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As one of the most appealing strategies for the synthesis of nanomaterials with various architectures, emulsion-directed methods have been rarely used to control the structure of metal-organic frameworks (MOFs). Herein, we report a versatile salt-assisted nanoemulsion-guided assembly to achieve continuous architecture transition of hierarchical Zr-based MOFs. The morphology of nanoemulsion can be facilely regulated by tuning the feed ratio of a dual-surfactant and the introduced amount of compatible hydrophobic compounds, which directs the assembly of MOFs with various architectures such as bowl-like mesoporous particle, dendritic nanospheres, walnut-shaped particles, crumpled nanosheets and nanodisks. The developed dendritic nanospheres with highly open and large mesochannels is successfully used as matrix for the co-immobilization of coenzymes and corresponding enzymes to realize the in situ heterogeneous regeneration of NAD + . This strategy is expected to pave a way for exploring sophisticated hierarchical MOFs which can be competent for practical applications with bulk molecules involved. Controlling the structure of hierarchical metal-organic frameworks via soft template remains a challenge. Here, the authors report a salt-assisted nanoemulsion-guided strategy to achieve continuous structure transition of hierarchical Zr-based MOFs.

Complex coumarins (CCs) represent characteristic metabolites found in Apiaceae plants, possessing significant medical value. Their essential functional role is likely as protectants against pathogens and regulators responding to environmental stimuli. Utilizing genomes and transcriptomes from 34 Apiaceae plants, including our recently sequenced Peucedanum praeruptorum , we conduct comprehensive phylogenetic analyses to reconstruct the detailed evolutionary process of the CC biosynthetic pathway in Apiaceae. Our results show that three key enzymes – p -coumaroyl CoA 2’-hydroxylase (C2’H), C-prenyltransferase (C-PT), and cyclase – originated successively at different evolutionary nodes within Apiaceae through various means of gene duplications: ectopic and tandem duplications. Neofunctionalization endows these enzymes with novel functions necessary for CC biosynthesis, thus completing the pathway. Candidate genes are cloned for heterologous expression and subjected to in vitro enzymatic assays to test our hypothesis regarding the origins of the key enzymes, and the results precisely validate our evolutionary inferences. Among the three enzymes, C-PTs are likely the primary determinant of the structural diversity of CCs (linear/angular), due to divergent activities evolved to target different positions (C-6 or C-8) of umbelliferone. A key amino acid variation (Ala161/Thr161) is identified and proven to play a crucial role in the alteration of enzymatic activity, possibly resulting in distinct binding forms between enzymes and substrates, thereby leading to different products. In conclusion, this study provides a detailed trajectory for the establishment and evolution of the CC biosynthetic pathway in Apiaceae. It explains why only a portion, not all, of Apiaceae plants can produce CCs and reveals the mechanisms of CC structural diversity among different Apiaceae plants. This study explores the origin and evolution of complex coumarin (CC) biosynthetic pathway in Apiaceae. The results explain why CCs are only detected in limited Apiaceae species, and clarify the mechanism underlying the linear and angular configurations of Apiaceae CCs.

Methoxylated coumarins represent a large proportion of officinal value coumarins while only one enzyme specific to bergaptol O-methylation (BMT) has been identified to date. The multiple types of methoxylated coumarins indicate that at least one unknown enzyme participates in the O-methylation of other hydroxylated coumarins and remains to be identified. Combined transcriptome and metabonomics analysis revealed that an enzyme similar to caffeic acid O-methyltransferase (COMT-S, S is short for similar) was involved in catalyzing all the hydroxylated coumarins in Peucedanum praeruptorum. However, the precise molecular mechanism of its substrate heterozygosis remains unsolved. Pursuing this question, we determined the crystal structure of COMT-S to clarify its substrate preference. The result revealed that Asn132, Asp271, and Asn325 govern the substrate heterozygosis of COMT-S. A single mutation, such as N132A, determines the catalytic selectivity of hydroxyl groups in esculetin and also causes production differences in bergapten. Evolution-based analysis indicated that BMT was only recently derived as a paralogue of caffeic acid O-methyltransferase (COMT) via gene duplication, occurring before the Apiaceae family divergence between 37 and 100 mya. The present study identified the previously unknown O-methylation steps in coumarin biosynthesis. The crystallographic and mutational studies provided a deeper understanding of the substrate preference, which can be used for producing specific O-methylation coumarins. Moreover, the evolutionary relationship between BMT and COMT-S was clarified to facilitate understanding of evolutionary events in the Apiaceae family.

This study proposes a balanced truck dispatching strategy for inter-terminal transportation (ITT) in large ports, incorporating proactive demand outsourcing to address stochastic and imbalanced ITT demand. A portion of ITT tasks are intentionally outsourced to third-party public trucks at a higher cost, so that self-owned trucks can be reserved for more critical tasks. The ITT system is modeled as a closed Jackson network, in which self-owned trucks circulate among terminals and routes. An optimization model is developed to determine the optimal proactive outsourcing ratios for origin–destination terminal pairs and the appropriate fleet size of self-owned trucks, aiming to minimize total transportation costs. Reactive outsourcing is also included to handle occasional truck shortages. A mean value analysis method is used to evaluate system performance with given decisions, and a differential evolution algorithm is employed for optimization. The case study of Shanghai Yangshan Port demonstrates that the proposed strategy reduces total system cost by 9.8% compared to reactive outsourcing. The results also highlight the importance of jointly optimizing outsourcing decisions and fleet size. This study provides theoretical insights and practical guidance for ITT system management under demand uncertainty.

Peucedanum praeruptorum Dunn is one of the main traditional Chinese medicines producing coumarins and plenty of literatures are focused on the biosynthesis of coumarins. Quantitative real-time reverse transcription PCR (qRT-PCR) is a widely used method in studying the biosynthesis pathway and the selection of reference genes plays a crucial role in accurate normalization. To facilitate biosynthesis study of coumarins, twelve candidate reference genes were selected from the transcriptome database of P. praeruptorum according to previous studies. Then, BestKeeper, geNoFrm and NormFinder were used for selecting stably expressed reference genes in different tissues and under various stress treatments. The results indicated that, among the twelve candidate reference genes, the SAND family protein (SAND), actin 2 (ACT2), ubiquitin-conjugating enzyme 9 (UBC9), protein phosphatase 2A gene (PP2A) and polypyrimidine tract-binding protein (PTBP1) were the most stable reference genes under different experimental treatments, while glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tubulin beta-6 (TUB6) were the least stable genes. In addition, the suitability of SAND, TIP41-like protein (TIP41), UBC9, ACT2, TUB6 and their combination as reference genes were confirmed by normalizing the expression of 1-aminocyclopropane-1-carboxylate oxidase (ACO) in different treatments. This work is the first survey of the stability of reference genes in P. praeruptorum and provides guidelines to obtain more accurate qRT-PCR results in P. praeruptorum and other plant species.

To address the poor effect of optical/visual guidance used in AUV terminal docking with strong background light and turbid water quality, an underwater electromagnetic guidance method based on the magnetic dipole model is proposed in this paper. According to the magnetic dipole model, the electromagnetic field of 1 kHz frequency generated by the coil in the range of terminal docking is the near field, where the position can be figured out through the amplitude and phase information of three orthogonal magnetic field intensity vectors. A triaxial-coil magnetometer with three orthogonal coils and a method for extracting the amplitude and phase information of the induced voltage are presented in this paper. According to Faraday’s law, the amplitude and phase information of the induced voltage of a triaxial-coil magnetometer replace the information of magnetic field intensity in relation to positioning. The underwater positioning results show that the average positioning error within 6 m can reach the centimeter level. Five underwater terminal docking tasks were carried out, and four of them were successfully completed, which verified the feasibility of the proposed method.

Earthworms have been extensively studied as ancient soil invertebrates, that are highly diverse. Previous studies of these invertebrates have mainly focused on their ecosystem services, medicinal value, and ecological habits. However, their genomic analysis remains inadequate. In this study, we generated the first high-quality telomere-to-telomere (T2T) assembly of the genome of the earthworm, Amynthas aspergillum (Perrier, 1872) , which belongs to the genus Amynthas of the family Megascolecidae. The T2T assembly was 758.86 Mb with an N50 contig size of 16.59 Mb. The sequences were anchored to 43 chromosomes (2n = 2x = 86) with a coverage of 98.43% (746.95 Mb), and 83 telomeres were detected. In addition, we also predicted 35,723 protein-coding genes with 97.02% being functionally annotated. This T2T genome assembly will establish substantial groundwork for exploring the evolutionary mechanisms of the earthworm genome and enhance the specificity of its pharmacological effects.

Background Coumarins play roles in many biological processes. Angelica decursiva is one of the major sources of coumarins in China. Due to increasing demand for coumarins in the marketplace, traditional extraction from plants is now considered economically insufficient and unsustainable. Microbial synthesis is a promising strategy for scalable production of coumarins. However, the biosynthetic pathway of coumarin remains poorly understood, and even more, the genes associated with this process have not been characterized in A. decursiva . Results RNA-seq was employed to elucidate the umbelliferone biosynthetic pathway. The results indicated that three enzymes, phenylalanine ammonia-lyase (PAL), 4-Coumarate: Coenzyme A Ligase (4CL), and p -coumaroyl CoA 2'-hydroxylase (C2’H) were involved in umbelliferone biosynthesis. Using the cloned genes, we generated a synthetic biology based microbial cell factory that produces coumarins from tyrosine utilizing Rhodotorula glutinis tyrosine ammonia lyase (RgTAL) to bypass cinnamic acid 4-hydroxylase (C4H). With metabolic engineering strategies, we deleted prephenate dehydratase (pheA), anthranilate synthase (trpE) and transcriptional regulatory protein (tyrR) and overexpressed six related genes involved in tyrosine biosynthesis, to drive the carbon flux from tyrosine. To overcome the limitation of 4CL, a virtual screening and site-specific mutagenesis-based protein engineering approach was applied. In addition, induction/culture conditions and different ions were employed to further improve the yield of umbelliferone. Finally, a yield of 356.59 mg/L umbelliferone was obtained. Conclusions The current study elucidated the umbelliferone biosynthesis pathway in A. decursiva . The results also demonstrated the feasibility of integrating gene mining with synthetic biology techniques to produce natural compounds.

Salvia plebeia (Lamiaceae) is a valuable medicinal plant widely distributed across Asia and Oceania. However, the composition and accumulation patterns of its active ingredients in different organs during the growth and their biosynthetic mechanism remain unknown. Therefore, we conducted metabolite profiling, transcriptomic analysis, and biological functional verification to explore the distribution, accumulation, and biosynthesis mechanisms of flavonoids in S. plebeia. We identified 70 metabolites including 46 flavonoids, 16 phenolic acids, seven terpenoids, and one organic acid, of which 21 were previously unreported in S. plebeia . Combining metabolomic-transcriptomic analysis and biological functional verification, we identified the key genes involved in biosynthesis of its main active ingredients, hispidulin and homoplantaginin, including SpPAL , SpC4H , Sp4CL2 , Sp4CL5 , SpCHS1 , SpCHI , SpFNS , SpF6H1 , SpF6OMT1 , SpF6OMT2 , SpUGT1 , SpUGT2 , and SpUGT3 . Using the identified genes, we reconstructed the hispidulin and homoplantaginin biosynthesis pathways in Escherichia coli , and obtained a yield of 5.33 and 3.86 mg/L for hispidulin and homoplantaginin, respectively. Our findings provide valuable insights into the changes in chemical components in different organs of S. plebeia during different growth and harvest stages and establishes a foundation for identifying and synthesizing its active components.

Corydalis yanhusuo W.T. Wang is a classic herb that is frequently used in traditional Chinese medicine and is efficacious in promoting blood circulation, enhancing energy, and relieving pain. Benzylisoquinoline alkaloids (BIAs) are the main bioactive ingredients in Corydalis yanhusuo . However, few studies have investigated the BIA biosynthetic pathway in C. yanhusuo , and the biosynthetic pathway of species-specific chemicals such as tetrahydropalmatine remains unclear. We performed full-length transcriptomic and metabolomic analyses to identify candidate genes that might be involved in BIA biosynthesis and identified a total of 101 full-length transcripts and 19 metabolites involved in the BIA biosynthetic pathway. Moreover, the contents of 19 representative BIAs in C. yanhusuo were quantified by classical targeted metabolomic approaches. Their accumulation in the tuber was consistent with the expression patterns of identified BIA biosynthetic genes in tubers and leaves, which reinforces the validity and reliability of the analyses. Full-length genes with similar expression or enrichment patterns were identified, and a complete BIA biosynthesis pathway in C. yanhusuo was constructed according to these findings. Phylogenetic analysis revealed a total of ten enzymes that may possess columbamine-O-methyltransferase activity, which is the final step for tetrahydropalmatine synthesis. Our results span the whole BIA biosynthetic pathway in C. yanhusuo . Our full-length transcriptomic data will enable further molecular cloning of enzymes and activity validation studies.