| Debaryomyces hansenii FBKL2.0130 is an industrial fungus isolated from Maotai Town Daqu,the core producing area of Maotai-flavor baijiu.It has the characteristics of high glucose tolerance and high yield of D-arabitol.D-arabitol is a kind of natural polyol,which has important application value,so it is widely used in food,pharmaceutical,chemical and other fields.At present,the study on the metabolism of D-arabitol mainly focuses on the isolation and screening of strains,optimization of fermentation conditions and fermentation performance.However,the understanding of metabolic mechanism is insufficient.Yeast uses D-glucose as sugar source to produce D-ribulose by oxidative decarboxylation via pentose pathway.It is reduced to D-arabitol by D-arabitol dehydrogenase.Therefore,this study takes Debaryomyces hansenii FBKL2.0130 as the research object,and explores the metabolism mechanism of D-arabitol by genomics,transcriptomics and metabolites,.The main research results are as follows:1.The whole genome of Debaryomyces hansenii FBKL2.0130 was determined,a total of 740 contigs and 13135015 BP sequence information were obtained after data assembly,and the GC content was 36.47 %.A total of 5983 genes were predicted to encode in FBKL2.0130,accounting for 74.26 % of the total genome size.Go function was annotated to 5886 genes,mainly to metabolic process(38.19 %)and cellular process(42.41 %),which maintained the normal genetic metabolism of yeast.At the same time,KEGG metabolic pathway analysis,which mainly annotates carbon metabolism,amino acid metabolism,MAPK signaling pathway and other genes related to energy metabolism and hypertonic stress response.2.RNA-seq was used to analyze the samples fermented for 14 h,24 h and 48 h under the concentration of 10 g/L and 50 g/L glucose.The results showed that there were many different genes in the fermentation process by GO and KEGG function enrichment analysis.Many biological metabolic pathways were designed,and the different genes in different fermentation time were more than those in different concentration.By focusing on the pathway of D-arabitol metabolism,it was found that some related genes such as rpi B and SORD were up-regulated by 3.06 times and6.77 times,respectively,during the D-arabitol synthesis stage.3.Gas Chromatography-Mass Spectrometer(GC-MS)was used to detect and analyze the metabolites of fermentation for 0 h,96 h,168 h and 240 h at 50 g/L glucose concentration.Through differential product analysis,it was found that polyols were mainly metabolized at 96 h of fermentation,in which D-arabitol and glycerol accounted for 38.89 % and 22.62 % of the total peak area of nonvolatile substances respectively.However,galactitol was rapidly accumulated and synthesized at 168 h,accounting for 46.80 % of the total nonvolatile substances;finally,phenylethanol accumulated the most at 240 h,accounting for 38.10 % of the total volatile substances.4.Established a Flow cytometry(FCM)and fluorescence microscope(FM)method to determine the activity of the strain,combined with the turbidimetric method to detect the fermentation growth performance of the strain under different glucose concentrations.The results showed that the strain had good osmotic resistance,only when the glucose concentration was greater than or equal to 200 g/L,the growth of microorganism lagged slightly,but there was no significant difference in biomass at each concentration in the late stage of fermentation.Meanwhile,the accumulation of D-arabitol and the residual glucose concentration of strain D-arabitol were determined by High Performance Liquid Chromatography-Evaporative Light-Scattering Detector(HPLC-ELSD).It was found that increasing the concentration of glucose was beneficial to the accumulation of D-arabitol,but the accumulation rate changed with the fermentation time and the concentration of glucose involved.The highest accumulation of D-arabitol was(34.38 ± 0.36)g/L after 228 h fermentation at 150g/L.5.Pyrthiol dihydrochloride was used to enhance the activity of glucose-6-phosphate dehydrogenase,The highest yield of D-arabitol was(17.43)g/L when the dosage was 3.5 g/L,which increased by 37.75 % compared with the control group.The addition of alcohol dehydrogenase inhibitor 4-methylpyrazole was 0.6 g/L,the accumulation of D-arabitol was(19.44)g/L,which was 63.90%higher than that of the control group.In addition,the use of non-ionic surfactants:Tween 80,Triton X-100 and glycerol for metabolic regulation,2 g/L Tween 80 and 7 g/L glycerol increased D-arabitol by 29.78%and 27.43%respectively. |
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