Metamorphosis of Magnetospirillum magneticum AMB-1 cells

Magnetospirillum magneticum strain AMB-1 belongs to the family of magnetotactic bacteria. It possesses a magnetosome chain aligning, with the assistance of cytoskeleton filaments MamK, along the long axis of the spiral cells. Most fresh M. magneticum AMB-1 cells exhibit spiral morphology. In addition, other cell shapes such as curved and spherical were also observed in this organism. Interestingly, the spherical cell shape increased steadily with prolonged incubation time. As the actin-like cytoskeleton protein MreB is involved in maintenance of cell shapes in rod-shaped bacteria such as Escherichia coli and Bacillus subtilis, the correlation between MreB protein levels and cell shape was investigated in this study. Immunoblotting analysis showed that the quantity of MreB decreased when the cell shape changed along with incubation time. As an internal control, the quantity of MamA was not obviously changed under the same conditions. Cell shape directs cell-wall synthesis during growth and division. MreB is required for maintaining the cell shape. Thus, MreB might play an essential role in maintaining the spiral shape of M. magneticum AMB-1 cells.

Asm8, a specific LAL-type activator of 3-amino-5-hydroxy-benzoate biosynthesis in ansamitocin production

The highly potent antitumor agent ansamitocin P3 is a macrolactam isolated from Actinosynnema pretiosum ATCC 31565. A 120-kb DNA fragment was previously identified as the ansamitocin biosynthetic gene cluster, and contains genes for polyketide assembly, precursor synthesis, post-polyketide synthesis modification, and regulation. Within the biosynthetic gene cluster, asm8 encodes an 1117-amino-acid protein with a high degree of similarity to the large ATP-binding LuxR family-type regulators. In the current study, we determined that inactivation of asm8 by gene replacement in ATCC 31565 resulted in the complete loss of ansamitocin production, and that complementation with a cloned asm8 gene restored ansamitocin biosynthesis. Interestingly, the disruption of asm8 decreased the transcription of genes responsible for 3-amino-5-hydroxybenzoate (AHBA) formation, the starter unit required for ansamitocin biosynthesis. Subsequently, feeding of exogenous AHBA to the asm8 mutant restored ansamitocin biosynthesis, which showed that Asm8 is a specific positive regulator in AHBA biosynthesis. In addition, investigation of asm8 homologs identified two new ansamitocin producers, and inactivation of the asm8 homolog in A. pretiosum ATCC 31280 abolished ansamitocin production in this strain. Characterization of the positive regulator Asm8 and discovery of the two new ansamitocin producers paves the way for further improving production of this important antitumor agent.

Conversion of the high-yield salinomycin producer Streptomyces albus BK3-25 into a surrogate host for polyketide production

An ideal surrogate host for heterologous production of various natural products is expected to have efficient nutrient utilization,fast growth,abundant precursors and energy supply,and a pronounced gene expression.Streptomyces albus BK3-25 is a high-yield industrial strain producing type-Ⅰ polyketide sahnomycin,with a unique ability of bean oil utilization.Its potential of being a surrogate host for heterologous production of PKS was engineered and evaluated herein.Firstly,introduction of a three-gene cassette for the biosynthesis of ethylmalonyl-CoA resulted in accumulation of ethylmalonyl-CoA precursor and sahnomycin,and subsequent deletion of the sahnomycin biosynthetic gene cluster resulted in a host with rich supplies of common polyketide precursors,including malonyl-CoA,methylmalonyl-CoA,and ethylmalonyl-CoA.Secondly,the energy and reducing force were measured,and the improved accumulation of ATP and NADPH was observed in the mutant.Furthermore,the strength of a series of selected endogenous promoters based on microarray data was assessed at different growth phases,and a strong constitutive promoter was identified,providing a useful tool for further engineered gene expression.Finally,the potential of the BK3-25 derived host ZXJ-6 was evaluated with the introduction of the actinorhodin biosynthetic gene cluster from Streptomyces coelicolor,and the heterologous production of actinorhodin was obtained.This work clearly indicated the potential of the high-yield sahnomycin producer as a surrogate host for heterologous production of polyketides,although more genetic manipulation should be conducted to streamline its performance.

Influence of counteranions on catalytic ability of immobilized laccase in Cu-alginate matrices: Inhibition of chloride and activation of acetate

Laccase is a promising oxidase with environmental applications, such as lignin degradation and chlorophenol detoxification. Laccase immobilization can significantly improve physiochemical stability and reusability compared to the free enzymes. In this work, anion effect was investigated in entrapment of Cu-alginate matrix with five types of anions, including perchlorate （ClO4）, nitrate （NO3）, sulfate （SO42 ）, chloride （Cl）, and acetate （CH3CO2-）. Accordingly, chloride inhibition and acetate activation were detected in the o-tolidine kinetic experiments, while effects of the other three anions were much smaller. Such counteranion effects were also observed in the laccase-catalyzed biodegradation of 2,4- dichlorophenol. The results indicated that counteranions in the enzyme immobilization process are crucial for catalytic capacity, probably due to the competition with the carboxylate groups in alginate. Our results also imply that these anions might coordinate the copper cations in laccase.

The Cloning and Expression of Renalase and the Preparation of Its Monoclonal Antibody

To obtain the recombination protein of renalase and prepare the monoclonal antibody, the human renalase gene was amplified from human kidney by specific primer and cloned the DNA fragments into the pET22b. After verification of the positive clones, the gene was transformed to E. coli BL21 to express the protein with 6His on C terminal. The Balb/c mouse was immunized with the purified protein to prepare the monoclonal antibody by hybidoma technique. The renalase protein was reconstructed and 2 strains of the hybidoma which can stable secrete renalase were obtained. The monoclonal antibody can both react with the both recombinant and human serum renalase.

Biochemical Characterization of Translesion Synthesis by Sulfolobus acidocaldarius DNA Polymerases

To study the DNA synthesis mechanism of Sulfolobus acidocaldarius, a thermophilic species from Crenarehaeota, two DNA polymerases of B family（polB1 and polB3）, and one DNA polymerase of Y family（polIV） were recombinantly expressed, purified and biochemically characterized. Both DNA polymerases polBl（Saci_1537） and polB3（Saci_0074） possessed DNA polymerase and 3＇ to 5＇ exonuclease activities; however, both the activities of B3 were very inefficient in vitro. The polIV（Saci_0554） was a polymerase, not an exonuclease. The activities of all the three DNA polymerases were dependent on divalent metal ions Mn1＋ and Mg2＋. They showed the highest activity at pH values ranging from 8.0 to 9.5. Their activities were inhibited by KC1 with high concentration. The optimal reaction temperatures for the three DNA polymerases were between 60 and 70 ℃. Deaminated bases dU and dI on DNA template strongly hindered primer extension by the two DNA polymerases of B family, not by the DNA polymerase of Y family. DNA polymerase of Y Family bypassed the two AP site analogues dSpacer and propane on template more easily than DNA polymerases of B family. Our results suggest that the three DNA polymerases coordinate to fulfill various DNA synthesis in Sulfolobus acidocaldarius cell.

Asymmetric Reduction of 3,5-Bistrifluoromethyl Acetophenone with NADH Regeneration by Immobilized Cells of Saccharomyces rhodotorula in Aqueous-Organic Solvent Biphasic System

Asymmetric reduction of 3,5-bistrifluoromethyl acetophenone to produce(S)-3,5-bistrifluoromethylphenyl ethanol was successfully carried out with sodium alginate immobilized Saccharomyces rhodotorula cells in an aqueous-organic solvent biphasic system.The possible influential factors were examined thoroughly according to their effects on conversion rate and e.e of the product.Organic solvents were rated by their biocompatibility and conversion potential.The immobilized cells [125 mg/mL in 20 mmol/L Tris-HCl buffer and 5%(j) octane at pH 8] showed the best conversion with a substrate concentration of 1.42 g/L at 30℃ with glucose as co-substrate for cofactor regeneration.Sequential 8-batch process was carried out with immobilized cells with a slow decrease in conversion and e.e.The immobilized cells showed stable catalytic activity with 50% reserved activity and are superior especially in reusability in comparison with resting cells.

Biosynthesis of the Central Piperidine Nitrogen Heterocycle in Series a Thiopeptides

Summary of main observation and conclusion Thiopeptides,arising from complex posttranslational modifications of a genetically encoded precursor peptide,are of great interest due to their structural complexity and important biological activities.All of these antibiotics share a macrocyclic peptidyl core that contains a central,six-membered nitrogen heterocycle and are classified into five series a-e based on the oxidation state of the central nitrogenous ring.Here,we report that the biosynthesis of the central piperidine heterocycle of series a thiopeptides relies on the activity of homologues of an F420H2-dependent reductase TppX4 by exploiting and characterizing the piperidine-containing thiopeptin biosynthetic gene (tpp)cluster in Streptomyces tateyamensis.In vitro reconstruction of TppX4 activity demonstrated that the piperidine heterocycle of thiopeptins was transformed from a dehydropiperidine heterocycle,and TppX4 tolerated the changes in the C-termini and macrocyclic peptidyl core of substrate and also tolerated dehyropiperidine-containing monocyclic or bicyclic thiopeptides.The identification of TppX4 and its substrate tolerance enriches the biosynthetic toolbox for development of additional thiopeptide analogs for clinical drug screening.

A quantitative analytical method for valienone and its application in the evaluation of valienone production by a breakthrough microbial process

Valienone is a significant natural carbasugar member of the C7-cyclitol family as a valuable precursor for glycosidase inhibitor drugs.It is an intermediate of validamycin A biosynthesis pathway and exhibits minimal accumulation in the fermentation broth of the natural Streptomyces producer.A quantitative analytical method is crucial for the development of a breakthrough microbial process overcoming the consumption of the natural metabolic flux.The present study was designed to develop a pre-column derivatization high-performance liquid chromatography method for quantification of valienone and to help establish a straightforward fermentation process for valienone production by metabolically engineered Streptomyces hygroscopicus 5008.Valienone was derivatized by 2,4-dinitrophenylhydrazine(DNPH)in 10 mmol×L^(-1) H_3PO_4 at 37℃ for 45 min and the derivatives were separated on Eclipse XDB-C18(5 μm,4.6 mm×150 mm)column at 30 °C eluted with 50% acetonitrile for 18 min.The derivatives were detected by diode array detector at 380 nm and the configurations of the derivatives were determined by computational studies.The method was shown to be effective,sensitive,and reliable.Good linearity was found in the range of 5–2 000 μg×mL^(-1).The intra-and inter-day precisions were 1.1%–2.7% and 1.7%–2.2%,respectively.The absolute recovery of the spiked samples was 97.2%–102.6%.To date,this is the first reversed-phase high-performance liquid chromatography detection method for valienone in microbial culture medium.This method successfully helped evaluate the valienone production capability of the engineered Streptomyces hygroscopicus 5008 and could be promising for C7-cyclitol profiling of different engineered mutants combined with the metabonomics methods.

Nine Pathogenic Fungi of Waterhyacinth Isolated in China

The native pathogens of waterhyacinth in China, were studied and compared on pathogenicity by Koch's postulate. Nine pathogenic fungi, YBH, YBB, YB, YYX, YY, YBA1, YBA2, YBA3 and YYB12, were isolated from diseased waterhyacinth plants, and collected from Zhejiang province and Shanghai. According to cultural characteristics, the nine isolates were preliminarily identified. Isolates YBH and YBB were Collectotrichum sp.; YB, YYX and YY were placed in fungi imperfecti; the isolates YBA1, YBA2, YBA3 and YYB12 were Alternaria sp. The isolate YBH was the highly virulent with a disease index (DI) of 65.28% after one month inoculation. The isolate YBA3 was equily virulent, with the disease index of 67% after 7 day introduction. These two pathogens appear to have the potential as biocontrol agents and they deserve further study.

Biomechanical Properties and Modeling of Skin with Laser Influence

This paper studied experimentally and theoretically the biomechanical properties of skin with laser influence. Different types of tensile tests of the porcine skin in vitro were conducted to study effect of the laser, tensile strength, stress-strain relationship, influence of skin's anisotropy and different regions, repetitive loading and stress-relaxation. A modeling of skin was developed according to the experimental results. The modeling provided insights into the important structure-function relationship in skin tissue with the laser effect. The nonlinear and anisotropic mechanical responses of skin are largely due to varying degree of fiber undulation which is effected by laser and outside forces. By introducing the laser factor into the constitutive modeling, the skin's biomechanical properties and the mechanism of the skin repair with laser were discussed.

Indole methylation protects diketopiperazine configuration in the maremycin biosynthetic pathway

The maremycin biosynthetic gene cluster has been identified in Streptomyces sp. B9173. Comparative metabolic profiling with knockout mutant strains led to the identification of new products correlated to the maremycin biosynthesis, in particular the"demethyl"-maremycins with an unexpected D-tryptophan unit. A biosynthetic pathway for the maremycins is proposed and plausible reasoning for tryptophan epimerization in the demethylmaremycin biosynthesis is also provided.

A turn-on fluorescent probe for Au^(3+) based on rodamine derivative and its bioimaging application

A new fluorescent probe RY was synthesized for the detection of Au3+ions based on a rhodamine B derivative.The fluorescent probe showed good selectivity and sensitivity to Au3+ions.Obvious color and fluorescence changes could be observed with the naked eye while the fluorescent probe reacted with the Au3+ions.The detection limit of the probe was determined to be 36 ppb by the fluorescence titration.The excellent linear relationship suggests that the probe is potentially useful for quantitative detection of Au3+in vitro.We also demonstrated its bioimaging application in both living cells and mice.This was the first time that a fluorescent probe was successfully applied to imaging Au3+in living animals.

An unusual UMP C-5 methylase in nucleoside antibiotic polyoxin biosynthesis

Polyoxin is a group of structurally-related peptidyl nucleoside antibiotics bearing C-5 modifications on the nucleoside skeleton. Although the structural diversity and bioactivity preference of polyoxin are, to some extent, affected by such modifications, the biosynthetic logic for their occurence remains obscure. Here we report the identification of PolB in polyoxin pathway as an unusual UMP C-5 methylase with thymidylate syn- thase activity which is responsible for the C-5 methyla- tion of the nucleoside skeleton. To probe its molecular mechanism, we determined the crystal structures of PolB alone and in complexes with 5-Br UMP and 5-Br dUMP at 2.15 A, 1.76 A and 2.28 A resolutions, respec- tively. Loop 1 （residues 117-131）, Loop 2 （residues 192- 201） and the substrate recognition peptide （residues 94- 102） of PolB exhibit considerable conformational flexi-bility and adopt distinct structures upon binding to different substrate analogs. Consistent with the structural findings, a PolB homolog that harbors an identical function from Streptomyces viridochromogenes DSM 40736 was identified. The discovery of UMP C5-methy-lase opens the way to rational pathway engineering for polyoxin component optimization, and will also enrich the toolbox for natural nucleotide chemistry.

Enhanced validamycin production and gene expression at elevated temperature in Streptomyces hygroscopicus subsp. jingangensis 5008

Cultivation shift from 30℃ to 37℃ significantly enhanced validamycin (VAL) production. Analyzed by reverse-transcription PCR, the transcription of three val genes, valA, valK and valG, representing the three operons of the cluster was simultaneously increased at elevated temperature. Furthermore, the transcription of valP and valQ, a pair of two-component regulators in validamycin biosynthetic gene cluster, was also increased at 37℃. Inactivation of valP and valQ reduced validamycin production at 37℃ to the yield level of wild type strain at 30℃, and the val genes showed reduced expression in the mutant LL-8 at 37℃. These results revealed that the two-component regulator valP and valQ contribute to the elevated validamycin production.

Correlating Expression Data with Gene Function Using Gene Ontology

Clustering is perhaps one of the most widely used tools for microarray data analysis. Proposed roles for genes of unknown function are inferred from clusters of genes similarity expressed across many biological conditions. However, whether function annotation by similarity metrics is reliable or not and to what extent the similarity in gene expression patterns is useful for annotation of gene functions, has not been evaluated. This paper made a comprehensive research on the correlation between the similarity of expression data and of gene functions using Gene Ontology. It has been found that although the similarity in expression patterns and the similarity in gene functions are significantly dependent on each other, this association is rather weak. In addition, among the three categories of Gene Ontology, the similarity of expression data is more useful for cellular component annotation than for biological process and molecular function. The results presented are interesting for the gene functions prediction research area.