Abstract:Salinomycin is a monocarboxylic acid polyether antibiotics produced by Streptomyces albus. It has strong inhibiting and killing activity against most gram-positive bacteria and various coccidiums with low adverse impact on environment. In addition, salinomycin can specifically inhibit the growth of a variety of cancer cells and cancer stem cells via targeting to multiple sites, and is a promising anti-tumor drug candidate. To obtain high yield salinomycinproducing strain, conventional mutation techniques and modern molecular genetic methods have been used. Meanwhile, bioactivity and selectivity of salinomycin could be improved by modifying the chemical structure and changing drug delivery methods. Here, we summarize the key strategies for enhancing salinomycin production and review the progresses in optimizing its drug activity and targeting properties. The future research focus is also addressed.

Abstract:Corn smut is a disease caused by Basidiomycetous fungus Ustilago maydis. This pathogen is a dimorphic fungus that needs to complete its sexual reproduction in living corn. We reviewed recent research reports of this disease, we divided the parasitization course of U. maydis into 7 stages in this paper, including the formation of pathogenic dikaryotic hyphae, attaching to the surface of host plant, penetrating the host epidermis, weakening host defense response, prolifering mycelium in host plant, inducing tumor in host tissue and the formation of chlamydospore. We also reviewed key genes involved in each stage and elaborate their function during pathogenesis. We present the sophisticated parasitic strategy of U. maydis in the process to achieve its sexual reproduction. The division of U. maydis parasitization course in this review will help understanding the interaction mechanisms between the pathogen and host plant, and provide new ideas for the prevention and control of such diseases.

Abstract:Glyceraldehyde 3-phosphosphate dehydrogenaseis (GAPDH) acts as an enzymes of glycolysis and participates in the glycolytic pathway to generate energy. As a "housekeeping" protein, GAPDH is involved in many cellular processes in addition to glycolysis. These functions include DNA repair, tRNA export, facilitating microtubule polymerization, regulating the expression of the protein, and cell autophagy. Many researches indicated that bacterial surface GAPDH is involved in adhesion. This article reviews the basis function of GAPDH, and the role and mechanism of GAPDH in adhesion of bacteria.

Abstract:Under the effects of a series of microbial infection and endogenous or exogenous stimuli, inflammasomes are assembled as multiple protein complexes in the cytoplasm, which mainly contain pattern recognition receptors (PRRs), apoptosis-associated speck-like protein containing a CARD (ASC), and pro-caspase-1. The inflammasomes are the platform for caspase-1 activation and subsequent proinflammatory cytokines secretion, and caspase-1 dependent cell death as well. As a key adaptor protein, ASC concatenates PRRs and pro-caspase-1 in the cytoplasm. During inflammasome activation, ASC molecules assemble into large molecule dimers, which is called ASC-speck. The formation of ASC-speck is critical for caspase-1 activation, and ASC-speck becomes a target for the therapy and prevention of inflammatory diseases. In this review, advances in molecular mechanism of ASC-speck formation and the regulation systems for ASC-speck are summarized from the aspects of phosphorylation, ubiquitination and iron channels etc. Finally, the research prospects in this field are discussed.

Abstract:[Objective] Paenibacillus sp. 1-49 is a nitrogen-fixing bacterium that has the potential as a fertilizer. However, it grows poorly (OD₆₀₀≤1) in both mineral medium and rich medium. To achieve high-yield biomass, we optimized fermentation medium for Paenibacillus sp. 1-49.[Methods] Plackett-Burman Design and Central Composite Design in response surface methodology were used to optimize medium composition.[Results] The optimal fermentation medium contained:(per liter) 36.22 g Sucrose, 5.31 g Tryptone, 10.92 g Yeast Extract, 0.51 g MgSO₄, 3.5 g NaCl, 0.02 g Na₂MoO₄ and 0.02 g FeSO₄. The maximum OD₆₀₀ of 10.280±0.009 was obtained in shake flask fermentation, reached 94.6% of the predicted value.[Conclusion] We succeeded in using a response surface methodology to optimize the fermentation medium for Paenibacillus sp. 1-49. Our results will be useful for largescale fermentation of this strain and other Paenibacillus spp..

Abstract:[Objective] The goal of this study was to identify uncultured actinobacterial diversity in Xinjiang Ebinur salt lake sediment.[Methods] Total DNA was extracted from 5 sediments of Ebinur lake and the actinobacterial 16S rRNA gene clone libraries were constructed through touchdown PCR program with the common primers. White clones were randomly selected after blue-white spot screening and detected by PCR. Positive clones were analyzed by restriction fragment length polymorphism (RFLP) with restriction enzyme Hha Ⅰ, and clones with unique RFLP were selected to analyze their sequences. After Chimera Check analysis, the sequence homology was analyzed by BLAST and phylogenetic tree was constructed by MEGA 5.0 software.[Results] In total 192 white clones were randomly selected and 166 clones were positive clones. The sequences of 51 clones with unique RFLP were analyzed and detected with Check Chimera program. The results show that 36 OTUs were obtained and GenBank accession numbers were KR182090-KR182131. The library coverage C value was 90.4% and 36 OTUs could be divided into 2 clusters by phylogenetic analysis. The first cluster accounted for 18.1% of the library and belonged to one class Actinobacteria of phylum Actinobacteria, which contained 4 orders including Actinomycetales, Propionibacteriales, Micrococcales and Corynebacteriales. Another cluster was the unclassified actinobacteria, divided into 3 groups and accounted for 81.9% of the library.[Conclusion] Ebinur Lake has a lot of unknown actinobacteria that need to be further investigated.

Abstract:[Objective] The objective of this study was to assess bacterial diversity in sediment samples from two stations (WBC1305 and WBC1316A) in the Pacific polymetallic nodule province.[Methods] The environmental total DNAs were extracted, and 6 bacterial 16S rRNA gene libraries were generated from 6 sediment layers. The Shannon diversity index and Simpson dominance index were calculated for each bacterial community and then compared. The bacterial community structure of each sediment sample was analyzed, and the results were used to construct phylogenetic trees.[Results] In total, 533 bacterial clones were obtained from 6 bacterial clone libraries. Among these 533 clones, 472 clones could be assigned to 16 phylogenetic groups (Acidobacteria, Actinobacteria, Alpha, Beta, Delta, gamma-Proteobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Gemmatimonadetes, Nitrospirae, Planctomycetes, Elusimicrobia, Hydrogenedentes, Chlorobi, and Nitrospinae), whereas the remaining 61 clones could not be classified into any known groups.[Conclusion] The bacterial communities in sediments from WBC1305 are dominated mainly by gamma-Proteobacteria and from WBC1316A by Firmicutes. In addition, the bacterial community structure at WBC1316A is more abundant and complex than that at WBC1305.

Abstract:[Objective] A high yield of ε-poly-L-lysine (ε-PL) producing strain was bred, and the effect of different carbon sources on fermentation performance was studied.[Methods] Genome shuffling and ribosome engineering were used to enhanced strain's productivity, and pH shock strategy was used to fermentation using different carbon sources.[Results] After four rounds of genome shuffling and ribosome engineering, we obtained a high yield mutant Streptomyces albulus GS114 with ε-PL productivity of 3.0 g/L, which was 1.7 folds than that of the initial strain. When we performed fed-batch fermentation using glucose and glycerol as carbon sources in a 5-L fermenter, ε-PL productions reached 43.4 and 45.7 g/L after 192 h fed-batch fermentations, which were increased by 11.0% and 14.9% than that of Streptomyces albulus M-Z18, respectively. Meanwhile, the dry cell weights decreased by 24.0% and 33.2%, and ε-PL yields increased by 34.2% and 30.7%, respectively.[Conclusion] Genome shuffling and ribosome engineering are effective to breed high yield strains.

Abstract:[Objective] To explore the feasibility of using enhancin as synergist to Bacillus thuringiensis (Bt), the truncated fragments of enhancin gene from Pseudaletia unipuncta granulovirus (PuGV-Ps) were optimized and the enhancing effects were studied.[Methods] Based on bioinformational analysis of the function domain of PuGV-Ps enhancin, the prokaryotic expression vectors were constructed, and the protein expression levels as well as their enhancing effects on the degradation of peritrophic membrane (PM) proteins were analyzed, and the function domains of PuGV-Ps enhancin were confirmed.[Results] Three domains were found in the enhancin of PuGV-Ps, including M60-like domain, Zincins catalytic domain and putative mucin or carbohydrate-binding domain. Thirteen predicted N-glycosylation sites were also identified. Based on the sequences of truncated M60-like domain (P69) and carbohydrate-binding domain (P77), two expression vectors, pET15b-P69 and pET15b-P77, were constructed. The expressed P69 and P77 abundance were higher than that of full length enhancing (P104). The degradation activity of purified P69 on the PM proteins of Spodoptera litura was higher than that of purified P77, but both showed lower degradation activities than P104. Both P69 and P77 improved the toxicity of Bt against larvae of Plutella xylostella. However, their synergistic effects were significantly lower than that of P104.[Conclusion] The results revealed that the M60-like domain in N-terminus and carbohydrate-binding domain in C-terminus of PuGV-Ps enhancin all contributed to the enhancing effects of enhancin as well as the maintenance of its native conformation. The truncated P69 fragment may function in keeping the activity of enhacin and improving prokaryotic expression levels. These results provide some useful guidance for the industrialized production of enhancin.

Abstract:[Objective] The complete genome of the agarolytic bacterium Pseudoalteromonas sp. NJ21 from Antarctic sample was analyzed by bioinformatics methods and putative agarase aga3311was screened. Expression and characterization of the putative agarase aga3311 were studied.[Methods] Gene aga3311 was cloned and expressed by genetic engineering method firstly; then, the recombinant enzyme was purified by Ni-NTA chromatography and the characterization of recombinant enzyme was determined by dinitrosalicylic acid method; the hydrolysis product of recombinant enzyme Aga3311 was analyzed by thin-layer chromatography (TLC) and mass spectrometry (MS).[Results] The recombinant expression vectors (pET-30(a)+aga3311) was overexpressed in E. coli BL21(DE3) and 30% of the recombinant protein was soluble. The purified agarase (Aga3311) revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 87 kDa. The optimum temperature of the recombinant agarase was 35℃, and it maintained higher activity between 30 and 45℃, but the activity declined rapidly above 50℃, typical of thermal instability enzyme. The optimum pH was 7.0, and it maintained 50% of its maximum activity between pH 4 and 10. Aga3311 was significantly activated by Fe³⁺, Be²⁺, Zn²⁺ and Ca²⁺, especially Ca²⁺ doubled the enzyme activity. The pattern of agar hydrolysis of Aga3311 is an exo-β-agarase, producing neoagarobiose (NA2) as the final main product.[Conclusion] Aga3311 is an exo-β-agarase of Glyco_hydro_42 family, producing neoagarobiose (NA2) as the final main product.

Abstract:[Objective] An acyl-CoA synthetase (ACS) was characterized by in vitro biochemical assays to get a potential biocatalyst for the generation of polyketide precursors such as propionyl-CoA and butyryl-CoA.[Methods] An ACS was identified in Caldicellulosiruptor owensensis OL genome by BLAST program using malonyl-CoA synthetase as the query sequence. The enzyme was heterologously expressed in Escherichia coli cells and purified to homogeneity by affinity purification. In vitro biochemical assays were used to reveal the substrate profile, the optimum reaction condition, the stability and the kinetics of the enzyme while the site-directed mutagenesis was used to assay the function of active site residues.[Results] The enzyme was promiscuous enough to accept propionic acid, butyric acid, 2-methyl-propionic acid, pentanoic acid, 2-methyl-butyric acid, 3-methyl-butyric acid and cyclohexanecarboxylic acid as the substrates. It was most active at 30℃ and pH 7.0 and relatively stable because 45% activity remained after 8 hours incubation at 70℃. Interestingly, the substrate specificity of the enzyme could be switched by engineering three residues of active site.[Conclusion] The ACS from C. owensensis OL was a potential biocatalyst for the generation of polyketide precursors.

Abstract:[Objective] In order to understand the regulatory mechanisms of chlortetracycline biosynthesis in an industrial strain, function of an Streptomyces antibiotic regulatory proteins (SARP) family transcriptional regulator ctcB in the biosynthetic gene cluster of chlortetracycline was studied.[Methods] By double crossover recombination, we constructed Streptomyces aureofaciens F3 with disrupted SARP family transcriptional regulator ctcB gene. The amplicons of RT-PCR were designed to cover the adjacent genes for verification of the operons in chlortetracycline biosynthetic cluster. Transcriptional level was analyzed in the chlortetracycline biosynthetic gene cluster in the wild type strain and the ctcB gene disrupted mutant LJIA02 by quantitative real-time RT-PCR.[Results] The disruption mutant LJIA02 abolished tetracycline and chlortetracycline production. In RT-PCR six operons were confirmed in chlortetracycline biosynthetic cluster. Quantitative real-time RT-PCR indicated that ctcB directly activated five promoters from ctcG-D, ctcH-K, ctcN-P, ctcW-T and ctcQ.[Conclusion] CtcB is an essential activator as an SARP family transcriptional regulator in the chlortetracycline biosynthetic gene cluster.

Abstract:[Objective] To identify differentially expressed proteins in Mycobacterium marinum wild-type (WT) and mkl::Tn mutant strains, and provide new clues for exploring the functions of mkl gene.[Methods] Cellular proteins were extracted from cultures of M. marinum WT and mkl::Tn strains, and labelled with isobaric tags for relative and absolute quantitation (iTRAQ) 4-plex. Differentially expressed proteins were identified with LC-MS/MS and subjected to biological information analysis.[Results] A total of 566 differentially expressed proteins were revealed, among which 232 proteins were up-regulated (ratio≥1.4) and 334 proteins were down-regulated (ratio≤0.7). These proteins are mainly associated with lipid metabolism, cell wall and cell processes, intermediary metabolism and respiration, and hypothetical proteins. The most down-regulated protein DesA3, is a fatty acid desaturase and involved in the synthesis of oleic acid. Further experiments showed that the growth of mkl::Tn strain was attenuated on 7H10-ADC agar plate without oleic acid, suggesting that mkl may play a role in the biosynthesis of oleic acid.[Conclusion] Differentially expressed proteins were identified in M. marinum mkl::Tn compared to WT, and these results shed light on the mechanisms of mkl gene in mycobacterial pathogenesis.

Abstract:[Objective] To study the effects of cell immobilization on sulfide degradation ability and microbial community structure.[Methods] Sulfide oxidizing microbiota was immobilized by entrapment on polyvinyl alcoholsodium alginate-activated carbon carrier. Sulfide degradation ability of the immobilized and free sulfide oxidizing microbiota was compared in sulfide-rich minimal medium. PCR-DGGE technique was used to reveal the effects of immobilization on microbial community structure.[Results] The maximum sulfide degradation ability of the sulfide oxidizing microbiota in 12 h decreased from 1000 to 600 mg/L after immobilization. Community structure of the sulfide oxidizing microbiota changed after immobilization, but Catenococcus thiocycli was little affected. Thioclava pacifica was even strengthened in the microbiota after immobilization and sulfide degrading.[Conclusion] In conclusion, limited by substrate diffusion and transfer in carrier material, sulfide degradation ability of the sulfide oxidizing microbiota under high sulfide concentration decreased after immobilization. Also, immobilization could affect the microbial community structure of sulfide oxidizing microbiota due to different adaptation ability to the microenvironment and adhesion ability to the carrier material.

Abstract:[Objective] To isolate the fungus with phytotoxic activity from the gut of Pantala flavescens larvae and find the phytotoxic lead compound from the fungal metabolites.[Methods] QTYC01 was isolated from the gut of P. flavescens larvae by means of spread plate and identified by 5.8S rRNA sequence analysis and morphologic observation. Phytotoxic activities of the fermentation broth and ethyl acetate extracts against the radical growth of weeds as well as the safety of crude extract to the selected crops were tested by Petri dish bioassay. The herbicidal activity of QTYC01 against Echinochloa crusgalli seedlings was carried out by potted bioassay. Fermentation product was purified by recrystallization and identified by extensive spectroscopic analysis.[Results] QTYC01 was identified as Curvularia crepinii. The fermentation broth of QTYC01 significantly inhibited the radical growth of E. crusgalli and Amaranthus retroflexus with the inhibition rate of 95.0% and 90.1%, respectively. The fermented liquid showed significant inhibitory activity to the seedling of E. crusgalli with the victimization rate of 71.1%. Under the concentration of 100 μg/mL, ethyl acetate extracts exhibited significant phytotoxic activities against the radical growth of E. crusgalli and A. retroflexus with inhibitory rates of 56.8% and 71.2%, respectively, and showed good security to the selected common crops with the inhibition rate of lower than 33%. Therewith, a bioactive compound was isolated from the ethyl acetate extract and determined as (5Z)-7-oxozeaenol. The compound showed good phytotoxic activity against A. retroflexus with the IC₅₀ value of 4.8 μg/mL.[Conclusion] Strain QTYC01 could be potentially developed as a new microbial herbicide.