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Atmospheric and room temperature plasma (ARTP) is an emerging artificial mutagenesis breeding technology. In comparison to traditional physical and chemical methods, ARTP technology can induce DNA damage more effectively and obtain mutation strains with stable heredity more easily after screening. It possesses advantages such as simplicity, safety, non-toxicity, and cost-effectiveness, showing high application value in microbial breeding. This article focuses on ARTP mutagenesis breeding of actinomycetes, specifically highlighting the application of ARTP mutagenesis technology in improving the performance of strains and enhancing the biosynthetic capabilities of actinomycetes. We analyzed the advantages and challenges of ARTP technology in actinomycetes breeding and summarized the common features, specific mutation sites and metabolic pathways of ARTP mutagenic strains, which could give guidance for genetic modification. It suggested that the future research work should focus on the establishment of high throughput rapid screening methods and integrate transcriptomics, proteomics, metabonomics and other omics to delve into the genetic regulations and synthetic mechanisms of the bioactive substances in ARTP mutated actinomycetes. This article aims to provide new perspectives for actinomycetes breeding through the establishment and application of ARTP mutagenesis technology, thereby promoting source innovation and the sustainable industrial development of actinomycetes.
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Actinobacteria , Mutagénesis , Actinobacteria/genética , MutaciónRESUMEN
Triacylglycerol (TAG) is crucial for antibiotic biosynthesis derived from Streptomyces, as it serves as an important carbon source. In this study, the supplementation of exogenous TAG led to a 3.92-fold augmentation in spinosad production. The impact of exogenous TAG on the metabolic network of Saccharopolyspora spinosa were deeply analyzed through comparative proteomics. To optimize TAG metabolism and enhance spinosad biosynthesis, the lipase-encoding genes lip886 and lip385 were overexpressed or co-expressed. The results shown that the yield of spinosad was increased by 0.8-fold and 0.4-fold when lip886 and lip385 genes were overexpressed, respectively. Synergistic co-expression of these genes resulted in a 2.29-fold increase in the yield of spinosad. Remarkably, the combined overexpression of lip886 and lip385 in the presence of exogenous TAG elevated spinosad yields by 5.5-fold, led to a drastic increase in spinosad production from 0.036 g/L to 0.234 g/L. This study underscores the modification of intracellular concentrations of free fatty acids (FFAs), short-chain acyl-CoAs, ATP, and NADPH as mechanisms by which exogenous TAG modulates spinosad biosynthesis. Overall, the findings validate the enhancement of TAG catabolism as a beneficial strategy for optimizing spinosad production and provide foundational insights for engineering secondary metabolite biosynthesis pathways in another Streptomyces.
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Pirin family proteins perform a variety of biological functions and widely exist in all living organisms. A few studies have shown that Pirin family proteins may be involved in the biosynthesis of antibiotics in actinomycetes. However, the function of Pirin-like proteins in S. spinosa is still unclear. In this study, the inactivation of the sspirin gene led to serious growth defects and the accumulation of H2O2. Surprisingly, the overexpression and knockout of sspirin slightly accelerated the consumption and utilization of glucose, weakened the TCA cycle, delayed sporulation, and enhanced sporulation in the later stage. In addition, the overexpression of sspirin can enhance the ß-oxidation pathway and increase the yield of spinosad by 0.88 times, while the inactivation of sspirin hardly produced spinosad. After adding MnCl2, the spinosad yield of the sspirin overexpression strain was further increased to 2.5 times that of the wild-type strain. This study preliminarily revealed the effects of Pirin-like proteins on the growth development and metabolism of S. spinosa and further expanded knowledge of Pirin-like proteins in actinomycetes. KEY POINTS: ⢠Overexpression of the sspirin gene possibly triggers carbon catabolite repression (CCR) ⢠Overexpression of the sspirin gene can promote the synthesis of spinosad ⢠Knockout of the sspirin gene leads to serious growth and spinosad production defects.
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Actinobacteria , Saccharopolyspora , Peróxido de Hidrógeno/metabolismo , Saccharopolyspora/metabolismo , Actinobacteria/metabolismo , Macrólidos/metabolismo , Combinación de MedicamentosRESUMEN
A high production mutated strain Bacillus thuringiensis X023PN (BtX023PN) was screened from the wild strain Bacillus thuringiensis X023 (BtX023) after atmospheric and room temperature plasma (ARTP) and nitrosoguanidine (NTG) mutation. BtX023PN grows faster than the wild strain, and its lysis of mother cell was 6 h ahead BtX023, but the ability of sporulation was significantly reduced. Bioassay indicated that compared with the wild type strain, the virulence of BtX023PN against Plutella xylostella (P. xylostella) and Mythimna seperata (M. seperata) increased to 2.33-fold and 2.13-fold respectively. qRT-PCR and SDS-PAGE demonstrated that the production of Cry1Ac increased by 61%. Resequence indicated that the mutated sites enriched on the key carbohydrate metabolism and amino acid metabolism. This study provides a new strain resource for the development of Bt insecticides and a feasible technical strategy for the breeding of Bt. KEY POINTS: ⢠Atmospheric and room temperature plasma used in breeding of Bacillus thuringiensis. ⢠Less stationary phase time with more ICP production. ⢠Semi-lethal concentration against Plutella xylostella reduced by about 57.
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Bacillus thuringiensis , Mariposas Nocturnas , Animales , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/metabolismo , Endotoxinas/genética , Endotoxinas/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Larva , Mutación , Nitrosoguanidinas , VirulenciaRESUMEN
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.
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Nitrógeno , Saccharopolyspora , Proteínas Bacterianas/genética , Genes Reguladores , Macrólidos/metabolismo , Nitrógeno/metabolismo , Saccharopolyspora/metabolismoRESUMEN
BACKGROUND: This study explored disparities in characteristics and mortalities among four major transmission groups on antiretroviral therapy in northwest China as well as the survival impact of each transmission route. METHODS: We first examined disparities in demographics and clinical characteristics of the four transmission populations. Kaplan Meier analysis was subsequently conducted to compare survival rates among all groups. At last, Cox proportional hazards regression model was employed to analyze the survival impact of a transmission route among seven main categories of survival factors associated with all-cause mortalities. RESULTS: Survival analysis showed significant differences in all-cause, AIDS- and non-AIDS-related deaths among four HIV populations (all P < 0.05). Using homosexuals as the reference, Cox proportional hazards model further revealed that the risk of all-cause death for blood and plasma donors was significantly higher than that of the reference (aHR: 5.21, 95%CI: 1.54-17.67); the risk of non-AIDS-related death for heterosexuals (aHR: 2.07, 95%CI: 1.01-4.20) and that for blood and plasma donors (aHR: 19.81, 95%CI: 5.62-69.89) were both significantly higher than that of the reference. CONCLUSIONS: Significant disparities were found in characteristics and mortalities among the four transmission groups where mortality disparities were mainly due to non-AIDS-related death. Suggestions are provided for each group to improve their survivorship.
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Síndrome de Inmunodeficiencia Adquirida , Infecciones por VIH , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Infecciones por VIH/tratamiento farmacológico , Humanos , Masculino , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Análisis de SupervivenciaRESUMEN
Understanding the metabolism of Saccharopolyspora pogona on a global scale is essential for manipulating its metabolic capabilities to improve butenyl-spinosyn biosynthesis. Here, we combined multiomics analysis to parse S. pogona genomic information, construct a metabolic network, and mine important functional genes that affect the butenyl-spinosyn biosynthesis. This research not only elucidated the relationship between butenyl-spinosyn biosynthesis and the primary metabolic pathway but also showed that the low expression level and continuous downregulation of the bus cluster and the competitive utilization of acetyl-CoA were the main reasons for reduced butenyl-spinosyn production. Our framework identified 148 genes related to butenyl-spinosyn biosynthesis that were significantly differentially expressed, confirming that butenyl-spinosyn polyketide synthase (PKS) and succinic semialdehyde dehydrogenase (GabD) play an important role in regulating butenyl-spinosyn biosynthesis. Combined modification of these genes increased overall butenyl-spinosyn production by 6.38-fold to 154.1 ± 10.98 mg/L. Our results provide an important strategy for further promoting the butenyl-spinosyn titer.
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Macrólidos , Saccharopolyspora , Proteínas Bacterianas/metabolismo , Macrólidos/metabolismo , Redes y Vías Metabólicas/genética , Saccharopolyspora/genética , Saccharopolyspora/metabolismoRESUMEN
Beneficial microorganisms to control bacterial diseases has been widely used in aquaculture, Bacillus amyloliquefaciens (BaX030) as a probiotic feed additive was a commonly biological control method. Added sucrose promoted the growth of BaX030, and the yield of its antibacterial substance macrolactin A was enhanced by 1.46-fold. A total of 2055 proteins were screened through proteomics, with 143 upregulated and 307 downregulated. Differential protein expression analysis and qRT-PCR verification showed that the pentose phosphate pathway and the fatty acid synthesis pathway were upregulated, thereby providing sufficient energy and precursors for the synthesis of macrolactin A. The influence of some potential regulatory factors (SecG, LiaI, MecG and ComG) on macrolactin A was discovered. After grass carp were fed with BaX030, the abundance of probiotics (Fusobacterium, Proteobacteria, Gemmobacter) were higher than the control group, and the abundance of potential pathogenic bacteria (Planctomycetes, Aeromonas) were significantly lower than the control group. The cell and challenge experiments showed that BaX030 can significantly increase the expression of C3 and IL8 in the liver and kidney, which decreases the risk of immune organ disease. Moreover, BaX030 effectively reduced the mortality of grass carp. The results revealed that BaX030 can significantly improve the structure of the intestinal flora, enhance immunity and it is beneficial to the control of grass carp Aeromonas.
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Aeromonas , Bacillus amyloliquefaciens , Carpas , Enfermedades de los Peces , Microbioma Gastrointestinal , Infecciones por Bacterias Gramnegativas , Probióticos , Aeromonas hydrophila , Animales , Antibacterianos/farmacología , Bacillus amyloliquefaciens/genética , Dieta , Enfermedades de los Peces/microbiología , Infecciones por Bacterias Gramnegativas/microbiología , Inmunidad Innata , Probióticos/farmacologíaRESUMEN
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.
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Eliminación de Gen , Familia de Multigenes , Naftoquinonas/química , Saccharopolyspora/genética , Saccharopolyspora/metabolismoRESUMEN
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.
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Proteómica , Saccharopolyspora , Proteínas Bacterianas/genética , Macrólidos , Saccharopolyspora/genéticaRESUMEN
PURPOSE: Keratin 17 (K17) is an embryonic keratin and overexpression is seen in psoriasis, which is a hyperproliferation skin disease. Nonetheless, whether it is also highly expressed in other proliferative skin diseases remains unclear. The aim of this study is to explore the expression of K17 in cutaneous lichen planus (CLP), lichen simplex chronicus (LSC), and prurigo nodularis (PN). METHODS: A total of 20 skin samples from CLP lesions, 20 from LSC lesions, 20 from PN lesions, and 10 healthy adult skin tissues were obtained. Then, the expression of K17 was analyzed using immunohistochemistry on paraffin-embedded tissue sections. Furthermore, quantitative and semi-quantitative immunohistochemical scores of K17 were independently evaluated under a microscope by 2 dermatologists. RESULTS: Immunohistochemical analysis revealed that in normal skin, K17 was minimally expressed. Nevertheless, it was highly expressed in all epidermal layers in CLP lesions (P-value <0.01), and negatively expressed in LSC and PN lesions (all P-value >0.05). The average gray value of K17 in CLP was 151.153±13.985 (P-value <0.001), while the average values of K17 in LSC and PN were 178.720±12.001 and 181.316±8.920, respectively (all P-value >0.05). CONCLUSION: K17 is potentially expressed in certain inflammatory skin diseases, including psoriasis and lichen planus. Besides, it is not always a marker of hyperproliferation of keratinocytes in skin diseases.
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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.
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Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Grupo Citocromo b/genética , Grupo Citocromo b/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Insecticidas/metabolismo , Hierro/metabolismo , Macrólidos/metabolismo , Saccharopolyspora/metabolismo , Proteínas Bacterianas/farmacología , Grupo Citocromo b/farmacología , Ferritinas/farmacología , Ingeniería Genética , Macrólidos/clasificación , Proteómica , Saccharopolyspora/efectos de los fármacos , Saccharopolyspora/genética , Saccharopolyspora/crecimiento & desarrolloRESUMEN
Bacillus amyloliquefaciens X030 (BaX030) has broad-spectrum antibacterial activity against the fish pathogens Aeromonas hydrophila and Aeromonas veronii. To improve its antibacterial effect, BaX030 was subjected to compound mutagenesis of atmospheric and room temperature plasma (ARTP) and nitrosoguanidine (NTG). The results showed that, compared with the original strain, the production of macrolactin A and oxydifficidin in mutated strain N-11 increased to 39 % and 268 %, respectively. The re-sequencing analysis suggested that there were SNPs and InDels in the gene clusters focused on the sucrose utilization pathway, glycolysis pathway and fatty acid synthesis pathway. Scanning electron microscopy revealed that strain N-11 became thin and long. The qRT-PCR results indicated that the expression of immune factors in the liver or kidney tissue of grass carp increased after feeding with N-11. H&E staining and protection experiments also showed that the mortality and surface symptoms of grass carp infected by the two pathogens were significantly reduced. The study identified a probiotic strain with potential application value in aquaculture production and provided a new strategy for the discovery of new strains with higher antibacterial biological activity.
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Aeromonas hydrophila/fisiología , Aeromonas veronii/fisiología , Bacillus amyloliquefaciens/genética , Carpas/microbiología , Interacciones Microbianas/genética , Mutación , Probióticos , Animales , Bacillus amyloliquefaciens/fisiología , Enfermedades de los Peces/microbiologíaRESUMEN
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.
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Sintasas Poliquetidas , Saccharopolyspora , Macrólidos , Familia de Multigenes , Sintasas Poliquetidas/genética , Saccharopolyspora/genéticaRESUMEN
Lysine metabolism plays an important role in the formation of the insecticidal crystal proteins of Bacillus thuringiensis (Bt). The genes lam, gabD and sucA encode three key enzymes of the lysine metabolic pathway in Bt4.0718. The lam gene mainly affects the cell growth at stable period, negligibly affected sporulation and insecticidal crystal protein (ICP) production. While, the deletion mutant strains of the gabD and sucA genes showed that the growth, sporulation and crystal protein formation were inhibited, cells became slender, and insecticidal activity was significantly reduced. iTRAQ proteomics and qRT-PCR used to analyse the differentially expressed protein (DEP) between the two mutant strains and the wild type strain. The functions of DEPs were visualized and statistically classified, which affect bacterial growth and metabolism by regulating biological metabolism pathways: the major carbon metabolism pathways, amino acid metabolism, oxidative phosphorylation pathways, nucleic acid metabolism, fatty acid synthesis and peptidoglycan synthesis. The gabD and sucA genes in lysine metabolic pathway are closely related to the sporulation and crystal proteins formation. The effects of DEPs and functional genes on basic cellular metabolic pathways were studied to provide new strategies for the construction of highly virulent insecticidal strains, the targeted transformation of functional genes.
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Bacillus thuringiensis , Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Endotoxinas , Técnicas de Inactivación de Genes , Proteínas Hemolisinas , LisinaRESUMEN
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.
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Proteínas Bacterianas/metabolismo , Saccharopolyspora/enzimología , Proteínas Bacterianas/genética , Perfilación de la Expresión Génica , Macrólidos/metabolismo , Mutación , Ácido Pirúvico/metabolismo , RNA-Seq , Saccharopolyspora/genética , Saccharopolyspora/metabolismo , TranscriptomaRESUMEN
One of the common shortcomings with Bacillus thuringiensis (Bt) biopesticides in field application is their instability under UV irradiation. In Bt, the leuB gene encodes the 3-isopropylmalate dehydrogenase. In addition to its role in leucine biosynthesis, LeuB would be likely recruited to catalyze the dehydrogenation of malate in the final step of tricarboxylic acid cycle during sporulation. In this study, we constructed a Bt recombinant strain in which the gene leuB was deleted by using the markerless gene deletion system. The ΔleuB mutant strain showed a conditionally asporogenous phenotype while overproducing insecticidal crystal proteins and retaining its insecticidal activity well in both fermentation and LB media. Furthermore, the metabolic regulation mechanisms of LeuB was elucidated by iTRAQ-based quantitative proteomics approach. Evidences from proteomics data suggested that the inhibited supply of pyruvate (carbon source) was an important factor related to the conditionally asporogenous feature of the mutant. Consistently, the mutant regained its ability to sporulate in LB medium by adding 1% glucose or 1% sodium pyruvate. Taken together, our study demonstrated that deletion of the leuB gene resulted in delayed or completely blocked mother cell lysis, allowing the crystals encapsulated within cells, which makes this recombinant strain a good candidate for developing Bt preparations with better UV-stability.
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Long noncoding RNA (lncRNA) FOXD2 adjacent opposite strand RNA 1 (FOXD2-AS1) has been shown to be dysregulated in several types of human cancer. However, the role of FOXD2-AS1 in cutaneous melanoma was still unclear. In our study, FOXD2-AS1 expression has been found to be upregulated in cutaneous melanoma tissue specimens and cell lines compared with that in normal tissue specimens and normal human epidermal melanocyte, respectively. Furthermore, high expression of FOXD2-AS1 was obviously correlated with deep Breslow thickness, present ulceration, high Clark level and distant metastasis in cutaneous melanoma patients. However, there were no statistical associations between FOXD2-AS1 expression and cutaneous melanoma patients' disease-free survival and overall survival. The results of loss-of-function study showed that inhibition of FOXD2-AS1 suppresses cutaneous melanoma cell proliferation, migration and invasion through regulating phospho-Akt expression. In conclusion, FOXD2-AS1 is associated with clinical progression in cutaneous melanoma patients, and functions as oncogenic lncRNA in cutaneous melanoma cells.
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Biomarcadores de Tumor/genética , Proliferación Celular/genética , Melanoma/genética , ARN Largo no Codificante/genética , Neoplasias Cutáneas/genética , Anciano , Carcinogénesis/genética , Línea Celular Tumoral , Movimiento Celular/genética , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Masculino , Melanoma/patología , Persona de Mediana Edad , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Transducción de Señal/genética , Neoplasias Cutáneas/patología , Melanoma Cutáneo MalignoRESUMEN
PURPOSE: This study examined the relationship between the mechanism of hepatitis B virus (HBV) father-infant transmission via reproductive cells and pregnancy outcome. METHODS: Abandoned in vitro fertilization (IVF) embryos of fathers with chronic HBV infection were taken as study objects. HBV mRNA in embryos was detected, and successfully transplanted embryos were followed up to determine the relationship between HBV-infected embryos and pregnancy outcome. RESULTS: HBV mRNA signals were detected in one embryo in the group with HBV-positive fathers; the positive rate was 1/18 (5.5%). IVF embryos of HBV-positive fathers with HBV mRNA signals were successfully implanted, but early abortion occurred. CONCLUSIONS: HBV mRNA was found in abandoned IVF embryos of HBV-infected fathers, which confirmed that HBV could not only enter early cleavage embryos via sperm but also replicate in embryos, resulting in HBV father-infant transmission. HBV may interfere with embryonic development and thus affect pregnancy outcome.
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Padre , Hepatitis B Crónica/transmisión , Transmisión Vertical de Enfermedad Infecciosa , Resultado del Embarazo , Adulto , Fase de Segmentación del Huevo/virología , Desarrollo Embrionario , Femenino , Fertilización In Vitro , Virus de la Hepatitis B/genética , Humanos , Lactante , Recién Nacido , Masculino , Embarazo , ARN Mensajero/genética , Espermatozoides/virología , Adulto JovenRESUMEN
The chemical constituents of Taxus chinensis var. mairei cell cultures were investigated by chromatographic methods, including silica gel column chromatography, Sephadex LH-20 and preparative HPLC. Thirteen compounds were isolated from the 80% ethanol extract of cultured cells and their structures were elucidated by spectral data and physicochemical properties, which were identified as 2α,4α,7ß,9α,10ß-pentaacetoxy-14ß-hydroxytax-11-ene (1), 2α,4α,7ß,9α,10ß-pentaacetoxytax-11-ene (2), 1ß-deoxybaccatin VI (3), 2α-acetoxytaxusin (4), taxuyunnanine C (5), yunnanxane (6), 2α,5α,10ß-triacetoxy-14ß-propionyloxy-4 (20), 11-taxadiene (7), 2α,5α,10ß-triacetoxy-14ß-isobutyryloxy-4 (20), 11-taxadiene (8), 2α,5α,10ß-triacetoxy-14ß-(2'-methyl)butyryloxy-4 (20), 11-taxadiene (9), 13-dehydroxylbaccatin III (10), 13-dehydroxy-10-deacetylbaccatin III (11), paclitaxel (12) and (13) ß-sitosterol. Among them, compound 1 is a new compound, and compounds 2, 4, 10 and 11 are isolated from the cell culture of Taxus chinensis var. mairei for the first time.