Biosynthesis Of Exopolysaccharides By Aureobasidium Pullulans And Its Mechanism

Aureobasidium pullulans,commonly known as black yeast,is a ubiquitous fungus widely distributed on plant surface and soil.Two different extracellular polysaccharides as pullulan and ?-glucan can be produced by this kind of black yeast.Pullulan is one of the most widely studied alpha-polysaccharide synthesized by A.pullulans.The polysaccharide is a water-soluble macromolecule polymer composed of 1,4-glycoside bonded maltotrisaccharide repetitive unit linking by a 1,6-glycoside bond.The unique structure of pullulan determines its wide application prospects in food processing,biomedicine,environmental protection,and other fields.In addition,A.pullulams can produce another water-insoluble gelatinous analog of ?-glucan,which is linked by?-1,3-glycoside bond and has biological activity.It has attracted much more attentions in food,medicine,cosmetics,and chemical industry.In this thesis,A.pullulans CCTCC M 2012259 was used as the starting strain to study the biosynthesis mechanism of microbial extracellular polysaccharides around the target products of pullulan and ?-glucan.The main research contents and results are as follows:1.The effects of sugars including glucose,sucrose,and xylose on the yield and molecular weight of pullulan were investigated under batch fermentation.The initial sugar concentrations were set at 50,100 and 150 g/L,respectively.It was shown that sucrose was more advantageous than glucose and xylose to obtain pullulan with higher production,higher yield and higher productivity.However,a higher molecular weight of pullulan can be obtained from glucose other than sucrose and xylose.With the increase in the initial sugar concentration and the extension of the batch culture,the molecular weight of pullulan decreases gradually.2.The effect of different divalent metal ions on extracellular polysaccharide production during batch culture of A.pullulans in shaking flasks was investigated.It was found that zinc sulfate could significantly promote the biosynthesis of ?-glucan.The highest yield of ?-glucan can be obtained when 0.1 g/L zinc sulfate was added to the fermentation medium.From the point of view of the physiological mechanism underlying the improvement,the addition of zinc sulfate increased the activity of key enzymes involved in biosynthesis of pullulan and ?-glucan,improved the intracellular levels of UDPG and ATP,favored to the regeneration of energy substances,which were all conducive to the biosynthesis of ?-glucan.The transcriptome analysis was carried out by RNA-seq to reveal the molecular mechanism of improved production of ?-glucan,results showed that 188 differentially expressed genes were screened after adding 0.1 g/L zinc sulfate,of which 90 genes were significantly up-regulated and 98 genes were significantly down-regulated.According to the bioinformatic analysis,these up-regulated genes were related to cell growth,energy,and substance metabolism.Further genes related to glycogen synthesis,and energy metabolism were also activated or up-regulated,which were closely related to the biosynthesis of ?-glucan.In particular,the addition of zinc sulfate significantly increased the expression levels of kre6 gene involved in ?-glucan biosynthesis and coxl gene directly involved in ATP generation,indicating that zinc sulfate was beneficial to the expression of genes encoding key enzymes involved in ?-glucan biosynthesis pathway and energy metabolism pathway,thus promoted the biosynthesis of ?-glucan.In addition,the metabolic network for pullulan and ?-glucan biosynthesis was successfully constructed and the metabolic fluxes of intermediates were calculated.In the cell growth phase,zinc sulfate addition resulted in more carbon flux towards the EMP pathway and then more energy substrate of ATP was formed for normal cell metabolism through the TCA cycle.In the polysaccharide synthesis phase,the carbon flux towards the precursor of UDPG for polysaccharides and the carbon flux from UDPG to polysaccharide biosynthesis were all increased.Therefore,the addition of zinc sulfate increased the carbon flux towards ?-glucan and in turn improved the yield of?-glucan.