Development of a bacterial gene transcription activating strategy based on transcriptional activator positive feedback.

INTRODUCTION: Transcription of biological nitrogen fixation (nif) genes is activated by the NifA protein which recognizes specific activating sequences upstream of σ54-dependent nif promoters. The large quantities of nitrogenase which can make up 20% of the total proteins in the cell indicates high transcription activating efficiency of NifA and high transcription level of nifHDK nitrogenase genes. OBJECTIVES: Development of an efficient gene transcription activating strategy in bacteria based on positive transcription regulatory proteins and their regulating DNA sequences. METHODS: We designed a highly efficient gene transcription activating strategy in which the nifA gene was placed directly downstream of its regulating sequences. The NifA protein binds its regulating sequences and stimulates transcription of itself and downstream genes. Overexpressed NifA causes transcription activation by positive reinforcement. RESULTS: When this gene transcription activating strategy was used to overexpress NifA in Pseudomonas stutzeri DSM4166 containing the nif gene cluster, the nitrogenase activity was increased by 368 folds which was 16 times higher than that obtained by nifA driven by the strongest endogenous constitutive promoter. When this strategy was used to activate transcription of exogenous biosynthetic genes for the plant auxin indole-3-acetic acid and the antitumor alkaloid pigment prodigiosin in DSM4166, both of them resulted in better performance than the strongest endogenous constitutive promoter and the highest reported productions in heterologous hosts to date. Finally, we demonstrated the universality of this strategy using the positive transcriptional regulator of the psp operon, PspF, in E. coli and the pathway-specific positive transcription regulator of the polyene antibiotic salinomycin biosynthesis, SlnR, in Streptomyces albus. CONCLUSION: Many positive transcription regulatory proteins and their regulating DNA sequences have been identified in bacteria. The gene transcription activating strategy developed in this study will have broad applications in molecular biology and biotechnology.

Systematic identification of endogenous strong constitutive promoters from the diazotrophic rhizosphere bacterium Pseudomonas stutzeri DSM4166 to improve its nitrogenase activity.

BACKGROUND: Biological nitrogen fixation converting atmospheric dinitrogen to ammonia is an important way to provide nitrogen for plants. Pseudomonas stutzeri DSM4166 is a diazotrophic Gram-negative bacterium isolated from the rhizosphere of cereal Sorghum nutans. Endogenous constitutive promoters are important for engineering of the nitrogen fixation pathway, however, they have not been systematically characterized in DSM4166. RESULTS: Twenty-six candidate promoters were identified from DSM4166 by RNA-seq analysis. These 26 promoters were cloned and characterized using the firefly luciferase gene. The strengths of nineteen promoters varied from 100 to 959% of the strength of the gentamicin resistance gene promoter. The strongest P12445 promoter was used to overexpress the biological nitrogen fixation pathway-specific positive regulator gene nifA. The transcription level of nitrogen fixation genes in DSM4166 were significantly increased and the nitrogenase activity was enhanced by 4.1 folds determined by the acetylene reduction method. The nifA overexpressed strain produced 359.1 µM of extracellular ammonium which was 25.6 times higher than that produced by the wild-type strain. CONCLUSIONS: The endogenous strong constitutive promoters identified in this study will facilitate development of DSM4166 as a microbial cell factory for nitrogen fixation and production of other useful compounds.

Improving spinosad production by tuning expressions of the forosamine methyltransferase and the forosaminyl transferase to reduce undesired less active byproducts in the heterologous host Streptomyces albus J1074.

BACKGROUND: Spinosad is a macrolide insecticide with the tetracyclic lactone backbone to which forosamine and tri-O-methylrhamnose are attached. Both the sugar moieties are essential for its insecticidal activity. In biosynthesis of spinosad, the amino group of forosamine is dimethylated by SpnS and then transferred onto the lactone backbone by SpnP. Because the spinosad native producer is difficult to genetically manipulate, we previously changed promoters, ribosome binding sites and start codons of 23 spinosad biosynthetic genes to construct an artificial gene cluster which resulted in a 328-fold yield improvement in the heterologous host Streptomyces albus J1074 compared with the native gene cluster. However, in fermentation of J1074 with the artificial gene cluster, the N-monodesmethyl spinosad with lower insecticidal activity was always produced with the same titer as spinosad. RESULTS: By tuning expression of SpnS with an inducible promotor, we found that the undesired less active byproduct N-monodesmethyl spinosad was produced when SpnS was expressed at low level. Although N-monodesmethyl spinosad can be almost fully eliminated with high SpnS expression level, the titer of desired product spinosad was only increased by less than 38%. When the forosaminyl transferase SpnP was further overexpressed together with SpnS, the titer of spinosad was improved by 5.3 folds and the content of N-desmethyl derivatives was decreased by ~ 90%. CONCLUSION: N-monodesmethyl spinosad was produced due to unbalanced expression of spnS and upstream biosynthetic genes in the refactored artificial gene cluster. The accumulated N-desmethyl forosamine was transferred onto the lactone backbone by SpnP. This study suggested that balanced expression of biosynthetic genes should be considered in the refactoring strategy to avoid accumulation of undesired intermediates or analogues which may affect optimal production of desired compounds.

Functional identification of moss PpGATA1 provides insights into the evolution of LLM-domain B-GATA transcription factors in plants.

B-GATA transcription factors with the LLM domain (LLM-domain B-GATAs) play important roles in developmental processes and environmental responses in flowering plants. Their characterization can therefore provide insights into the structural and functional evolution of functional gene families. Phylogenetic and sequence analysis suggests that LLM-domain B-GATAs evolved from ancestral GATA transcription factors before the divergence of chlorophyte algae and Streptophyta. We compared the function of PpGATA1, a LLM-domain B-GATA gene in moss Physcomitrium patens, with Arabidopsis thaliana counterparts and showed that, in P. patens, PpGATA1 controls growth and greening in haploid gametophytes, while in transgenic Arabidopsis it affects germination, leaf development, flowering time, greening and light responses in diploid sporophytes. These PpGATA1 functions are similar to those of Arabidopsis counterparts, AtGNC, AtGNL and AtGATA17. PpGATA1 was able to complement the role of GNC and GNL in a gnc gnl double mutant, and the LLM domains of PpGATA1 and GNC behaved similarly. The functions of LLM-domain B-GATAs regulating hypocotyl elongation and cotyledon epinasty in flowering plants pre-exist before the divergence of mosses and the lineage leading to flowering plants. This study sheds light on adaption of PpGATA1 and its homologs to new developmental designs during the evolution.

A triple-source CT system for micro-scale investigation of geological materials: A simulation study.

A triple-source CT system is proposed for micro-scale testing of geological materials. This study aims at reducing the projection acquisition time by two-thirds compared to a conventional single-source CT system. The proposed system with different positioning errors in the source-to-object distance (SOD) was simulated and tested using the Shepp-Logan phantom model, as well as slices of sand, glass beads, and concrete samples. Furthermore, the imaging quality of a single-source and the triple-source CT system with different dead detector pixels was compared. The results showed that within the maximum allowable positioning error, the pixel differences between the simulated and the original images are close to zero, and the structural similarities are greater than 0.96. In the presence of dead detector pixels, the quality of the simulated images in the triple-source CT system is superior to that of a single-source CT system. The presented triple-source CT system performs well in high-quality image reconstruction.

Architectural Glazed Tiles Used in Ancient Chinese Screen Walls (15th-18th Century AD): Ceramic Technology, Decay Process and Conservation.

The glazed tile is an important building material used throughout the history of traditional Chinese architecture. Architectural glazed tiles used to decorate the screen walls of ancient China are studied scientifically for the first time. More than 30 glazed tile samples from the screen walls of the 15th to 18th century AD of the Hancheng Confucian Temple and Town God's Temple in Shaanxi Province were carefully investigated using SEM-EDS and XRD. Microstructure and chemistry indicated the raw materials, the recipes and the technological choices used to produce the paste and glaze of the glazed tile samples studied. The causes for the key degradation processes of these glazed tiles used as building materials in the screen walls have also been discussed. This work has clear implications for the restoration and conservation treatments on these kinds of ancient Chinese building materials.

Establishing an efficient salinomycin biosynthetic pathway in three heterologous Streptomyces hosts by constructing a 106-kb multioperon artificial gene cluster.

Salinomycin is a promising anticancer drug for chemotherapy. A highly productive biosynthetic gene cluster will facilitate the creation of analogs with improved therapeutic activity and reduced side effects. In this study, we engineered an artificial 106-kb salinomycin gene cluster and achieved efficient heterologous expression in three hosts: Streptomyces coelicolor CH999, S. lividans K4-114, and S. albus J1074. The six-operon artificial gene cluster consists of 25 genes from the native gene cluster organized into five operons and five fatty acid ß-oxidation genes into one operon. All operons are driven by strong constitutive promoters. For K4-114 and J1074 harboring the artificial gene cluster, salinomycin production in shake flask cultures was 14.3 mg L-1 and 19.3 mg L-1 , respectively. The production was 1.3-fold and 1.7-fold higher, respectively, than that of the native producer S. albus DSM41398. K4-114 and J1074 harboring the native gene cluster produced an undetectable amount of salinomycin and 0.5 mg L-1 , respectively. CH999 harboring the artificial gene cluster produced 10.3 mg L-1 of salinomycin, which was 92% of the production by DSM41398. The efficient heterologous expression system based on the 106-kb multioperon artificial gene cluster established in this study will facilitate structural diversification of salinomycin, which is valuable for drug development and structure-activity studies.

RedEx: a method for seamless DNA insertion and deletion in large multimodular polyketide synthase gene clusters.

Biosynthesis reprograming is an important way to diversify chemical structures. The large repetitive DNA sequences existing in polyketide synthase genes make seamless DNA manipulation of the polyketide biosynthetic gene clusters extremely challenging. In this study, to replace the ethyl group attached to the C-21 of the macrolide insecticide spinosad with a butenyl group by refactoring the 79-kb gene cluster, we developed a RedEx method by combining Redαß mediated linear-circular homologous recombination, ccdB counterselection and exonuclease mediated in vitro annealing to insert an exogenous extension module in the polyketide synthase gene without any extra sequence. RedEx was also applied for seamless deletion of the rhamnose 3'-O-methyltransferase gene in the spinosad gene cluster to produce rhamnosyl-3'-desmethyl derivatives. The advantages of RedEx in seamless mutagenesis will facilitate rational design of complex DNA sequences for diverse purposes.

Enhanced Heterologous Spinosad Production from a 79-kb Synthetic Multioperon Assembly.

Refactoring biosynthetic pathways for enhanced secondary metabolite production is a central challenge for synthetic biology. Here we applied advanced DNA assembly methods and a uniform overexpression logic using constitutive promoters to achieve efficient heterologous production of the complex insecticidal macrolide spinosad. We constructed a 79-kb artificial gene cluster in which 23 biosynthetic genes were grouped into 7 operons, each with a strong constitutive promoter. Compared with the original gene cluster, the artificial gene cluster resulted in a 328-fold enhanced spinosad production in Streptomyces albus J1074. To achieve this goal, we applied the ExoCET DNA assembly method to build a plasmid from 13 GC-rich fragments with high efficiency in one step. Together with our previous direct cloning and recombineering tools, we present new synthetic biology options for refactoring large gene clusters for diverse applications.

Effects of Senegenin against hypoxia/reoxygenation-induced injury in PC12 cells / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effect and the potential mechanism of Senegenin (Sen) against injury induced by hypoxia/reoxygenation (H/R) in highly differentiated PC12 cells.</p><p><b>METHODS</b>The cultured PC12 cells were treated with H/R in the presence or absence of Sen (60 μmol/L). Four groups were included in the experiment: control group, H/R group, H/R+Sen group and Sen group. Cell viability of each group and the level of lactate dehydrogenase (LDH) in culture medium were detected for the pharmacological effect of Sen. Hoechst 33258 staining and annexin V/propidium iodide double staining were used to analyze the apoptosis rate. Moreover, mitochondrial membrane potential (△Ψm), reactive oxygen species (ROS) and intracellular free calcium ([Ca(2+)]i) were measured by fluorescent staining and flow cytometry. Cleaved caspase-3 and activity of NADPH oxidase (NOX) were determined by colorimetric protease assay and enzyme linked immunosorbent assay, respectively.</p><p><b>RESULTS</b>Sen significantly elevated cell viability (P<0.05), decreased the leakage of LDH (P<0.05) and apoptosis rate (P<0.05) in H/R-injured PC12 cells. Sen maintained the value of △Ψm (P<0.05) and suppressed the activity of caspase-3 (P<0.05). Moreover, Sen reduced ROS accumulation P<0.05) and [Ca(2+)]i increment (P<0.05) by inhibiting the activity of NOX (P<0.05).</p><p><b>CONCLUSION</b>Sen may exert cytoprotection against H/R injury by decreasing the levels of intracellular ROS and [Ca(2+)]i, thereby suppressing the mitochondrial pathway of cellular apoptosis.</p>

Mitochondrial DNA genetic polymorphism in thirteen rice cytoplasmic male sterile lines.

KEY MESSAGE: Thirteen rice CMS lines derived from different cytoplasms were classified into eight groups by PCR amplification on mtDNA. The orf79 gene, which causes Boro II CMS, possibly results in Dian1-CMS. Thirteen rice cytoplasmic male sterile (CMS) lines derived from different cytoplasms are widely used for hybrid rice breeding. Based on 27 loci on mitochondrial DNA, including single nucleotide polymorphisms and segmental sequence variations between typical indica and japonica as well as high-polymorphism segmental sequence variations and single nucleotide polymorphisms among rice CMS lines, the 13 rice CMS lines were classified into eight groups: (I) wild-abortive CMS, Indonesian Shuitiangu CMS, K-CMS, Gang CMS, D-CMS and dwarf abortive CMS; (II) Maxie-CMS; (III) Honglian CMS; (IV) Boro II CMS; (V) Dian1-CMS; (VI) Liao-CMS; (VII) Lead CMS; and (VIII) Chinese wild rice CMS. According to their pollen abortion phenotypes, groups I and II (including 7 CMS lines) were classified as sporophytic CMS lines, the cytoplasmic genetic relationships among which were very close. They could have originated from similar, or even the same, cytoplasm donors. Groups III-VIII (including 6 CMS lines) were categorized as gametophytic CMS lines, the cytoplasms of which differed from one another, with some having relatively far genetic relationships. Dian1-CMS was found to harbor the orf79 gene, which causes Boro II CMS, whereas Liao-CMS had an orf79 structure that does not result in Lead CMS. Therefore, we speculated that orf79 is associated with Dian1-CMS but not with Liao-CMS. The atp6-orf79 structure related to sterility was also found to experience multiple evolutionary turnovers. All sporophytic CMS lines were indica-like. Except the Honglian CMS line, which was indica-like, all gametophytic CMS lines were japonica-like.