Measurement of Hyperfine Structure and the Zemach Radius in

We investigate the 2^{3}S_{1}-2^{3}P_{J} (J=0, 1, 2) transitions in ^{6}Li^{+} using the optical Ramsey technique and achieve the most precise values of the hyperfine splittings of the 2^{3}S_{1} and 2^{3}P_{J} states, with smallest uncertainty of about 10 kHz. The present results reduce the uncertainties of previous experiments by a factor of 5 for the 2^{3}S_{1} state and a factor of 50 for the 2^{3}P_{J} states, and are in better agreement with theoretical values. Combining our measured hyperfine intervals of the 2^{3}S_{1} state with the latest quantum electrodynamic (QED) calculations, the improved Zemach radius of the ^{6}Li nucleus is determined to be 2.44(2) fm, with the uncertainty entirely due to the uncalculated QED effects of order mα^{7}. The result is in sharp disagreement with the value 3.71(16) fm determined from simple models of the nuclear charge and magnetization distribution. We call for a more definitive nuclear physics value of the ^{6}Li Zemach radius.

Identification and characterization of a novel diacylglycerol acyltransferase gene from Mortierella alpina.

OBJECTIVES: To clone and express a diacylglycerol acyltransferase (DGAT) gene from Mortierella alpina in Saccharomyces cerevisiae and characterize oil production and fatty acid composition of the resulting recombinant RESULTS: A new, full-length cDNA, putatively encoding a DGAT, was cloned from M. alpina. We subsequently cloned the gene, except the transmembrane-encoding region, termed MaDGAT, its molecular mass was 31.3 kDa. MaDGAT shares 75% identity with a DGAT from Mortierella verticillata NRRL 6337. A recombinant vector expressing MaDGAT, pYES2-DGAT, was constructed and transformed into S. cerevisiae H1246, a neutral, lipid-deficient quadruple mutant. TLC analysis showed that the recombinant vector restored triacylglycerol biosynthesis and its content in the recombinant strain was 3.9%. CONCLUSION: MaDGAT is a novel DGAT gene and could increase TAG biosynthesis in M. alpina or other filamentous fungi, thereby promoting the synthesis of polyunsaturated fatty acids.

Agrobacterium tumefaciens-mediated transformation of a taxol-producing endophytic fungus, Cladosporium cladosporioides MD2.

In this study, an Agrobacteriurn tumefaciens-mediated transformation (ATMT) protocol was successfully developed for the genetic transformation of a taxol-producing fungus, Cladosporium cladosporioides MD2, and the co-cultivation conditions affecting the transformation efficiency were optimized. The optimal transformation conditions were that 1 ml of C. cladosporioides MD2 spore suspension (10(8) spores/ml) was mixed with an equal volume of A. tumefaciens cultures, which contained 400 µl of A. tumefaciens LBA4404 (OD(660) ≈ 0.6) and 600 µl LB medium that were used to make up difference in volume, and the mix cultures were supplemented with 300 µM acetosyringone (AS) and co-cultivated at 26°C and 50 rpm for 48 h. Stable transformants were obtained through analysis of the mitotic stability of inserted T-DNA and the presence of hygromycin resistance gene (hpt II). This study laid a fine groundwork for development of transgenic C. cladosporioides MD2 strains.

Isolation of eicosapentaenoic acid-producing fungi from soil based on polymerase chain reaction amplification.

A method was developed for rapid screening and isolation of eicosapentaenoic acid (EPA)-producing soil fungi through polymerase chain reaction (PCR) amplification. Genes coding for delta6 fatty acid desaturase and delta5 fatty acid desaturase were used as molecular markers for screening these EPA-producing fungi from soil. Three out of 65 soil fungi gave positive results through PCR amplification. Two out of these three strains were found to produce EPA when they had grown in 80 ml potato/dextrose liquid medium at (25 +/- 1) degrees C for 144 h. The EPA yields were 215.81 mg 1(-1) and 263.80 mg 1(-1), respectively. The other positive strain was detected to produce arachidonic acid (AA). This study indicates that molecular detection of genes encoding delta6 and delta5 desaturases is an efficient method for primary screening of EPA- or its related polyunsaturated fatty acids (PuFAs)-producing fungi, which can improve the screening efficiency prominently.

Microbial production of docosahexaenoic acid by a low temperature-adaptive strain Thraustochytriidae sp. Z105: screening and optimization.

As an alternative source in addition to fish oil, microbial production of docosahexaenoic acid has been recieved more and more attentions owing to their culture advantage. A unicellular eukaryotic microbe with high DHA production and capable of low temperature-adaptive growth was isolated from seawater and identified as Thraustochytriidae sp. Z105. The siginificant effect of temperature on cell growth and DHA synthesis by the strain was revealed. It could grow and produce DHA even at 4 degrees C, but hardly grow above 35 degrees C. Low temperature (15-25 degrees C) was favorable for formation of biomass, lipids and DHA, but DHA synthesis was completely blocked above 30 degrees C. Conditions for high level DHA production by Thraustochytriidae sp. Z105 in flask culture were optimized as follows: medium containing glucose 80 g/l, yeast extract 5.0 g/l, K2HPO(4) . 3 H2O 1.0 g/l, MgSO4 . 7 H2O 0.5 g/l, seawater crystal 20 g/l, pH 6.0, liquid volume 30 ml/250 ml, temperature 20 degrees C, agitation speed of 200 r/min, and culture for 120 h. Under the optimal conditions, biomass of 16.72 g/l, total lipids of 5.35 g/l, DHA yield of 1.71 g/l (accounting for 32% of the total lipids) were achieved, respectively. In flask cluture level, the DHA productivity of Thraustochytriidae sp. Z105 was higher than most reported results, which suggested the wild type strain was a potential superior candidate for industrialization of DHA production. Moreover, the strain is an unique and valuable resource for investigation of the low temperature adaptive mechanism related to DHA synthesis.

[Vulnerability assessment of agro-ecological environment in Karst mountain based on set pair analysis model of grey trigonometrically whitening weight]. / 基于灰色三角白化权集对分析模型的喀斯特山区农业生态环境脆弱性评价.

Ecological vulnerability is a hot issue in the study of global change and sustainable development. Understanding the vulnerability of agro-ecological environment is conducive to rational utilization of regional agricultural resources, which could put forward effective measures for prote-cting agro-ecological environment. Given that the evaluation of agricultural eco-environment vulnerability generally does not consider the relationships among different indicators in different evaluation levels, we used the grey trigonometrically whitening weight set pair analysis (SPA) model to evaluate the vulnerability of agricultural eco-environment in Karst mountain by selecting 11 indicators such as population density, per capita arable land area and per capita afforestation area from the external vulnerability of ecological environment. The results showed that the vulnerability degree of agro-ecological environment in the study area was very high, mainly at the extreme, high and medium vulnerability grades. The proportion of extremely, highly, moderately, mildly and slightly vulnerable areas was 32.4%, 14.1%, 17.7%, 23.6% and 12.2% respectively. This result was consistent with the status of agricultural ecological environment vulnerability in the study area. It was feasible to evaluate the vulnerability of agro-ecosystem with the SPA Model of grey trigonometrically whitening weight, which provided a new method for evaluating agricultural ecological environment vulnerability.

An endophytic taxol-producing fungus from Taxus media, Cladosporium cladosporioides MD2.

Fermentation processes using taxol-producing fungi other than Taxus spp. may be an alternative way to produce taxol, which is an important antitumor agent used widely in the clinic setting. In this study, a taxol-producing endophytic fungus strain MD2 was isolated from the inner bark of Taxus media. Strain MD2 produced taxol when grown in potato dextrose liquid medium. The fungal taxol-which was analyzed by ultraviolet, high-performance liquid chromatography and mass spectrometry-was shown to be identical to authentic taxol and 10-deacetylbaccatin III. Further analysis with nuclear magnetic resonance (NMR) spectroscopy to show the chemical structure of the fungal taxol indicated that the fungal taxol produced an NMR spectrum identical to that of authentic taxol. Strain MD2 was identified as Cladosporium cladosporioides according to morphology of the fungal culture, characteristics of the spores, and analysis of 18S rDNA sequence. In addition, 10-deacetylbaccatin III-10-O-acetyl transferase gene of C. cladosporioides MD2 was cloned for the first time and was shown to share 99% identity with that of T. x media and 97% identity with that of T. wallichiana var. mairei.

Improvement of arachidonic acid and eicosapentaenoic acid production by increasing the copy number of the genes encoding fatty acid desaturase and elongase into Pichia pastoris.

Genes encoding Delta6 desaturase, Delta6 fatty acid elongase, and Delta5 desaturase from the alga, Phaeodactylum tricornutum, were co-expressed in Pichia pastoris to produce arachidonic acid (ARA; 20:4 Delta(5, 8, 11, 14)) and eicosapentaenoic acid (EPA; 20:5 Delta(5, 8, 11, 14, 17)). A panel of Pichia clones carrying progressively increasing copies of the heterologous gene expression cassette was created using an in vitro multimerization approach. ARA and EPA accumulated up to 0.3 and 0.1% of total fatty acids, respectively, in the recombinant P. pastoris carrying with double copies of these three heterologous genes, as compared to 0.1 and 0.05%, respectively, in the recombinant P. pastoris with single copy.

Screening of Taxol-producing fungi based on PCR amplification from Taxus.

Genes coding for 10-deacetylbaccatin III-10-O-acetyl transferase and C-13 phenylpropanoid side chain-CoA acyltransferase were used as molecular markers for screening of Taxol-producing endophytic fungi. Using PCR, three out of 90 endophytic fungi, isolated from Taxus x media and Taxus yunnanensis, gave positive results. These 3 strains, when grown in 300 ml potato/dextrose liquid medium at 25 degrees C for 10 days, contained 100-160 microg Taxol/g dry wt of mycelium.

Four-stage dissolved oxygen strategy based on multi-scale analysis for improving spinosad yield by Saccharopolyspora spinosa†ATCC49460.

Dissolved oxygen (DO) is an important influencing factor in the process of aerobic microbial fermentation. Spinosad is an aerobic microbial-derived secondary metabolite. In our study, spinosad was used as an example to establish a DO strategy by multi-scale analysis, which included a reactor, cell and gene scales. We changed DO conditions that are related to the characteristics of cell metabolism (glucose consumption rate, biomass accumulation and spinosad production). Consequently, cell growth was promoted by maintaining DO at 40% in the first 24 h and subsequently increasing DO to 50% in 24 h to 96 h. In an in-depth analysis of the key enzyme genes (gtt, spn A, spn K and spn O), expression of spinosad and specific Adenosine Triphosphate (ATP), the spinosad yield was increased by regulating DO to 30% within 96 h to 192 h and then changing it to 25% in 192 h to 240 h. Under the four-phase DO strategy, spinosad yield increased by 652.1%, 326.1%, 546.8%, and 781.4% compared with the yield obtained under constant DO control at 50%, 40%, 30%, and 20% respectively. The proposed method provides a novel way to develop a precise DO strategy for fermentation.

Influence of initial pH on the precipitation and crystal morphology of calcium carbonate induced by microbial carbonic anhydrase.

The dynamics of calcium carbonate (CaCO(3)) precipitation induced by microbial intracellular or extracellular carbonic anhydrase (CA) at initial pH 6.0, 6.5, 7.0 and 8.0 were investigated through the gaseous diffusion method. The results indicated that both the intracellular and extracellular CA could promote CaCO(3) precipitation. The Ca(2+) ions in the enzymatic systems at initial pH 8.0 were completely deposited at 48 h, which were respectively 21 h, 15 h and 14 h earlier compared with that at initial pH 6.0, pH 6.5 and pH 7.0, indicating that higher pH favored CaCO(3) precipitation in the experimental pH range, and was beneficial to the catalytic action of microbial CA on CaCO(3) precipitation. In addition, XRD analysis indicated that the CaCO(3) precipitates were mainly calcite crystals in the presence of microbial CA. With increasing deposition time, the crystals gradually changed from prism shape to pyramid-like or irregular polyhedral shape based on FESEM analysis.

An endophytic taxol-producing fungus from Taxus x media, Aspergillus candidus MD3.

An endophytic taxol-producing fungus (strain MD3) isolated from the inner bark of Taxus x media was identified as Aspergillus candidus according to its morphological characteristics, physiological and biochemical characteristics, and 18S rRNA gene sequence analysis. Taxol produced by A. candidus MD3 was shown to be identical to authentic taxol analyzed by UV, HPLC, MS and nuclear magnetic resonance spectroscopy. The gene encoding the 10-deacetylbaccatin III-10-O-acetyl transferase, which catalyzes formation of the last diterpene intermediate in the taxol biosynthetic pathway, has been cloned from A. candidus MD3 for the first time and possesses high homology to the same gene found in Taxus spp.

Limestone dissolution induced by fungal mycelia, acidic materials, and carbonic anhydrase from fungi.

Microorganisms influence the dissolution of a number of minerals. Limestone is one of the most abundant rock types in karst areas, and is predominantly calcium carbonate. Two types of experimental systems were designed in this paper, to make comparisons of limestone dissolution rate among the acidic materials and extracellular carbonic anhydrase (CA) excreted by fungi and the enwrapping effect of fungal mycelia. One was the simulated experimental system containing microorganisms. Another was the simulated experimental system without microorganisms. Results of previous experiment indicated that the acidic materials and CA like enzymatic materials excreted by fungi and the enwrapping effect of fungal mycelia were important factors influencing limestone dissolution. In the three factors mentioned above, the dissolution effect was mycelia enwraping effect>acidic dissolution effect>CA enzymatic effect. The results of the second experiment demonstrated further that the limestone dissolution effect of the acidic materials excreted by fungi was stronger than that of CA excreted by fungi. Nevertheless, CA still played an important role in promoting the dissolution of limestone.