| Candida glycerinogenes is an osmotic pressure industrial yeast which can be used for environmentally friendly and energy efficient high gravity fermentation.Especially at a high glucose concentration of 500 g·L-1,it has the characteristics of strong anti-stress ability,rapid sugar metabolism and high conversion rate of target products.Yeast plasma membrane is an important place for material,energy exchange and signal transmission with the external environment.A series of dynamic responses will occur under stress.The relevant theoretical mechanism can provide important scientific basis for guiding actual production.Based on the establishment of a method for extracting C.glycerinogenes plasma membrane,the composition of the plasma membrane and the global transcriptional response mechanism of related genes under high glucose stress were investigated in this study.The main results are as follows:?1?The preparation method of protoplasts,and the two-phase separation and purification system of plasma membrane were optimized.The crude membrane extract was obtained by protoplast preparation and low osmotic cracking.Then the cude plasma membrane was purified by two-phase separation.A high-efficiency plasma membrane extraction method suitable for C.glycerinogenes was obtained,under which the average purity of plasma membrane can reach more than 65%.?2?The relative contents of unsaturated fatty acids?especially oleic acid?and ergosterol in plasma membrane of C.glycerinogenes increased significantly under high glucose stress,and the fluidity and permeability of plasma membrane decreased to varying degrees.Therefore,the plasma membrane fluidity of C.glycerinogenes can be stabilized by increasing ergosterol content,and the plasma membrane homeostasis may be maintained by increasing oleic acid content under high glucose stress.?3?The global transcriptional response of plasma membrane associated genes in the cytoplasmic membrane of C.glycerinogenes under high glucose stress were investigated by RNA-Seq sequencing.Eighty-five genes of plasma membrane-associated differentially expressed were obtained,of which 68 genes were up-regulated and 17 genes were down-regulated.The ergosterol synthesis related genes of ERG6,ERG2,ERG3,ERG5,and ERG4 were significantly up-regulated,which resulting in increasing of ergosterol content.Most of the genes in the unsaturated fatty acid synthesis pathway were significantly up-regulated.The highest differential expression multiple was found in a glucose transporter gene?log2=9.55?.And the genes of glycolipid synthesis,MAPK pathway and mTOR pathway were also significantly up-regulated.In addition,GPD1,a key glycerol synthesis gene regulated by HOG pathway,was not up-regulated.Saccharomyces cerevisiae was more dependent on intracellular glycerol synthesis under high glucose stress.In summary,C.glycerinogenes under high glucose stress is more dependent on the regulatory responses of cytoplasmic membrane and cell growth related genes.?4?Based on the results of RNA-Seq sequencing,six plasmalemma-related genes of C.glycerinogenes with high glucose resistance were identified by RT-qPCR,gene overexpression,heterologous expression and high glucose tolerance test of recombinant strains.The above six plasmalemma-related genes are the glucose transporters Cg2615?A0A1V2LT11?and Cg5001?A0A2U9R987?,the putative cytochrome P450 gene Cg4756?A9YUC7?,the Cg3824?A0A099P0V3?with C-5 sterol desaturase activity,the Cg2914?A0A099P2M9?with mannosyltransferase activity,and the Cg2071?A0A1Z8JUV2?with urea transmembrane transporter activity.And these six genes can enhance the high glucose tolerance of S.cerevisiae and provide a genetic source for engineering strain transformation. |
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