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

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.

2-chloronicotinic acid as an important building block has been widely used in pharmaceutical and pesticide synthesis. Chemical synthesis of 2-chloronicotinic acid suffers from poor specificity, high waste salt, and low yields. The synthesis of 2-chloronicotinic acid from 2-chloronicotinonitrile via nitrilase-catalyzed hydrolysis demonstrates high atom economy and environmental friendliness, representing a promising route for industrial production of 2-chloronicotinic acid. However, the o-substituted Cl of the pyridine ring causes a strong electronic effect and steric hindrance, resulting in low activity and catalytic efficiency. Focusing on the nitrilase GiNIT_M11, we employed semi-rational design to modify distal amino acid residues from the active center for efficient synthesis of 2-chloronicotinic acid. Two distal residues affecting activity, W66 and T107, were identified. A double mutant GiNIT_M11^W66F/T107S was constructed by iterative mutation, demonstrating 78.6% higher activity and 146.1% higher catalytic efficiency than GiNIT_M11. Molecular dynamics simulations revealed that the enhanced catalytic performance of GiNIT_M11^W66F/T107S resulted from synergistic effects of shortened nucleophilic attack, enhanced hydrogen bonding networks, and reduced hydrophobic environment and steric hindrance. This study is of great significance for elucidating the structure-function relationship of nitrilase and developing the industrial production of 2-chloronicotinic acid via nitrilase.

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.