| Aureobasidium pullulans,commonly known as black yeast,is a yeast-like fungus.Extracellular polysaccharides such as pullulan can be produced by A.pullulans during batch fermentation.The unique physicochemical properties of pullulan determine its broad application prospects in food processing,medicine,cosmetics and environmental protection etc.As a member of the glucose transporter protein family and Triose phosphate translocator protein family,RCO3 and YDB1 play important roles in sugar transportation in cells.The biosynthesis of pullulan desires that the intracellular precursors formed inside the cell must be secreted to outside of cell with the help of sugar transporters.RCO3 may be involved in the translocation of pullulan biosynthetic precursors,while YDB 1 may affect the biosynthesis of pullulan precursors.In order to ascertain the role of rco3-2 and ydb1 in pullulan biosynthesis,A.pullulans CCTCC M 2012259 was used as the parental strain to construct mutants by disruption and overexpression of the rco3-2 and ydb1 genes.The cell growth,pullulan production and molecular weight,key enzyme activities,intracellular precursor and energy levels in parental strain and mutants were measured to investigate the function and it’s intrinsic physiological mechanism of rco3-2 and ydb1 in biosynthesis of pullulan in batch culture.The main contents and results are summarized as follows:(1)The deletion and overexpression vectors of rco3-2 were successfully constructed.Then the rco3-2 gene was deleted or overexpressed,resultant strains were screened and then named as PUL-1 and PUL-2,respectively.Flask culture results showed that the pullulan titer of PUL-1 decreased by 50.1%,while the maximum biomass increased by 78.4%,compared with the parental strain.However,the pullulan titer of PUL-2 increased by 11.3%,while no significant differences in maximum biomass was observed.(2)Construction of knockout and overexpression vectors of ydb1.The PUL-3 was obtained by knocking out the ydb1 gene of parental strain,while PUL-4 was constructed by inserting ydb1 into genome of A.pullulans by Agrobacterium tumefaciens-mediated transformation.Flask culture results revealed that PUL-3 increased the titer of pullulan by 8.6%.While no significant differences in biomass and the titer of pullulan between PUL-4 and had no difference with the parental strain PUL-0.Another strain PUL-5 was obtained by knocking out the ydb1 of PUL-2,and the flask culture results showed a 12.5%increase in the titer of pullulan compared to the parental strain.(3)The parental strain and all mutants were cultured in a 5 L fermentor.Based on the results of fermentation kinetic parameters and physiological indexes,the biosynthetic rate of pullulan of the PUL-1 significantly decreased when compared with parental strain,the final titer of pullulan also decreased by 63.5%.At the same time,the activity of α-amylase was significantly decreased,which greatly increased the molecular weight of pullulan.Besides,the expression levels of pgm1,ugp and ags2 in PUL-2,PUL-3 and PUL-5 were up-regulated to different degrees,and intracellular energy levels and ratios were significantly increased when compared with the parental strain,and the biosynthetic rate of pullulan was significantly boosted,and the pullulan titers was raised by 10.6%、8.4%and 12.8%,respectively.The above results indicated that the sugar transporter RCO3 and YDB1 is closely related to biosynthesis of pullulan and involved in the transmembrane transportation of precursors.The results of this study provide favorable evidence for further genetic modification of the strain at molecular level to improve the efficiency of extracellular polysaccharide pullulan synthesis. |
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