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In the context of the rapid development of Internet technology, the integration of the world economy has been strengthened. The continuous innovation of technology and foreign trade business forms has promoted the rapid development of cross-border e-commerce (CBE). Based on the research on the development status of CBE, this paper analyzes the reasons for the above problems in the development of CBE and discusses the innovation of CBE business models based on blockchain. Its purpose is to study the CBE business model based on big data and blockchain. This paper proposes to build a CBE framework system based on blockchain. It also uses blockchain technology (BT) to provide solutions to the problems of trust, customs supervision, cross-border payment, cross-border logistics, and cross-border data flow encountered in CBE. According to experimental statistics, China’s CBE exports were 7.29 trillion yuan, an increase of 40.1%, and imports were 3.76 trillion yuan, an increase of 16.5%.

Reduction and optimization of the microbial genome is an important strategy for constructing synthetic biological chassis cells and overcoming obstacles in natural product discovery and production. However, it is of great challenge to discover target genes that can be deleted and optimized due to the complicated genome of actinomycetes. Saccharopolyspora pogona can produce butenyl-spinosyn during aerobic fermentation, and its genome contains 32 different gene clusters. This suggests that there is a large amount of potential competitive metabolism in S. pogona, which affects the biosynthesis of butenyl-spinosyn. By analyzing the genome of S. pogona, six polyketide gene clusters were identified. From those, the complete deletion of clu13, a flaviolin-like gene cluster, generated a high butenyl-spinosyn-producing strain. Production of this strain was 4.06-fold higher than that of the wildtype strain. Transcriptome profiling revealed that butenyl-spinosyn biosynthesis was not primarily induced by the polyketide synthase RppA-like but was related to hypothetical protein Sp1764. However, the repression of sp1764 was not enough to explain the enormous enhancement of butenyl-spinosyn yields in S. pogona-Δclu13. After the comparative proteomic analysis of S. pogona-Δclu13 and S. pogona, two proteins, biotin carboxyl carrier protein (BccA) and response regulator (Reg), were investigated, whose overexpression led to great advantages of butenyl-spinosyn biosynthesis. In this way, we successfully discovered three key genes that obviously optimize the biosynthesis of butenyl-spinosyn. Gene cluster simplification performed in conjunction with multiomics analysis is of great practical significance for screening dominant chassis strains and optimizing secondary metabolism. This work provided an idea about screening key factors and efficient construction of production strains.

After entering the 21st century, the electronic commerce system has affected all aspects of our lives. Whether we read news on our mobile phones or computers or purchase items on our online websites, it greatly facilitates our lives. With the rapid development of short videos, many people like to watch small videos that interest them. The rapid development of e-commerce has facilitated our lives, so that we no longer have to go to many shopping malls to buy our favorite items, and we also no need to change TV stations one by one after watching a program to find our favorite programs. However, due to the rapid development of electronic commerce, there has been a lot of information overload. When users browse the website, items they are not interested in will appear, and even information about online fraud appears. How to filter this information and how to intelligently recommend to users more favorite items is the main research direction of this article. The research of this article is mainly divided into four parts. The first part analyzes the current situation of intelligent recommendation technology research and puts forward the idea of this article. The second part introduces the commonly used collaborative filtering algorithm and the principle and process of the fuzzy clustering algorithm used in this experiment, analyzes the shortcomings of the traditional collaborative filtering algorithm and illustrates the adaptability of the fuzzy clustering algorithm in practical applications. The third part introduces an intelligent recommendation system based on fuzzy clustering, which comprehensively analyzes the characteristics of users and products, makes full use of users’ evaluation information of products, and realizes intelligent recommendations based on content and collaborative filtering. At the end of the article, the comparative analysis experiment with the intelligent recommendation system of collaborative recommendation algorithm further proves the superiority of the intelligent recommendation system of electronic commerce based on fuzzy clustering algorithm in this paper and improves the accuracy of intelligent recommendation.

Reduction and optimization of the microbial genome is an important strategy for constructing synthetic biological chassis cells and overcoming obstacles in natural product discovery and production. However, it is of great challenge to discover target genes that can be deleted and optimized due to the complicated genome of actinomycetes.

PII signal transduction proteins are widely found in bacteria and plant chloroplast, and play a central role in nitrogen metabolism regulation, which interact with many key proteins in metabolic pathways to regulate carbon/nitrogen balance by sensing changes in concentrations of cell-mediated indicators such as α-ketoglutarate. In this study, the knockout strain Saccharopolyspora pogona-ΔpII and overexpression strain S. pogona-pII were constructed using CRISPR/Cas9 technology and the shuttle vector POJ260, respectively, to investigate the effects on the growth and secondary metabolite biosynthesis of S. pogona. Growth curve, electron microscopy, and spore germination experiments were performed, and it was found that the deletion of the pII gene inhibited the growth to a certain extent in the mutant. HPLC analysis showed that the yield of butenyl-spinosyn in the S. pogona-pII strain increased to 245% than that in the wild-type strain while that in S. pogona-ΔpII decreased by approximately 51%. This result showed that the pII gene can promote the growth and butenyl-spinosyn biosynthesis of S. pogona. This research first investigated PII nitrogen metabolism regulators in S. pogona, providing significant scientific evidence and a research basis for elucidating the mechanism by which these factors regulate the growth of S. pogona, optimizing the synthesis network of butenyl-spinosyn and constructing a strain with a high butenyl-spinosyn yield. KEY POINTS: • pII key nitrogen regulatory gene deletion can inhibit the growth and development of S. pogona. • Overexpressed pII gene can significantly promote the butenyl-spinosyn biosynthesis. • pII gene can affect the amino acid circulation and the accumulation of butenyl-spinosyn precursors in S. pogona.

Background Butenyl-spinosyn, produced by Saccharopolyspora pogona, is a promising biopesticide due to excellent insecticidal activity and broad pesticidal spectrum. Bacterioferritin (Bfr, encoded by bfr) regulates the storage and utilization of iron, which is essential for the growth and metabolism of microorganisms. However, the effect of Bfr on the growth and butenyl-spinosyn biosynthesis in S. pogona has not been explored. Results Here, we found that the storage of intracellular iron influenced butenyl-spinosyn biosynthesis and the stress resistance of S. pogona, which was regulated by Bfr. The overexpression of bfr increased the production of butenyl-spinosyn by 3.14-fold and enhanced the tolerance of S. pogona to iron toxicity and oxidative damage, while the knockout of bfr had the opposite effects. Based on the quantitative proteomics analysis and experimental verification, the inner mechanism of these phenomena was explored. Overexpression of bfr enhanced the iron storage capacity of the strain, which activated polyketide synthase genes and enhanced the supply of acyl-CoA precursors to improve butenyl-spinosyn biosynthesis. In addition, it induced the oxidative stress response to improve the stress resistance of S. pogona. Conclusion Our work reveals the role of Bfr in increasing the yield of butenyl-spinosyn and enhancing the stress resistance of S. pogona, and provides insights into its enhancement on secondary metabolism, which provides a reference for optimizing the production of secondary metabolites in actinomycetes.

Butenyl-spinosyn produced by Saccharopolyspora pogona exhibits strong insecticidal activity and broad pesticidal spectrum. However, its synthetic level was low in the wild-type strain. At present, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficiently editing its genome to improve the butenyl-spinosyn yield. To accelerate the genetic modification of S. pogona, we conducted comparative proteomics analysis to screen differentially expressed proteins related to butenyl-spinosyn biosynthesis. A TetR family regulatory protein was selected from the 289 differentially expressed proteins, and its encoding gene (SP_1288) was successfully deleted by CRISPR/Cas9 system. We further deleted a 32-kb polyketide synthase gene cluster (cluster 28) to reduce the competition for precursors. Phenotypic analysis revealed that the deletion of the SP_1288 and cluster 28 resulted in a 3.10-fold increase and a 35.4% decrease in the butenyl-spinosyn levels compared with the wild-type strain, respectively. The deletion of cluster 28 affected the cell growth, glucose consumption, mycelium morphology, and sporulation by controlling the expression of ptsH, ptsI, amfC, and other genes related to sporulation, whereas SP_1288 did not. These findings confirmed not only that the CRISPR/Cas9 system can be applied to the S. pogona genome editing but also that SP_1288 and cluster 28 are closely related to the butenyl-spinosyn biosynthesis and growth development of S. pogona. The strategy reported here will be useful to reveal the regulatory mechanism of butenyl-spinosyn and improve antibiotic production in other actinomycetes. KEY POINTS: • SP_1288 deletion can significantly promote the butenyl-spinosyn biosynthesis. • Cluster 28 deletion showed pleiotropic effects on S. pogona. • SP_1288 and cluster 28 were deleted by CRISPR/Cas9 system in S. pogona.

Summary Butenyl-spinosyn produced by Saccharopolyspora pogona exhibits strong insecticidal activity and a broad pesticidal spectrum. Currently, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficient understanding of its regulatory mechanism and improving its production by metabolic engineering. Here, we present data supporting a role of the SenX3-RegX3 system in regulating the butenyl-spinosyn biosynthesis. EMSAs and qRT-PCR demonstrated that RegX3 positively controls butenyl-spinosyn production in an indirect way. Integrated proteomic and metabolomic analysis, regX3 deletion not only strengthens the basal metabolic ability of S. pogona in the mid-growth phase but also promotes the flow of the acetyl-CoA produced via key metabolic pathways into the TCA cycle rather than the butenyl-spinosyn biosynthetic pathway, which ultimately leads to continued growth but reduced butenyl-spinosyn production. The strategy demonstrated here may be valuable for revealing the regulatory role of the SenX3-RegX3 system in the biosynthesis of other natural products., Graphical Abstract, Highlights • Butenyl-spinosyn biosynthesis is highly sensitive to Pi control • RegX3 regulates polyP accumulation in S. pogona • RegX3 may indirectly regulate butenyl-spinosyn production • RegX3 plays an important role in the normal growth development of S. pogona, Genetics; Microbiology

Background Saccharopolyspora pogona is a prominent industrial strain due to its production of butenyl-spinosyn, a high-quality insecticide against a broad spectrum of insect pests. TetR family proteins are diverse in a tremendous number of microorganisms and some are been researched to have a key role in metabolic regulation. However, specific functions of TetR family proteins in S. pogona are yet to characterize. Results In the present study, the overexpression of the tetR-like gene sp1418 in S. pogona resulted in marked effects on vegetative growth, sporulation, butenyl-spinosyn biosynthesis, and oxidative stress. By using qRT-PCR analysis, mass spectrometry, enzyme activity detection, and sp1418 knockout verification, we showed that most of these effects could be attributed to the overexpression of Sp1418, which modulated enzymes related to the primary metabolism, oxidative stress and secondary metabolism, and thereby resulted in distinct growth characteristics and an unbalanced supply of precursor monomers for butenyl-spinosyn biosynthesis. Conclusion This study revealed the function of Sp1418 and enhanced the understanding of the metabolic network in S. pogona, and provided insights into the improvement of secondary metabolite production.

Additional file 2: Table S1. Heterologously expressed protein Sp1418 identified by 1D-LC–MS/MS.

Additional file 3: Table S2. Biological insecticidal activity of S. pogona, S. pogona-Δsp1418 and S. pogona-Sp1418.

Additional file 4: Table S3. Primers, plasmids and strains used in this study. Table S4. qRT-PCR primers used in this study.

Additional file 1: Figure S1. Mass spectrum identification of butenyl-spinosyns. MS identification results showed that MS parent ion [M+H]+ = 633 (black arrow) contained 617 (M+H+) (m/z) ion data and a rhamnose ion fragment of 189 molecular mass, which was confirmed as a butenyl-spinosyn component. Figure S2. The sporulation phenotypes in wild type, S. pogona-Δsp1418 and S. pogona-Sp1418. S. pogona-Sp1418 did not produce spores or was almost invisible, and the amount of spores of S. pogona-Δsp1418 is much more than that of S. pogona, but there was no significant difference in the spore morphology of S. pogona and S. pogona-Δsp1418. Figure S3. Tricine-SDS-PAGE analysis of heterologously expressed protein Sp1418. Coomassie Brilliant Blue staining of Tricine-SDS-PAGE showing heterologous protein Sp1418 expressed in the supernatants of E. coli BL21 bearing recombinant plasmid after IPTG induction and ultrasonication. M: 66 kDa protein marker; 1: Samples from E. coli BL21 as a negative control 2: Samples from E. coli BL21 contained the recombinant plasmid. Figure S4. SDS-PAGE gel analysis of total proteins. M: Protein marker; 1: Samples from 96 h S. pogona; 2: Samples from 96 h S. pogona-Sp1418 cells; 3: Samples from 96 h S. pogona-Δsp1418 cells. Compared with the three strains, there are many distinctive bands, and totally 9 proteins were identified via 1D-LC–MS/MS. Figure S5. Construction of pKCcas9d-sgRNA-UHA-DHA and pOJ260-PermE-sp1418. A. Construction of plasmid pKCcas9d-sgRNA-UHA-DHA; B. Construction of plasmid pOJ260-PermE-sp1418.Figure S6. Recombination schematic diagram of vetor pOJ260-PermE-sp1418 and pKCcas9d-sgRNA-UHA-DHA. A. Recombination schematic diagram of vetor pOJ260-PermE-sp1418; B. Recombination schematic diagram of vetor pKCcas9d-sgRNA-UHA-DHA. Figure S7. Identification of S. pogona-Δsp1418 and S. pogona-Sp1418. A: Identification of PermE-sp1418 fragment in S. pogona and S. pogona-Sp1418. M: DL 2000 DNA marker; 1: PCR products of S. pogona with primer pair perm-F/tetR-R; 2: PCR products of S. pogona-Sp1418 with primer pair perm-F/tetR-R; B: PCR amplification of aac(3)IV gene in S. pogona and S. pogona-Sp1418. M: DL 2000 DNA marker; 1–2: PCR products of S. pogona with primer pair Apr-F/Apr-R; 3: PCR products of S. pogona-Sp1418 with primer pair Apr-F/Apr-R; C: Identification of aac(3)IV gene in S. pogona and S. pogona-Δsp1418. M: DL 2000 DNA marker; 1: PCR products of S. pogona with primers Apr-F/Apr-R; 2: PCR products of S. pogona-Δsp1418 with primers Apr-F/Apr-R; D: Identification of sp1418 gene in S. pogona and S. pogona-Δsp1418 M: DL 2000 DNA marker; 1: PCR products of S. pogona with primers tetR-P-F/tetR-P-R; 2: PCR products of S. pogona-Δsp1418 with primers tetR-P-F/tetR-P-R.

The LytTR family two-component system widely exists in bacterial cells and plays an important role in metabolic regulation. The lytS-L gene that encodes for a LytTR family sensor kinase was knocked out to study its influence on the growth, phenotype, and the biosynthesis of the insecticidal polyketide butenyl-spinosyn in Saccharopolyspora pogona NRRL 30141 (S. pogona). High performance liquid chromatography (HPLC) results showed that the butenyl-spinosyn yield of the lytS-L knockout mutant decreased by 58.9% compared with that of the parental strain. This is manifested by a weak toxicity of the mutant against the insect Helicoverpa assulta (H. armigera). Comparative proteomic analysis revealed the expression characteristics of the proteins in S. pogona and S. pogona-ΔlytS-L: a total of 14 proteins involved in energy metabolism were down-regulated, 9 proteins related to carbon metabolism such as glycolysis, and tricarboxylic acid cycle (TCA) were up-regulated, while 13 proteins involved in the biosynthesis of butenyl-spinosyn were down-regulated (fold change >1.2 or< 0.83). The qRT-PCR (Quantitative Real-time PCR) analysis illustrated that the changes in the expression levels of transcription and translation of the identified genes were consistent. This study explores the function of the two-component system of the LytTR family in S. pogona and shows that the lytS-L gene has an important influence on regulating primary metabolism and butenyl-spinosyn biosynthesis of S. pogona.

Due to the property of high energy density, temperature and density, the plasma prodced by the coaxial gun is usually untilized to simulate the Type- I ELM in tokamak. In this work, the plasma characteristics of a tapered coaxial gun was studied and its effect on the tungsten target was investigated. By using current probe, photodetector, spectrometer and calorimeter, it is found that the plasma energy density, density and velocity increase obviously with the discharge voltage. The maximum energy density could reach 1.4 MJ/m2, the maximum electron density could reach 3 × 1015 /cm3, and the maximum ion kinetic energy could reach 113 eV at the voltage of 16 kV, which is suitable for simulating the type I Elms in Tokamak. After one shot, there are many cracks and ablative spots on the surface of W target. Whose size could reach ∼ μm by scanning electron microscope(SEM), this is because W material will become brittle after the frequent heating and cooling alternations under thermal shock and the melt motion driven by high plasma pressure gradient. With the Energy Dispersive Spectrometer(EDS), it can be seen that no matter from the cracks and other area of the surface after one shot, the impurity can be ignored. These results help us to better understand the damage effect of tungsten target for further research.