| Polysaccharide is the main active substance of Pleurotus tuber-regium,which not only has excellent antioxidant,antitumor and immunomodulatory activities,but also has important research value and application potential in the fields of medicine,cosmetics,food and biomaterials due to its unique hyperbranched structure.The polysaccharides content in fruiting body and sclerotia of P.tuber-regium are substantially higher than that in mycelia,but their long growth period and harsh cultivation limit the industrial applicability of these polysaccharides.There is a lack of research on the metabolic control strategies and related mechanisms to improve the polysaccharide production in the mycelia of P.tuber-regium.This study aims to improve polysaccharides production in submerged fermentation of P.tuber-regium by introducing exogenous substances that efficiently promote polysaccharide synthesis and examine the underlying process.The key research findings are as follows:1.Corn oil was screened to be the most significant exogenous factors for the development of mycelial biomass and polysaccharide among particulates and plant oil factors.The optimization of the corn oil amount showed that the extracellular polysaccharide（EPS）production and mycelial biomass of P.tuber-regium was enhanced with the increase of corn oil addition and reached the maximum value of 0.48±0.00 g·L-1 and 19.49±0.04 g·L-1,respectively,at 4%corn oil addition,which were 1.78 and 2.42 times of the control group.2.The phenotypic changes were examined in the experimental and control groups,and the results showed that corn oil caused the mycelia to thicken and become less entangled.The results of transmission electron microscopy and laser scanning confocal microscopy revealed that large amounts of lipid droplets existed in the cells of the experimental group.The number of mitochondria has also grown considerably,and the cell wall thickness had increased by15.39%.In addition,the glucose content of EPSs decreased significantly,while the amount of mannose and galactose increased.The glucose,mannose and fucose content of cell wall polysaccharides reduced significantly,while the glucuronic acid and glucosamine content increased significantly.This indicates that adding corn oil to the fermentation broth not only altered the cell morphology,but also generated an intracellular accumulation of lipid droplets,which in turn affected the metabolic flux of polysaccharide production.3.In order to further explore the key inducing factor,five fatty acids and methyl jasmonate（Me JA,fatty acid derivative）were selected as exogenous additive substances for submerged fermentation of P.tuber-regium.The results showed that the addition of five fatty acids and appropriate amount of Me JA had a certain promotion effect on EPS production,with linolenic acid having the most significant effect for EPS production up to 0.73±0.01 g·L-1 when the added amount was 2.5 g·L-1,which was 2.70 times of the control group.Moreover,the maximum amount of P.tuber-regium of mycelial biomass(13.01±0.29 g·L-1)and the EPS production(0.63±0.03 g·L-1)were reached at the addition of Me JA at 33×10-3 g·L-1.4.Transcriptome sequencing was used to identify the differential genes,and the mechanisms of their impacts were analyzed in terms of polysaccharide metabolism,lipid metabolism and membrane,respectively,according to the annotation analysis from KEGG database and GO database.In the experimental group,the expression levels of most glycoside hydrolases were down-regulated in polysaccharide biosynthetic pathway,with 20 genes encoding chitinase down-regulated and 5 up-regulated.The expression levels of genes involved in fatty acid oxidation,organelle membrane synthesis and transmembrane transport were dramatically up-regulated in the experimental group,particularly those implicated in peroxisome membrane synthesis.In addition,the up-regulated genes related to membrane metabolism were primarily enriched in the GPI-anchor biosynthesis pathway,whereas the down-regulated genes related to membrane metabolism were mostly enriched in the MAPK signaling pathway-yeast. |
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