Genetic evidences for the core biosynthesis pathway, regulation, transport and secretion of liamocins in yeast-like fungal cells.

So far, it has been still unknown how liamocins are biosynthesized, regulated, transported and secreted. In this study, a highly reducing polyketide synthase (HR-PKS), a mannitol-1-phosphate dehydrogenase (MPDH), a mannitol dehydrogenase (MtDH), an arabitol dehydrogenase (ArDH) and an esterase (Est1) were found to be closely related to core biosynthesis of extracellular liamocins in Aureobasidium melanogenum 6-1-2. The HR-PKS was responsible for biosynthesis of 3,5-dihydroxydecanoic acid. The MPDH and MtDH were implicated in mannitol biosynthesis and the ArDH was involved in arabitol biosynthesis. The Est1 catalyzed ester bond formation of them. A phosphopantetheine transferase (PPTase) activated the HR-PKS and a transcriptional activator Ga11 activated expression of the PKS1 gene. Therefore, deletion of the PKS1 gene, all the three genes encoding MPDH, MtDH and ArDH, the EST1, the gene responsible for PPTase and the gene for Ga11 made all the disruptants (Δpks13, Δpta13, Δest1, Δp12 and Δg11) totally lose the ability to produce any liamocins. A GLTP gene encoding a glycolipid transporter and a MDR1 gene encoding an ABC transporter took part in transport and secretion of the produced liamocins into medium. Removal of the GLTP gene and the MDR1 gene resulted in a Δgltp1 mutant and a Δmdr16 mutant, respectively, that lost the partial ability to secrete liamocins, but which cells were swollen and intracellular lipid accumulation was greatly enhanced. Hydrolysis of liamocins released 3,5-dihydroxydecanoic acid, mannitol, arabitol and acetic acid. We proposed a core biosynthesis pathway, regulation, transport and secretion of liamocins in A. melanogenum.

Fatty acids from oleaginous yeasts and yeast-like fungi and their potential applications.

PURPOSE: Oleaginous yeasts, fatty acids biosynthesis and regulation in the oleaginous yeasts and the fatty acids from the oleaginous yeasts and their applications are reviewed in this article. RESULTS: Oleaginous yeasts such as Rhodosporidium toruloides, Yarrowia lipolytica, Rhodotorula mucilaginosa, and Aureobasidium melanogenum, which can accumulate over 50% lipid of their cell dry weight, have many advantages over other oleaginous microorganisms. The fatty acids from the oleaginous yeasts have many potential applications. Many oleaginous yeasts have now been genetically modified to over-produce fatty acids and their derivatives. The most important features of the oleaginous yeasts are that they have special enzymatic systems for enhanced biosynthesis and regulation of fatty acids in their lipid particles. Recently, some oleaginous yeasts such as R. toruloides have been found to have a unique fatty acids synthetase and other oleaginous yeasts such as A. melanogenum have a unique highly reducing polyketide synthase (HR-PKS) involved in the biosynthesis of hydroxyl fatty acids. CONCLUSIONS: It is necessary to further enhance lipid biosynthesis using metabolic engineering and explore new applications of fatty acids in biotechnology.

Multi-pathways-mediated mechanisms of selenite reduction and elemental selenium nanoparticles biogenesis in the yeast-like fungus Aureobasidium melanogenum I15.

Selenium (Se) plays a critical role in diverse biological processes and is widely used across manufacturing industries. However, the contamination of Se oxyanions also poses a major public health concern. Microbial transformation is a promising approach to detoxify Se oxyanions and produce elemental selenium nanoparticles (SeNPs) with versatile industrial potential. Yeast-like fungi are an important group of environmental microorganisms, but their mechanisms for Se oxyanions reduction remain unknown. In this study, we found that Aureobasidium melanogenum I15 can reduce 1.0 mM selenite by over 90% within 48 h and efficiently form intracellular or extracellular spherical SeNPs. Metabolomic and proteomic analyses disclosed that A. melanogenum I15 evolves a complicated selenite reduction mechanism involving multiple metabolic pathways, including the glutathione/glutathione reductase pathway, the thioredoxin/thioredoxin reductase pathway, the siderophore-mediated pathway, and multiple oxidoreductase-mediated pathways. This study provides the first report on the mechanism of selenite reduction and SeNPs biogenesis in yeast-like fungi and paves an alternative avenue for the bioremediation of selenite contamination and the production of functional organic selenium compounds.

Multi-layered e-feedback anxiety: An action research study among Chinese learners using peer feedback activities in an academic writing course.

There is a gradual increase in the use of e-feedback in higher education, but issues regarding learners' anxiety remain unresolved. In light of the learners' anxiety, e-feedback would essentially become a formality if they are not proactive in providing constructive feedback. This action research examines three cycles of e-feedback activities performed by 12 doctoral students in an academic writing course in a public university in Macau, China. Specifically, the e-feedback activity involved a comprehensive use of various new educational technology tools, namely Moodle, WeChat and Rain Classroom. This study reveals that the causes of students' anxiety when using e-feedback are multi-layered, mainly from the use of smartphones as a communication medium for conducting formal learning activities and the lack of interpersonal and English skills for conveying their thoughts when providing e-feedback. The traditional Chinese culture about the importance of "face" and interpersonal harmony also has impacts on learners' e-feedback delivery. These findings shed new lights on pedagogical practice in higher education.

Morphological characterization and genetic diversity of a new microsporidium, Neoflabelliforma dubium n. sp. from the adipose tissue of Diaphanosoma dubium (Crustacea: Sididae).

We reported a new microsporidium Neoflabelliforma dubium n. sp. from the adipose tissue of Diaphanosoma dubium in China. The infected daphnids generally appeared opaque due to the presence of numerous spore aggregates located in the adipose tissue. All developmental stages were in direct contact with the host cell cytoplasm. Multinucleate sporogonial plasmodia developed into uninucleate sporoblasts by rosette-like fashion. Mature spores were pyriform and monokaryotic, measuring 4.02 ± 0.24 (3.63-4.53) µm long and 2.27 ± 0.15 (2.12-2.57) µm wide (N = 40). The polaroplast was bipartite with a tightly packed anterior lamellae and a loosely aligned posterior lamellae. Isofilar polar filament was coiled 9-11 turns and arranged in 2-3 rows. The phylogenetic analysis based on the obtained SSU rDNA sequence indicated that the N. dubium n. sp. clustered with the freshwater oligochaete-infecting N. aurantiae to form an independent monophyletic group, positioned at the base of Clade 4. In addition, we analyzed the genetic diversity in three N. dubium n. sp. isolates based on the rDNA (SSU rDNA, ITS and LSU rDNA) and Rpb1 gene. The genetic variation among the rDNA sequences was not distinct, however, high nucleotide diversity could be observed in Rpb1 gene, and a wide variety of Rpb1 haplotypes were identified within each isolate. Genetic recombination detected in the Rpb1 sequences presumes cryptic sexual process occurring in N. dubium n. sp. Statistical evolutionary analyses further indicated that the purifying selection eliminated mutations in the Rpb1 gene.

Intraspecific genetic diversity of the fish-infecting microsporidian parasite Pseudokabatana alburnus (Microsporidia).

Pseudokabatana alburnus is a xenoma-forming fish microsporidium, firstly described from the liver of the Culter alburnus from Poyang Lake in China. In the present study, P. alburnus was firstly reported from the ovary of 6 other East Asian minnows, including Squaliobarbus curriculus, Hemiculter leucisculus, Cultrichthys erythropterus, Pseudolaubuca engraulis, Toxabramis swinhonis, and Elopichthys bambusa. Genetic analysis revealed high sequence diversity in the ribosomal internal transcribed spacer region (ITS) and the largest subunit of RNA polymerase II (Rpb1) loci of P. alburnus isolated from different hosts and locations. The variation of Rpb1 mainly occurred in the 1,477-1737 bp regions. The presence of a wide variety of Rpb1 haplotypes within a single fish host, together with evidence of genetic recombination suggested that P. alburnus may have the intergenomic variation and sexual reproduction might be present in other hosts (possibly freshwater shrimp). Phylogenetic analysis and population genetic analysis showed that there was no geographical population divergence for P. alburnus. Homogeneity and high variability of ITS sequences indicates that ITS may be a suitable molecular marker to distinguish different P. alburnus isolates. Our data confirm the broad geographical distribution and host range of P. alburnus in the middle and lower reaches of the Yangtze River. Additionally, we emendated the genus Pseudokabatana to exclude the infection site, liver as one of the taxonomic criteria, and proposed that fish ovary was be the general infection site of P. alburnus.

Diversity investigation of cultivable yeasts associated with honeycombs and identification of a novel Rhodotorula toruloides strain with the robust concomitant production of lipid and carotenoid.

Oleaginous yeasts-derived microbial lipids provide a promising alternative feedstock for the biodiesel industry. However, hyperosmotic stress caused by high sugar concentration during fermentation significantly prevents high cell density and productivity. Isolation of new robust osmophilic oleaginous species from specific environment possibly resolves this issue to some extent. In this study, the cultivable yeast composition of honeycombs was investigated. Totally, 11 species of honeycomb-associated cultivable yeast were identified and characterized. Among them, an osmophilic yeast strain, designated as Rhodotorula toruloides C23 was featured with excellent lipogenic and carotenogenic capacity and remarkable cell growth using glucose, xylose or glycerol as feedstock, with simultaneous production of 24.41 g/L of lipids and 15.50 mg/L of carotenoids from 120 g/L glucose in 6.7-L fermentation. Comparative transcriptomic analysis showed that C23 had evolved a dedicated molecular regulation mechanism to maintain their high simultaneous accumulation of intracellular lipids and carotenoids and cell growth under high sugar concentration.

First Natural Yeast Strain Trichosporon asahii HZ10 with Robust Flavonoid Productivity and Its Potential Biosynthetic Pathway.

Flavonoids are generally thought to be essential plant natural products with diverse bioactivities and pharmacological effects. Conventional approaches for the industrial production of flavonoids through plant extraction and chemical synthesis face serious economic and environmental challenges. Searching for natural robust flavonoid-producing microorganisms satisfying green and sustainable development is one of the good alternatives. Here, a natural yeast, Trichosporon asahii HZ10, isolated from raw honeycombs, was found to accumulate 146.41 mg/L total flavonoids intracellularly. Also, T. asahii HZ10 represents a broad flavonoid metabolic profiling, covering 40 flavonoids, among which nearly half were silibinin, daidzein, and irigenin trimethyl ether, especially silibinin occupying 21.07% of the total flavonoids. This is the first flavonoid-producing natural yeast strain worldwide. Furthermore, T. asahii HZ10-derived flavonoids represent favorable antioxidant activities. Interestingly, genome mining and transcriptome analysis clearly showed that T. asahii HZ10 possibly evolves a novel flavonoid synthesis pathway for the most crucial step of flavonoid skeleton synthesis, which is different from that in plants and filamentous fungi. Therefore, our results not only enrich the diversity of the natural flavonoid biosynthesis pathway but also pave an alternative way to promote the development of a synthetic biology strategy for the microbial production of flavonoids.

Effects of goal orientation on environment management in technology-based physics learning.

The purpose of the current study is to propose and examine a comprehensive model that uses motivational and self-regulated variables to explain factors affecting environment management in technology-based physics learning among Chinese secondary school students. Data were collected from 726 grade-eight secondary school students in Southeast China, who were learning physics. Structural equation modeling was used to analyze the relationships among students' goal orientations, environment management, and time management. Results suggest that students were more likely to manage their environment if they had learning-oriented goals and if they managed their time, but they were less likely to do so if they had social-oriented goals. Implications for teachers' technology integration in physics class were discussed.

(+)-[1-(4-Methoxy-benz-yl)pyrrolidin-2-yl]diphenyl-methanol.

The title compound, C(25)H(27)NO(2), was obtained as the product of a Grignard reagent and an inter-mediate ester synthesized from L-(-)-proline. The asymmetric unit contains two independent mol-ecules, both of which feature an intra-molecular O-H⋯N hydrogen bond. In one of the mol-ecules, the pyrrolidine ring is disordered over two orientations in a 0.63 (3):0.37 (3) ratio.

Efficient Conversion of Cane Molasses into Fructooligosaccharides by a Glucose Derepression Mutant of Aureobasidium melanogenum with High ß-Fructofuranosidase Activity.

Fructooligosaccharides (FOSs) are excellent food ingredients or feed additives by stimulating probiotics. In this paper, a CREA gene encoding a glucose repressor in the ß-fructofuranosidase producer Aureobasidium melanogenum 33 with high-level FOS biosynthesis was disrupted, and glucose repression in disruptant D28 was relieved. The disruptant D28 produced up to 2100 U/mL of ß-fructofuranosidase activity, whereas the enzyme activities produced by parent strain 33 and complemented strain C11 were below 600 U/mL. The whole cells of the disruptant D28 was used to convert cane molasses into FOSs, and 0.58 g of FOSs/g of molasses sugar was synthesized from 350 g/L cane molasses sugar within 4 h. Results demonstrated that the industrial waste cane molasses can be efficiently converted into FOSs by the glucose derepression mutant D28 with high ß-fructofuranosidase activity. This low-cost and environmentally friendly bioprocess has great potential applications in bioengineering and biotechnology for FOS production.

Improved pullulan production by a mutant of Aureobasidium melanogenum TN3-1 from a natural honey and capsule shell preparation.

Aureobasidium melanogenum TN3-1 isolated from a natural honey was a highly genome-duplicated yeast-like fungal strain and a very high pullulan producer. In this study, simultaneous removal of both duplicated AMY1 genes encoding α-amylase and duplicated PKS1 genes responsible for melanin biosynthesis in A. melanogenum TN3-1 rendered a mutant AMY-PKS-11 to transform 140.0 g/L of glucose to produce 103.50 g/L of pigment-free pullulan with molecular weight (Mw) of 3.2 × 105 g/mol. α-Amylase activity produced by the mutant AMY-PKS-11 and expression of the AMY1 genes and PKS genes in it was reduced, but expression of various genes responsible for pullulan biosynthesis in the mutant AMY-PKS-11 was up-regulated. The produced pullulan was used to make the capsule shells successfully and the prepared pullulan capsule shells had various advantages such as high strength, good oxygen barrier properties, raw materials availability, tightness, lightness and high water resistance and may be suitable for all the consumers. Therefore, the prepared capsule shells had highly potential applications in food and pharmaceutical industries.

Over-expression of Vitreoscilla hemoglobin (VHb) and flavohemoglobin (FHb) genes greatly enhances pullulan production.

Overexpression of the optimized Vitreoscilla hemoglobin (VHb) gene and the native flavohemoglobin (FHb) gene in Aureobasidium melanogenum P16 rendered a V6 strain and a F44 strain to overproduce pullulan compared to that produced by their wild type strain P16. The capacity to bind CO and oxygen in the V6 strain and the F44 strain was also obviously enhanced. At the same time, the transcriptional levels of the relevant genes were also increased in the V6 strain and the F44 strain and the fused vgbop + the gene encoding GFP and FHb gene + the gene encoding GFP were also actively expressed. During a 10-liter fermentation, the P16 strain produced only 72.0 ±â€¯1.0 g/L pullulan, the yield was 0.77 g/g of sucrose, the productivity was 0.5 ±â€¯0.01 g/L/h and only 79.4% of the total sugar was used. In contrast, the strain V6 yielded 102.3 ±â€¯1.8 g/L of pullulan, the yield was 0.89 g/g of sucrose, the productivity was 0.7 ±â€¯0.01 g/L/h and 96.0% of total sugar was used while 101.4 ±â€¯2.9 g/L of pullulan was accumulated in the culture of the strain F44, the yield was 0.88 g/g of sucrose, the productivity was 0.7 ±â€¯0.02 g/L/h and 96.4% of total sugar was utilized. These data strongly demonstrated that the concentration of pullulan, yield, productivity and sugar utilization were greatly enhanced by overexpression of the VHb and FHb. But their cell growth was almost the similar.

High pullulan biosynthesis from high concentration of glucose by a hyperosmotic resistant, yeast-like fungal strain isolated from a natural comb-honey.

A novel, yeast-like fungal strain, Aureobasidium melanogenum TN3-1, that was isolated from natural honey can actively transform 140.0 g/L of glucose into 110.29 ±â€¯2.17 g/L of pullulan during fermentation, whereas A. melanogenum P16 and TN1-2 converted 140.0 g/L of glucose into only 45.81 ±â€¯1.7 g/L and 48.7 ±â€¯2.6 g/L of pullulan, respectively. It was noted that most of the cells in the culture of the strain TN3-1 were arthroconidia, while all of the yeast-like fungal cells of A. melanogenum P16 cultivated under the same conditions were blastoconidia. The cell sizes, cell walls and the number of small vacuoles of A. melanogenum TN3-1 were also much larger, thicker and higher, respectively, than those of A. melanogenum P16. The glycerol, trehalose and glycogen content in the A. melanogenum TN3-1 cells was higher than that of the A. melanogenum P16 and TN1-2 cells.

Efficient transformation of sucrose into high pullulan concentrations by Aureobasidium melanogenum TN1-2 isolated from a natural honey.

A very high pullulan producing yeast-like fungus, Aureobasidium melanogenum TN1-2 isolated from a natural honey was found to be able to produce 97.0 g/L of pullulan from 140.0 g/L sucrose at a flask level while it could yield 114.0 g/L of pullulan within 134 h during a 10-liter fermentation, the yield was 0.81 g/g and the productivity was 0.86 g/L/h. The high ability to biosynthesize pullulan by this yeast-like fungal strain TN1-2 was related to high glucosyltransferase activity, high phosphofructo-2-kinase activity, high content of its cellular glycerol and low glucose repressor. The Mw of the produced pullulan was 1.42 × 105 g/mol. The low Mw may be due to the high α-amylase, glucoamylase and isopullulanase activities. The intracellular level of trehalose had no influence on high pullulan production by the yeast-like fungal strain TN1-2.

A trifluoromethyl substituted organoimido derivative of the hexametalate cluster: synthesis, crystal structure and bioactivity of [Mo6O17(NAr)2]2- (Ar=o-CF3C6H4).

The hexamolybdate cluster has been functionalized with fluorine-containing aromatic amine by the DCC (N,N'-dicyclohexylcarbodiimide) dehydrating protocol and a novel bifunctionalized arylimido derivative of hexamolybdate bearing the electron-withdrawing trifluoromethyl group, (Bu(4)N)(2)[Mo(6)O(17)(NAr)(2)] (Ar=o-CF(3)C(6)H(4)), has been prepared. Complete assignments were achieved for the title compound by elemental analysis, IR, (1)H NMR, UV/visible and single-crystal X-ray diffraction analyses. The preliminary agricultural biological activity test indicated that it has some herbicidal and insecticidal activities.

A new class of functionalized polyoxometalates: synthesis, structure and preliminary antitumor activity studies of three arylimido substituted hexamolybdates bearing a strong electron-withdrawing nitro group, (Bu4N)2[Mo6O18([triple bond]NAr)] (Ar = 3-NO2-C6H4, 2-CH3-4-NO2-C6H3, 2-CH3-5-NO2-C6H3).

Three novel mono-functionalized arylimido derivatives of hexamolybdate bearing the strongest electron-withdrawing nitro group, (Bu(4)N)(2)[Mo(6)O(18)([triple bond]NAr)] (1, 2 and 3), have been synthesized for the first time by an improved reaction of octamolybdate ion and 3-nitroaniline hydrochloride, 2-methyl-4-nitroaniline hydrochloride and 2-methyl-5-nitroaniline hydrochloride respectively with DCC (N,N'-dicyclohexylcarbodiimide) as a dehydrating agent. Complete assignments were achieved for the title compounds by elemental analysis, IR, (1)H NMR, UV/visible and single-crystal X-ray diffraction analyses. The preliminary antitumor activity test indicated that the title compounds have some effects on the cellular growth inhibition of K562 cells.