy4 within the Cas6 protein family, along with RNA scaffolding, we constructed a protein-RNA complex for enzyme assembly. Sixteen plasmids (LYC-1 to LYC-16) were designed, and the assembly strategy was systematically optimized by varying the gene arrangement, linker length, and RNA scaffold expression. The performance of RNA scaffold-based enzyme assembly was compared with conventional protein linker-based approaches. Lycopene production was quantified via high-performance liquid chromatography (HPLC) to evaluate system performance. The recombinant strain LYC-3-4, which co-localized CrtB and CrtI via EcCas6e-Csy4 protein-RNA complexes, achieved the highest lycopene yield (4.02 mg/L), 58% higher than the control strain LYC-3-5 (2.55 mg/L) with mismatched RNA hybridization regions, and 41% higher than strain LYC-6 (2.86 mg/L), in which the enzymes were expressed separately. This result indicates that protein-RNA-mediated spatial co-localization significantly enhanced the substrate channeling effect, whereas other assembly configurations either failed to improve or even reduced lycopene production. In summary, we exploited the protein assembly capability of CRISPR-Cas6 proteins in combination with RNA scaffolds to achieve efficient enzyme co-localization within the lycopene biosynthetic pathway. This approach offers a convenient, flexible, and scalable tool for enzyme assembly regulation in metabolic engineering, with potential applications in microbial production of lycopene and other valuable metabolites.

Liquid-liquid phase separation (LLPS) is a crucial mechanism regulating the formation of biomolecular condensates in eukaryotic cells. Recent studies have revealed that LLPS plays a central role in the biogenesis of small RNAs (sRNAs, including miRNA and siRNA) and the regulation of RNA silencing pathway in plants. This review systematically summarizes the regulatory roles and molecular mechanisms of LLPS in the production of miRNAs and siRNAs in plants. In the nucleus, the SE (SERRATE) protein mediates LLPS through its intrinsically disordered region (IDR), forming dicing bodies (D-bodies) that recruit DCL1 (DICER-LIKE 1), HYL1 (HYPONASTIC LEAVES), pri-miRNAs (primary miRNA) and other newly identified proteins, significantly enhancing miRNA processing efficiency. Additionally, researchers have uncovered the critical role of m⁶A (N6-methyadenosine) modification of pri-miRNA in LLPS and the biogenesis of miRNA. In the cytoplasm, the SGS3 (SUPPRESSOR OF GENE SILENCING 3) protein undergoes LLPS to form siRNA bodies, enriching RDR6 (RNA-DEPENDENT RNA POLYMERASE), DCL2/4, and other factors to promote the production of siRNA, which participates in antiviral defenses and developmental regulation. New studies indicate that the phase separation ability of SGS3 is regulated by phosphorylation. These findings provide novel insights into the precise regulation of RNA silencing in plants and lay a theoretical foundation for developing new antiviral strategies.

Acid proteases are extensively applied in various industries, including food, animal feed, and leather production. They are ubiquitously present in animals, fungi, plants, protozoa, bacteria, and viruses. The filamentous fungus Aspergillus niger has become the primary producer of acid proteases due to its high protein secretion efficiency and safety. Although multiple acid proteases with distinct characteristics from A. niger have been well characterized, numerous others remain insufficiently studied. In view of the growing demands for productivity across industries, it is essential to develop acid proteases with enhanced activities and wide environmental adaptability. This review summarizes the classification, structural and enzymatic properties of acid proteases produced by A. niger, regulatory factors influencing their secretion, and different optimization strategies for enhancing the acid protease production by A. niger, aiming to provide references for future research.

Microbial energy conversion refers to the process of converting raw materials such as organic matter (sugars, acids, waste biomass, organic wastewater, etc.) or inorganic substrates (carbon dioxide, ammonia, sulfides, etc.) into renewable energy products, such as hydrogen, methane, ethanol, and electrical energy, through microbial metabolic processes. With the rapid development of synthetic biology and enzyme engineering, researchers can perform targeted modifications on microorganisms and their functional enzyme systems, thereby enhancing the conversion efficiency of substrates to energy products. However, in practical applications, microbial energy conversion still generally faces common bottlenecks such as limited electron transfer, complex metabolic regulation, and low energy conversion efficiency, which severely restrict the energy efficiency improvement and engineering promotion of the system. Iron-based materials, with excellent electron transfer ability, potential as enzyme cofactors, and good magnetic separation performance, are widely used in microbial energy conversion to synergistically improve the energy conversion efficiency and operational stability of the system. This paper systematically reviews the research progress in the applications of iron-based materials in representative microbial energy conversion technologies (such as hydrogen production, methane production, electricity production, ethanol production, and lipid production) and analyzes the key mechanisms by which different types of iron-based materials promote microbial energy conversion. This paper aims to provide theoretical support and technical reference for the construction, optimization, and practical application of efficient iron-based material-microbial coupling systems.

To obtain full-length recombinant human type I and type II collagen α1 chains with biological activity, potential cleavage sites and surrounding amino acid sequences that may exist during heterologous expression of native human type I and type II collagen α1 chains in Komagataella phaffii were mutated. Two variants were constructed to enable their intact expression in the Komagataella phaffii. Through purification techniques, complete full-length recombinant human type I and type II collagen α1 chain variants were obtained. Both variants demonstrated excellent stability under high-density fermentation conditions, with expression levels quantified by UV reaching 18.7 g/L (type I) and 17.3 g/L (type II), respectively. High-purity products were obtained after purification. These results provide an important reference for the development of highly stable full-length recombinant collagen α chains. The recombinant collagen hydrogels prepared from the two types of collagen exhibited similar biological functions to those of hydrogels prepared from natural collagen and can be utilized as novel biological materials in the medical device field.

To establish the capillary isoelectric focusing (cIEF) method for determining the isoelectric point (pI) of recombinant respiratory syncytial virus vaccine (CHO cell) bulk antigen (ReRSVA-PreF bulk antigen), thereby enhancing the quality control system for vaccine production. According to the structural characteristics of the ReRSVA-PreF bulk antigen, we pretreated the samples with the denaturing agent RapiGest SF and the reducing agent dithiothreitol to ensure stable monomerization. A robust cIEF method was developed through systematic optimization of key parameters including protein solubilizer type, addition ratio, and sample loading volume. Furthermore, its main validation parameters and robustness were evaluated. The optimized conditions were as follows: sample buffer containing 34% SimpleSol Protein Solubilizer, 0.35% methylcellulose (MC), 4% ampholytes, and 1% pI marker mixture; focusing program set at 1 500 V for 1 min followed by 3 000 V for 7 min. Under these conditions, the ReRSVA-PreF bulk antigen exhibited well-resolved peaks with high resolution in the cIEF profile. We successfully established a cIEF method for determining the pI of ReRSVA-PreF bulk antigen. The method provides reliable technical support for molecular characterization and quality control during manufacturing, and serves as a valuable reference for the development and standardized analysis of similar recombinant protein vaccines.

N6,2'-O-dimethyladenosine (m6Am), as a critical RNA epigenetic modification, has become a hot topic in current research due to its unique chemical structure and biological functions. m6Am not only regulates mRNA stability and half-life but also modulates its interactions with translation initiation factors and RNA-binding proteins, thereby playing a crucial role in post-transcriptional regulation. Recent medical research indicates that m6Am is closely associated with metabolic disorders such as obesity and type 2 diabetes mellitus, as well as multiple viral infections and tumor development. Therefore, predicting m6Am site holds significant importance for early diagnosis and precision treatment of diseases. Although the biological functions of m6Am modification are gradually attracting attention, related research is still limited by the limitations of detection technology and high costs. Computational biology methods provide new ideas for large-scale identification of m6Am sites. This paper proposes an imbalanced m6Am site prediction model-im6Am-DC. The model employs a DenseNet architecture to extract high-level local features and introduces a CA attention mechanism to highlight crucial feature information. Furthermore, to solve the data imbalance, the model utilizes the focal loss function. Experimental results showed that on the full transcript dataset, the im6Am-DC model achieved the sensitivity (Sn) of 0.523 7, specificity (Sp) of 0.988 4, accuracy (Acc) of 0.946 1, and Matthews correlation coefficient (MCC) of 0.629 8. On the mature RNA dataset, the model demonstrated equally strong performance across these metrics. Simultaneously, we employed the im6Am-DC model to predict across multiple distinct datasets, including unbalanced m6Am site datasets, balanced m6Am site datasets, and other types of RNA modification sites. This comprehensive, multidimensional approach enabled us to evaluate the performance and application potential of the model. The results demonstrate that the im6Am-DC model exhibits significant advantages in predicting unbalanced m6Am sites, providing effective technical support for studying the roles of m6Am modifications and exploring related disease mechanisms.