A strategy to reduce the byproduct glucose by simultaneously producing levan and single cell oil using an engineered Yarrowia lipolytica strain displaying levansucrase on the surface.

Currently, levan is attracting attention due to its promising applications in the food and biomedical fields. Levansucrase synthesizes levan by polymerizing the fructosyl unit in sucrose. However, a large amount of the byproduct glucose is produced during this process. In this paper, an engineered oleaginous yeast (Yarrowia lipolytica) strain was constructed using a surface display plasmid containing the LevS gene of Gluconobacter sp. MP2116. The levansucrase activity of the engineered yeast strain reached 327.8 U/g of cell dry weight. The maximal levan concentration (58.9 g/l) was achieved within 156 h in the 5-liter fermentation. Over 81.2 % of the sucrose was enzymolyzed by the levansucrase, and the byproduct glucose was converted to 21.8 g/l biomass with an intracellular oil content of 25.5 % (w/w). The obtained oil was comprised of 91.3 % long-chain fatty acids (C16-C18). This study provides new insight for levan production and comprehensive utilization of the byproduct in levan biosynthesis.

Functional Characterization of Verticillium dahliae Race 3-Specific Gene VdR3e in Virulence and Elicitation of Plant Immune Responses.

Verticillium dahliae is a soilborne fungal pathogen that causes disease on many economically important crops. Based on the resistance or susceptibility of differential cultivars in tomato, isolates of V. dahliae are divided into three races. Avirulence (avr) genes within the genomes of the three races have also been identified. However, the functional role of the avr gene in race 3 isolates of V. dahliae has not been characterized. In this study, bioinformatics analysis showed that VdR3e, a cysteine-rich secreted protein encoded by the gene characterizing race 3 in V. dahliae, was likely obtained by horizontal gene transfer from the fungal genus Bipolaris. We demonstrate that VdR3e causes cell death by triggering multiple defense responses. In addition, VdR3e localized at the periphery of the plant cell and triggered immunity depending on its subcellular localization and the cell membrane receptor BAK1. Furthermore, VdR3e is a virulence factor and shows differential pathogenicity in race 3-resistant and -susceptible hosts. These results suggest that VdR3e is a virulence factor that can also interact with BAK1 as a pathogen-associated molecular pattern (PAMP) to trigger immune responses. IMPORTANCE Based on the gene-for-gene model, research on the function of avirulence genes and resistance genes has had an unparalleled impact on breeding for resistance in most crops against individual pathogens. The soilborne fungal pathogen, Verticillium dahliae, is a major pathogen on many economically important crops. Currently, avr genes of the three races in V. dahliae have been identified, but the function of avr gene representing race 3 has not been described. We investigated the characteristics of VdR3e-mediated immunity and demonstrated that VdR3e acts as a PAMP to activate a variety of plant defense responses and induce plant cell death. We also demonstrated that the role of VdR3e in pathogenicity was host dependent. This is the first study to describe the immune and virulence functions of the avr gene from race 3 in V. dahliae, and we provide support for the identification of genes mediating resistance against race 3.

Identification of HSP90C as a substrate of E3 ligase TaSAP5 through ubiquitylome profiling.

Protein ubiquitination is a major post-translational modification important for diverse biological processes. In wheat (Triticum aestivum) and Arabidopsis thaliana, STRESS-ASSOCIATED PROTEIN 5 (SAP5) is involved in drought tolerance, acting as an E3 ubiquitin ligase to target DRIP and MBP-1 for degradation. To identify further target proteins of SAP5, we implemented two independent approaches in this work. We used ubiquitylome capture with a di-Gly-Lys antibody-based peptide enrichment and affinity purification with a polyubiquitin antibody coupled with mass spectrometry to elucidate the SAP5-dependent ubiquitylation of its target proteins in response to osmotic stress. Wild type or TaSAP5-overexpressing Arabidopsis line, which was more tolerant to osmotic stress according to our previous study, were used here. We identified HSP90C (chloroplast heat shock protein 90) as a substrate of TaSAP5. Further biochemical experiments indicated that TaSAP5 interacts with HSP90C and mediates its degradation by the 26S proteasome. Our work also demonstrates that ubiquitylome profiling is an effective approach to search for substrates of the TaSAP5 E3 ubiquitin ligase when heterologously expressed in Arabidopsis.

Cytoprotective effects of galacto-oligosaccharides on colon epithelial cells via up-regulating miR-19b.

AIMS: Despite the protective effect of galacto-oligosaccharides (GOS) on human colon has been widely-reported, the mechanism of its beneficial effect is still unclear. This paper aims to reveal the internal mechanism underlined the anti-colitis effect of GOS by studying its regulatory effect on miRNAs. MAIN METHODS: An in vitro model of colitis was constructed by using human colon epithelial FHC cells and lipopolysaccharide (LPS). An in vivo colitis model was established as well, by injecting Rag2-/- Sprague-Dawley (SD) rats with helicobacter hepaticus. The effects of GOS pre-treatment on these two models were tested, and the miRNAs involved in these effects were studied. KEY FINDINGS: The expression of miR-19b, miR-590-5p and miR-495 was up-regulated, and the expression of miR-29a, miR-31 and miR-142-5p was down-regulated by GOS treatment in both normal and LPS-stimulated FHC cells. Among which, miR-19b was the most varied miRNA. GOS pre-treatment significantly attenuated LPS-induced cell injury, as evidenced by the increase of cell viability, the decrease of apoptosis, as well as the suppressed release of TNF-α, IFN-γ and IL-1ß. GOS pre-treatment could also prevent Rag2-/- rats against helicobacter hepaticus injection induced diarrhea and inflammation, as the body weight and colon organ weight were recovered, diarrhea score was declined, and the release of pro-inflammatory cytokines was inhibited. The in vitro and in vivo effects of GOS abovementioned were all impeded when miR-19b was silenced. SIGNIFICANCE: In vitro and in vivo experiments showed that GOS have certain anti-colitis effect, and this effect may be achieved by up-regulating miR-19b.

Simultaneous production of single cell oil and fumaric acid by a newly isolated yeast Aureobasidium pullulans var. aubasidani DH177.

Microbial oils can be used for biodiesel production and fumaric acid (FA) is widely used in the food and chemical industries. In this study, the production of lipids and FA by Aureobasidium pullulans var. aubasidani DH177 was investigated. A high initial carbon/nitrogen ratio in the medium promoted the accumulation of lipids and FA. When the medium contained 12.0% glucose and 0.2% NH4NO3, the yeast strain DH177 accumulated 64.7% (w/w) oil in its cells, 22.4 g/l cell biomass and 32.3 g/l FA in a 5-L batch fermentation. The maximum yields of oil and FA were 0.12 g/g and 0.27 g/g of consumed sugar, respectively. The compositions of the produced fatty acids were C14:0 (0.6%), C16:0 (24.9%), C16:1 (4.4%), C18:0 (2.1%), C18:1 (57.6%), and C18:2 (10.2%). Biodiesel obtained from the extracted oil burned well. This study provides the pioneering utilization of the yeast strain DH177 for the integrated production of oil and FA.

Single Cell Oil Production from Hydrolysates of Inulin by a Newly Isolated Yeast Papiliotrema laurentii AM113 for Biodiesel Making.

Microbial oils are among the most attractive alternative feedstocks for biodiesel production. In this study, a newly isolated yeast strain, AM113 of Papiliotrema laurentii, was identified as a potential lipid producer, which could accumulate a large amount of intracellular lipids from hydrolysates of inulin. P. laurentii AM113 was able to produce 54.6% (w/w) of intracellular oil in its cells and 18.2 g/l of dry cell mass in a fed-batch fermentation. The yields of lipid and biomass were 0.14 and 0.25 g per gram of consumed sugar, respectively. The lipid productivity was 0.092 g of oil per hour. Compositions of the fatty acids produced were C14:0 (0.9%), C16:0 (10.8%), C16:1 (9.7%), C18:0 (6.5%), C18:1 (60.3%), and C18:2 (11.8%). Biodiesel obtained from the extracted lipids could be burnt well. This study not only provides a promising candidate for single cell oil production, but will also probably facilitate more efficient biodiesel production.

Comparative transcriptome analysis reveals multiple functions for Mhy1p in lipid biosynthesis in the oleaginous yeast Yarrowia lipolytica.

Yarrowia lipolytica is considered as a promising microbial cell factory for bio-oil production due to its ability to accumulate a large amount of lipid. However, the regulation of lipid metabolism in this oleaginous yeast is elusive. In this study, the MHY1 gene was disrupted, and 43.1% (w/w) intracellular oil based on cell dry weight was obtained from the disruptant M-MHY1, while only 30.2% (w/w) lipid based on cell dry weight was obtained from the reference strain. RNA-seq was then performed to analyze transcriptional changes during lipid biosynthesis after MHY1 gene inactivation. The expression of 1597 genes, accounting for 24.7% of annotated Y. lipolytica genes, changed significantly in the disruptant M-MHY1 during lipid biosynthesis. Differential gene expression analysis indicated that Mhy1p performs multiple functions and participates in a wide variety of biological processes, including lipid, amino acid and nitrogen metabolism. Notably, data analysis revealed increased carbon flux through lipid biosynthesis following MHY1 gene inactivation, accompanied by decreased carbon flux through amino acid biosynthesis. Moreover, Mhy1p regulates the cell cycle, and the cell cycle rate was enhanced in the disruptant M-MHY1. These results suggest that Mhy1p plays critical regulatory roles in diverse aspects of various biological processes, especially in lipid biosynthesis, amino acid and nitrogen metabolism and cell cycle. Our dataset appears to elucidate the crucial role of Mhy1p in lipid biosynthesis and serves as a resource for exploring physiological dimorphic growth in Y. lipolytica.

High-Level Production of Exopolysaccharides by a Cosmic Radiation-Induced Mutant M270 of the Maitake Medicinal Mushroom, Grifola frondosa (Agaricomycetes).

A new Grifola frondosa mutant, M270, was successfully isolated for high production of exopolysaccharides (EPSs) using cosmic radiation-induced mutagenesis. We found that the mutant M270 had a clearer and thicker EPS layer (~10 µm) adhering to mycelia than those of its parent strain 265 after Congo red staining. In the 20-L batch fermentation for M270, 10.3 g/L of EPS and 17.9 g/L of dry mycelia biomass were obtained after 204 hours of fermentation. Furthermore, a main water-soluble fraction (EP1) in the EPS was purified from M270 and then confirmed to be heteroglycan-protein complex with 91% (w/w) total carbohydrates and 9% (w/w) total proteins. Four kinds of monosaccharide-D-mannose, D-glucosamine, D-glucose, and D-xylose-were detected in EP1 with a molar ratio of 17.6:1.8:100:2.5. The molecular mass of the main component in EP1 was 8.9 kDa. The EPS from M270 significantly inhibited the growth of sarcoma 180 solid tumors in mice. This G. frondosa M270 mutant could serve as a better candidate strain for polysaccharide production.

Salicylic acid confers enhanced resistance to Glomerella leaf spot in apple.

Glomerella leaf spot (GLS) caused by Glomerella cingulata is a newly emergent disease that results in severe defoliation and fruit spots in apple. Currently, there are no effective means to control this disease except for the traditional fungicide sprays. Induced resistance by elicitors against pathogens infection is a widely accepted eco-friendly strategy. In the present study, we investigated whether exogenous application of salicylic acid (SA) could improve resistance to GLS in a highly susceptible apple cultivar (Malus domestica Borkh. cv. 'Gala') and the underlying mechanisms. The results showed that pretreatment with SA, at 0.1-1.0 mM, induced strong resistance against GLS in 'Gala' apple leaves, with SA treated leaves showing significant reduction in lesion numbers and disease index. Concurrent with the enhanced disease resistance, SA treatment markedly increased the total antioxidant capacity (T-AOC) and defence-related enzyme activities, including catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO). As expected, SA treatment also induced the expression levels of five pathogenesis-related (PR) genes including PR1, PR5, PR8, Chitinase and ß-1,3-glucanase. Furthermore, the most pronounced and/or rapid increase was observed in leaves treated with SA and subsequently inoculated with G. cingulata compared to the treatment with SA or inoculation with the pathogen. Together, these results suggest that exogenous SA triggered increase in reactive oxygen species levels and the antioxidant system might be responsible for enhanced resistance against G. cingulata in 'Gala' apple leaves.