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Butenyl-spinosyn is a highly effective and broad-spectrum biopesticide produced by Saccharopolyspora pogona. However, the yield of this compound is difficult to increase because the regulatory mechanism of secondary metabolism is still unknown. Here, the transcriptional regulator Sp13016 was discovered to be highly associated with butenyl-spinosyn synthesis and bacterial growth. Overexpression of sp13016 improved butenyl-spinosyn production to a level that was 2.84-fold that of the original strain, while deletion of sp13016 resulted in a significant decrease in yield and growth inhibition. Comparative proteomics revealed that these phenotypic changes were attributed to the influence of Sp13016 on the central carbon metabolism pathway to regulate the supply of precursors. Our research helps to reveal the regulatory mechanism of butenyl-spinosyn biosynthesis and provides a reference for increasing the yield of natural products of Actinomycetes.

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.

Extensive efforts to enhance the oxygen evolution reaction (OER) catalytic performance of transition metal oxides mainly concentrate on the extrinsic morphology tailoring, lattice doping, and electrode interface optimizing. Nevertheless, little room is left for performance improvement using these methods and an obvious gap still exists compared to the precious metal catalysts. In this work, a novel "mixed-valent cobalt modulation" strategy is presented to enhance the electrocatalytic OER of perovskite LaCoO3 (LCO) oxide. The valence transition of cobalt is realized by ethylenediamine post reduction procedure at room temperature, which further induces the variation of magnetic properties for LCO catalyst. The optimized LCO catalyst with Co2+/Co3+ of 1.98% exhibits the best OER activity, and the overpotential at 10 mA cm-2 current density is decreased by 170 mV compared pristine LCO. Impressively, the ferromagnetic LCO catalyst can perform magnetic OER enhancement. By application of an external magnetic field, the overpotential of LCO at 10 mA cm-2 can be further decreased by 20 mV compared to that of under zero magnetic field, which arises from the enhanced energy states of electrons and accelerated electron transfer process driven by magnetic field. Our findings may provide a promising strategy to break the bottleneck for further enhancement of OER performance.

Spinosyns, the secondary metabolites produced by Saccharopolyspora spinosa, are the active ingredients in a family of novel biological insecticides. Although the complete genome sequence of S. spinosa has been published, the transcriptome of S. spinosa remains poorly characterized. In this study, high-throughput RNA sequencing (RNA-seq) technology was applied to dissect the transcriptome of S. spinosa. Through transcriptomic analysis of different periods of S. spinosa growth, we found large numbers of differentially expressed genes and classified them according to their different functions. Based on the RNA-seq data, the CRISPR-Cas9 method was used to knock out the PEP phosphonomutase gene (orf 06952-4171). The yield of spinosyns A and D in S. spinosa-ΔPEP was 178.91 mg/L and 42.72 mg/L, which was 2.14-fold and 1.76-fold higher than that in the wild type (83.51 and 24.34 mg/L), respectively. The analysis of the mutant strains also verified the validity of the transcriptome data. The deletion of the PEP phosphonomutase gene leads to an increase in pyruvate content and affects the biosynthesis of spinosad. The replenishment of phosphoenol pyruvate in S. spinosa provides the substrate for the production of spinosad. We envision that these transcriptomic analysis results will contribute to the further study of secondary metabolites in actinomycetes.

Butenyl-spinosyn is a highly effective and broad-spectrum biopesticide produced by

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.

This study evaluated the inhibition and interaction of Bacillus velezensis BvL03 as a probiotic agent against Aeromonas hydrophila. Strain BvL03 isolated from sediment samples of fish ponds had excellent antimicrobial activity against several fish pathogenic bacteria, especially Aeromonas, including A. hydrophila, A. veronii, A. caviae, and A. sobria. The successful amplification of lipopeptide antimicrobial chemical biosynthetic genes, including iturin family (ituA, ituB, and ituD), bacillomycin family (bacA, bacD, and bacAB), surfactin family (srfAB, srfC, and srfAA), and subtilosin family (albF and sunT) from the genome of BvL03 strain, confirmed its predominant antimicrobial activity. The challenge test suggested that BvL03 significantly decreased fish mortality when challenged with A. hydrophila, which had a cumulative mortality of 12.5% in the treatment group. Toxicity and hemolytic activity of A. hydrophila after co-cultured with BvL03 were relieved as confirmed by the cell experiments, when the initial inoculated concentration of BvL03 was 109 cfu/mL or higher. Moreover, the BvL03 strain labeled with GFP protein (BvL03-GFP) and AhX040 strain labeled with mCherry protein (AhX040-mCherry) were injected into grass carps. The fluorescence levels were monitored by using In Vivo Imaging System (IVIS), in which the green color was steadily increasing, whereas the red color was gradually weakening. Whole genome sequencing revealed that strain BvL03 possesses 15 gene clusters related to antibacterial compounds, including 5 NRPS gene clusters and 3 PKS gene clusters. These results suggested that B. velezensis BvL03 has the potential to be developed as a probiotic candidate against A. hydrophila infection in aquaculture.

To generate a AfsR-like (AfsR-L) overexpression strain Saccharopolyspora pogona-AfsR-L and investigate its effects on the morphology and metabolism of S. pogona. Firstly, we generated the overexpression vector pOJ260-PermE-afsR-L via overlap extension PCR. Then, the recombination strain S. pogona-AfsR-L was constructed via conjugal transfer. To monitor the growth and morphology, mycelia and sporulation were observed. The distinctive proteins and butenyl-spinosyn biosynthesis were investigated by SDS-PAGE, HPLC, and mass spectrometry. And the transcriptional level of afsR-L and other relative functional genes in S. pogona-AfsR-L was analyzed by qRT-PCR. Western blot verified the increased amount of AfsR-L protein in the overexpression strain. Growth curve and mycelia observation showed that afsR-L overexpression make the stationary phase of S. pogona-AfsR-L longer than that of wild S. pogona by approximate 3 days. Moreover, S. pogona-AfsR-L exhibited a more obvious white phenotype on the solid medium, which means afsR-L overexpression affects the sporulation ability of S. pogona. HPLC analysis revealed that the peak area of the butenyl-spinosyn yield of S. pogona-AfsR-L was 293.6, while that of S. pogona was 250.9. SDS-PAGE analysis showed that the two strains had different whole protein expression profiles, and the distinctive proteins were further identified by LC-MS/MS identification, which showed the possible control mechanism of afsR-L gene in S. pogona. We concluded that AfsR could directly or indirectly positively regulate the biosynthesis of butenyl-spinosyn and affect the growth features of S. pogona. We envisioned that this result can be expanded to other Streptomyces for strain improvement.

The Streptomyces virginiae strain W18 was screened from soil, which exhibited broad-spectrum antibacterial activity against fish pathogens. Safety assays showed that strain W18 had no toxicity to fish. Additionally, strain W18 promoted the growth performance of Carassius auratus after feeding in feed mixed with bacteria for one month. Moreover, the activities of AKP, ACP, and SOD in the serum of C. auratus were significantly increased, while the activity of LZM did not greatly change. To detect the expression levels of the genes related to immune factors in the livers, kidneys, and spleens of C. auratus, qRT-PCR was performed. The expression levels of KEAP1, IL-8, TNF-α, IL-β, and C3 were upregulated in all three organs compared to the control, but LZM expression was downregulated in the kidney. The challenge experiment illustrated that the probability of infection with Aeromonas veronii was reduced by 60% and 40% when C. auratus was fed with two different doses of strain W18 in advance. The whole genome of strain W18 was sequenced, and the gene clusters of secondary metabolites in strain W18 were analyzed by AntiSMASH. The results showed that strain W18 contained a total of 26 gene clusters, and functional annotation analysis was conducted by using the non-coding databases COG and KEGG. All of the above results indicated that the use of strain W18 as a feed additive could enhance the resistance of C. auratus toward pathogenic bacteria and disease. In conclusion, an antagonistic strain (W18) against fish pathogenic bacteria was obtained in this study, which is of great significance for finding new treatment methods for bacterial diseases in the aquaculture industry.

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.

Spinosyns, the secondary metabolites produced by

In this paper, a discrete-time sliding mode control method for multi-cylinder hydraulic presses is proposed, which can be applied exactly for computer real-time discrete control in practical engineering. Proposed method bases on discretetime multi-cylinder hydraulic press model and adopts the design method of the distributed controller. First, the expected virtual force is designed according to the overall output demand, and the discrete-time sliding mode controller is designed to obtain the virtual force. Then the virtual force is separated by control allocation method to obtain the desired output force of each cylinder, finally, the second discrete-time sliding mode controller is designed to control the actual output force of each cylinder. The simulation results show that the proposed method has a good effect on output and leveling control of multi-cylinder hydraulic press and solves its problems of non-linear control and redundancy in discrete-time model.

According to the characteristics of parabola function, a new discrete-time sliding mode repetitive control method based on parabola approach law is proposed for the periodic trajectory tracking of uncertain discrete-time systems in modern industrial production. The discrete sliding mode controller based on parabola approach law can effectively suppress chattering, and the discrete sliding mode repetitive controller based on ideal switching dynamics can solve the problem of periodic disturbance. The convergence characteristics of parabola approach law are analyzed, including absolute convergence layer, monotone convergence layer and steady-state error band, which are used to describe the dynamic performance of system error under different parameters. The simulation results verify the effectiveness of the proposed control method.

Using the Latour system, a hybrid NRPS‐T1PKS cluster (˜20 kb) which was responsible for phthoxazolin biosynthesis was efficiently deleted in S. pogona. A TetR family transcriptional regulatory gene that could regulate the butenyl‐spinosyn biosynthesis has been identified from the phthoxazolin biosynthetic gene cluster., Summary Butenyl‐spinosyn, a promising biopesticide produced by Saccharopolyspora pogona, exhibits stronger insecticidal activity and a broader pesticidal spectrum. However, its titre in the wild‐type S. pogona strain is too low to meet the industrial production requirements. Deletion of non‐target natural product biosynthetic gene clusters resident in the genome of S. pogona could reduce the consumption of synthetic precursors, thereby promoting the biosynthesis of butenyl‐spinosyn. However, it has always been a challenge for scientists to genetically engineer S. pogona. In this study, the Latour gene knockout system (linear DNA fragment recombineering system) was established in S. pogona. Using the Latour system, a hybrid NRPS‐T1PKS cluster (˜20 kb) which was responsible for phthoxazolin biosynthesis was efficiently deleted in S. pogona. The resultant mutant S. pogona‐Δura4‐Δc14 exhibited an extended logarithmic phase, increased biomass and a lower glucose consumption rate. Importantly, the production of butenyl‐spinosyn in S. pogona‐Δura4‐Δc14 was increased by 4.72‐fold compared with that in the wild‐type strain. qRT‐PCR analysis revealed that phthoxazolin biosynthetic gene cluster deletion could promote the expression of the butenyl‐spinosyn biosynthetic gene cluster. Furthermore, a TetR family transcriptional regulatory gene that could regulate the butenyl‐spinosyn biosynthesis has been identified from the phthoxazolin biosynthetic gene cluster. Because dozens of natural product biosynthetic gene clusters exist in the genome of S. pogona, the strategy reported here will be used to further promote the production of butenyl‐spinosyn by deleting other secondary metabolite synthetic gene clusters.

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

Escherichia coli Nissle 1917 (EcN) is non-pathogenic probiotic bacteria. Previous studies have indicated that EcN can accumulate and proliferate selectively in solid tumours in BALB/c mouse models. In this study, EcN was engineered to express human interleukin 2 (hIL-2), which is known to enhance immune responses to tumours by activating a variety of immune cells. IL-2 expressed by EcN was proven to activate PBMCs in vitro. Compared to control EcN, intraperitoneally injected EcN expressing hIL-2 (EcN(hIL-2)) was selectively distributed in the tumour microenvironment and inhibited the growth of CT26 tumours in a tumour-bearing mouse model. Antitumour activity was achieved without toxicity to key normal organs and tissues, such as liver, spleen and kidneys. The antitumour mechanism was associated with the infiltration of inflammatory cells, such as T cells, neutrophils and macrophages. These findings provide evidence that the combination of tumour-targeting EcN bacteria and delivery of the immunostimulatory factor IL-2 can be exploited as a promising tumour immunotherapy.

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.

In this study, based on the computer vision technology, we developed a recognition system for nuts positioning to complete the automatic bolt assembly part of the automatic production line. The actual image of the nut was captured by an industrial camera, which would be processed by the following edge detection and Hough circle transformation. After that, the coordinates of the nuts were obtained in the pixel scale. Finally, the real position of nuts would be fed back to the robot arm, according to which the automatic assembly of the bolt would be completed. This computer vision based recognition system is an indispensable part for the efficiency and accuracy improvement of automatic production line.

The fcl gene encodes GDP-fucose synthase, which catalyzes two-step differential isomerase and reductase reactions in the synthesis of GDP-L-fucose from GDP-D-mannose. It also participates in the biosynthesis of amino sugar and ribose sugar, and is one of the key enzymes to regulate the metabolism of sugar and nucleotides in organisms. The presence of fcl gene in Saccharopolyspora pogona was found through sequencing result of genome. The mutant S. pogona-fcl and S. pogona-Δfcl were constructed by gene engineering technology. The results showed that the gene had an effects on growth and development, protein expression and transcriptional level, insecticidal activity, and biosynthesis of butenyl-spinosyn of Saccharopolyspora pogona. The results of HPLC analysis showed that the yield of butenyl-spinosyn in S. pogona-Δfcl was 130% compared with that in S. pogona, which reduced by 25% in S. pogona-fcl. The results of determination of insecticidal activity showed that S. pogona-Δfcl had a stronger insecticidal activity against Helicoverpa armigera than that of S. pogona, while the S. pogona-fcl had a lower insecticidal activity against Helicoverpa armigera compared with S. pogona. Scanning electron microscopy (SEM) was used to observe the morphology of the mycelia. It was found that the surface of the S. pogona-Δfcl was wrinkled, and the mycelium showed a short rod shape. There was no significant difference in mycelial morphology between S. pogona-fcl and S. pogona. Aboved all showed that deletion of fcl gene in S. pogona hindered the growth and development of mycelia, but was beneficial to increase the biosynthesis of butenyl-spinosyn and improve insecticidal activity. Whereas the fcl gene over-expression was not conducive to the biosynthesis of butenyl-spinosyn and reduced their insecticidal activity. SDS-PAGE results showed that the difference of protein expression among the three strains was most obvious at 96 hours, which was identified by real-time fluorescence quantitative polymerase chain reaction, the results showed that there were significant differences of related genes in transcriptional levels among the three strains. Based on the results of the study, a network metabolic control map was constructed to analyze the effect of fcl gene on growth and the regulation pathway of butenyl-spinosyn biosynthesis, which provided an experimental basis for revealing the regulation mechanism of butenyl-spinosyn biosynthesis and related follow-up studies.fcl 基因编码的GDP-岩藻糖合成酶 (GDP fucose synthetase，GFS)，能催化由GDP-D-甘露糖合成GDP-L-岩藻糖过程中的两步差向异构酶和还原酶反应；还参与氨基糖和核糖的生物合成，是调控生物体糖代谢、核苷酸代谢的关键酶之一。通过前期基因组测序表明须糖多孢菌Saccharopolyspora pogona 中存在fcl 基因。利用基因工程技术构建了fcl 基因的过表达菌株S. pogona-fcl 和敲除菌株S. pogona-Δfcl。结果表明该基因对菌株生长发育、蛋白表达及其转录水平、杀虫活性、丁烯基多杀菌素的生物合成均存在影响。经HPLC 分析显示，S. pogona-Δfcl的丁烯基多杀菌素产量增加为野生型菌株的130%，S. pogona-fcl 的丁烯基多杀菌素产量降低了25%。生测结果显示，与野生型菌株相比S. pogona-Δfcl 对棉铃虫的杀虫活性明显增强，而S. pogona-fcl 的杀虫活性降低。利用扫描电镜观察发现，S. pogona-Δfcl 菌丝体表面出现褶皱，呈现短棒状，S. pogona-fcl 菌丝形态与野生型菌株一致。以上结果表明，fcl 基因的敲除影响菌丝体的生长发育，能促进丁烯基多杀菌素的生物合成和增强杀虫活性，该基因的过表达抑制了丁烯基多杀菌素的生物合成和降低了杀虫活性。SDS-PAGE 结果表明，三株菌株在96 h 时蛋白表达差异最为明显。对差异蛋白通过实时荧光定量聚合酶链式反应结果显示，三菌株蛋白的转录水平存在显著表达差异。通过研究结果构建了网络代谢调控图，分析fcl 基因对须糖多孢菌生长发育及丁烯基多杀菌素生物合成代谢调控网络途径的影响，初步构建了fcl 基因调控的代谢途径，为揭示丁烯基多杀菌素生物合成的调控机制及相关后续研究提供了实验依据。.

Traditional cancer therapies, such as surgery treatment, radiotherapy, and chemotherapy, often fail to completely eliminate tumor cells in an anaerobic microenvironment of tumor regions. In contrast to these traditional cancer therapies, the use of targeted delivery vectors to deliver anticancer genes or antitumor drugs to hypoxic areas in tumors is the most clinically promising cancer treatment with rapid development in recent years. In this study, E.coli Nissle 1917 (EcN), an intestinal probiotic, was utilized as a targeted transport vector to deliver p53 and Tum-5 protein to tumor hypoxic regions. The tumor-targeting characteristics of EcN were investigated using luciferase LuxCDABE operon, and the results demonstrated that EcN could specifically accumulate in the solid tumor areas of SMMC-7721 tumor-bearing BALB/c nude mice. The Tum 5-p53 bifunctional proteins were initially constructed and then delivered to solid tumor regions by using the targeted transporter EcN for cancer therapy. The antitumor effect and safety of three engineered bacteria, namely, EcN (Tum-5), EcN (p53), and EcN (Tum 5-p53), were also examined. The calculated tumor volume and tumor weight indicated that these three engineered bacteria could inhibit the growth of human hepatoma SMMC-7721 cells, and the antitumor effect of EcN (Tum 5-p53) expressing the Tum 5-p53 fusion protein was significantly better than those of EcN (Tum-5) and EcN (p53) alone. Immunofluorescence demonstrated that the expression of Ki-67, a nuclear proliferation-related protein, was inhibited in the tumor areas of the groups treated with the engineered bacteria, whereas the expression of caspase-3 was upregulated. The expression trends of Ki-67 and caspase-3 were consistent with the different antitumor efficacies of these three engineered bacteria. EcN did not elicit obvious side effects on mice. This research not only provids a foundation for tumor-targeted therapy but also contributes greatly to the development of antitumor agents and anticancer proteins. Electronic supplementary material The online version of this article (10.1186/s13036-019-0189-9) contains supplementary material, which is available to authorized users.

Probiotic-feeding continues to be a promising strategy to control the bacterial pathogens in aquaculture. A new Pediococcus pentosaceus strain (SL001) was isolated from 1000s of soil samples, which exhibited wide antimicrobial spectrum of against fish pathogens, involving Aeromonas hydrophila, Aeromonas veronii, Aeromonas sobria, Edwardsiella tarda, Lactococcus garvieae, and Plesiomonas shigelloide. The challenge test against A. hydrophila showed that the survival rate of SL001-supplemented group was significantly higher than that of control group (P < 0.05). Moreover, SL001 could stably colonize in gut of grass carp and increased mucus-secreting goblet cells and extended intestinal villi could be observed in SL001-supplemented group (P < 0.05). Feeding with SL001 supplemented diet could significantly enhance the growth rate (P < 0.05) and markedly affect gut microbiota structure of grass carps, resulting in reduced potential pathogens and increased potential probiotics. Furthermore, feeding grass carps with SL001 caused the up-regulated expression of insulin-like growth factor (IGF-1 and IGF-2) and down-regulated expression of myostatin (MSTN-1 and MSTN-2) (P < 0.05), which probably also account for the increased growth rate of SL001-fed group. Meanwhile, relative mRNA expression levels of immune-related genes in liver, spleen, and head kidney were analyzed in grass carps after feeding for 30 days with SL001 supplemented diets. In all three immune organs, the expression levels of immunoglobulin M (IgM) and complement 3 (C3) were significantly increased (P < 0.05), whereas the interleukin-8 (IL-8) was down-regulated (P < 0.05). Besides, whole genome sequencing revealed several probiotics properties of SL001, including organic acid synthesis, bacteriocin synthesis (coagulin), superoxide dismutase, and digestive enzymes. In conclusion, P. pentosaceus SL001 which could enhance immunity and promoter growth rate of grass carps, is prospective to be used as a dietary probiotic in freshwater fish aquaculture.

Table S1. Primers used in PCR amplification. Figure S1. Agarose gel electrophoresis of human p53 gene amplification products. Figure S2. Construction and identification of recombinant plasmid pET28a-p18. Figure S3. Construction and identification of plasmid pET28a-p18-p53. Figure S4. Purification and identification of recombinant p53 protein. Figure S5. Inhibitory effect of recombinant p53 protein on different tumor cells. Figure S6. The construction program of hypoxia expression vector pET28a-Pvhb-pelB-p18-p53. Figure S7. Construction of p53 hypoxia expression vector pET28a-Pvhb-pelB-p18-p53. Figure S8. Construction of p53 and Tum-5 fusion expression vector. Figure S9. Construction of Tum-5 and p53 fusion expression vector. Figure S10. Morphological changes of different tumor cells after recombinant Tum 5-p53 treated (100Ă ). Figure S11. The construction process diagram of hypoxia expression vector pET28a-Pvhb-pelB-SUMO-Tum 5-MMP-p53. Figure S12. Construction of recombinant plasmid pET28a-Pvhb-pelB-SUMO-Tum 5-MMP-p53 (DOC 1943 kb)

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.