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.

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.

Metabolome and Transcriptome Profiling Reveal Carbon Metabolic Flux Changes in Yarrowia lipolytica Cells to Rapamycin.

Yarrowia lipolytica is an oleaginous yeast for the production of oleochemicals and biofuels. Nitrogen deficiency is beneficial to lipids biosynthesis in Y. lipolytica. Target of rapamycin (TOR) regulates the utilization of nutrients, which is inhibited in nitrogen starvation or by rapamycin treatment. However, under nitrogen-rich conditions, the lipids biosynthesis in Y. lipolytica after inhibition of TOR by rapamycin is elusive. Combining metabolomics and transcriptomics analysis, we found that rapamycin altered multiple metabolic processes of Y. lipolytica grown in nitrogen-rich medium, especially the metabolisms of amino acids and lipids. A total of 176 differentially accumulated metabolites were identified after rapamycin treatment. Rapamycin increased the levels of tryptophan, isoleucine, proline, serine, glutamine, histidine, lysine, arginine and glutamic acid, and decreased the levels of threonine, tyrosine and aspartic acid. Two fatty acids in lipid droplets, stearic acid (down-regulated) and stearidonic acid (up-regulated), were identified. The expression of 2224 genes changed significantly after rapamycin treatment. Further analysis revealed that rapamycin reduced carbon flux through lipids biosynthesis, accompanied by increased carbon flux through fatty acids degradation and amino acid (especially glutamic acid, glutamine, proline and arginine) biosynthesis. The dataset provided here is valuable for understanding the molecular mechanisms of amino acid and lipids metabolisms in oleaginous yeast.

BcMettl4-Mediated DNA Adenine N6-Methylation Is Critical for Virulence of Botrytis cinerea.

DNA adenine N6-methylation (6mA) plays a critical role in various biological functions, but its occurrence and functions in filamentous plant pathogens are largely unexplored. Botrytis cinerea is an important pathogenic fungus worldwide. A systematic analysis of 6mA in B. cinerea was performed in this study, revealing that 6mA is widely distributed in the genome of this fungus. The 2 kb regions flanking many genes, particularly the upstream promoter regions, were susceptible to methylation. The role of BcMettl4, a 6mA methyltransferase, in the virulence of B. cinerea was investigated. BcMETTL4 disruption and point mutations of its catalytic motif "DPPW" both resulted in significant 6mA reduction in the genomic DNA and in reduced virulence of B. cinerea. RNA-Seq analysis revealed a total of 13 downregulated genes in the disruption mutant ΔBcMettl4 in which methylation occurred at the promoter sites. These were involved in oxidoreduction, secretory pathways, autophagy and carbohydrate metabolism. Two of these genes, BcFDH and BcMFS2, were independently disrupted. Knockout of BcFDH led to reduced sclerotium formation, while disruption of BcMFS2 resulted in dramatically decreased conidium formation and pathogenicity. These observations indicated that 6mA provides potential epigenetic markers in B. cinerea and that BcMettl4 regulates virulence in this important plant pathogen.

Acetylation of BcHpt Lysine 161 Regulates Botrytis cinerea Sensitivity to Fungicides, Multistress Adaptation and Virulence.

BcHpt is a core element of the high-osmolarity glycerol (HOG) transduction pathway in Botrytis cinerea. In contrast to other elements of the pathway, which have been characterized and proven to play important roles in vegetative differentiation, fungicide resistance, the multistress response, and virulence in B. cinerea, BcHpt (Histidine-containing phosphotransfer) is essential but uncharacterized in B. cinerea. Our previous study reported the first lysine acetylation site (Lys161) in BcHpt. In this study, the functions of this lysine acetylation site in BcHpt were characterized using site-directed mutagenesis. To mimic Lys161 acetylation, we generated the mutant strain ΔBcHPt + BcHptK161Q-GFP, which exhibited a slower growth rate; lower pathogenicity; higher sensitivity to multiple stresses, including osmotic and oxidative stresses, dicarboximides, and demethylation inhibitors (DMIs); and lower BcSak1 phosphorylation levels than wild-type B. cinerea. Constitutive acetylation of BcHpt Ly161 apparently inhibits hyphal growth, the multistress response, and sensitivity to fungicides in B. cinerea. Moreover, the lysine acetylation site affected phosphorylation of the MAPK BcSak1.