Prediction,Clone And Knockout Of Genes Encoding Sugar Transporters In Aureobasidium Pullulans

Aureobasidium pullulans,also known as black yeast,is a yeast-like fungus that produces pullulan,during its fermentation.Pullulan is a macromolecular polymer with good biological activity.It has important application value in many fields such as food,medicine,environmental protection and cosmetics.Its precursor materials are produced in the body of the cell and secreted extracellularly with the help of sugar transporters to participate in the synthesis of pullulan.Sugar transporter plays an important role in microbial fermentation,not only participating in the absorption of extracellular sugars,but also assisting in the release of sugar metabolites in cells.In this thesis,A.pullulans CCTCC M 2012259 was used to study sugar transporters by predicting the gene information related to the sugar transporters,cloning and knocking out these genes.The mutant strains were compared with the starting strain from the aspects of fermentation process,and pullulan production,and the role of sugar transporter in the synthesis of extracellular polysaccharides by A.pullulans.The main contents and results of this study are summarized as follows:1.On the basis of sequencing the whole genome of A.pullulans CCTCC M 2012259,three different bioinformatics methods namely homology prediction,de novo prediction,and RNA-seq data analysis were used.By comparing genomic information with sugar transporter information from all known fungal in the database,the possible sugar transporter coding genes in A.pullulans were screened out.Then GENS CAN software was used to accurately model the preliminary prediction results,and obtain complete gene information including promoter,exon,intron and terminator.Finally,the predicted genes were translated into protein sequences and verified with the blastp program.Finally,it was determined that 13 gene sequences in A.pullulans could encode sugar transporters.The obtained structural models were visualized with gene sequences and protein sequences using ProcessOn software.2.The predicted 13 sequences that could encode sugar transporters were cloned and the corresponding knockout vectors were constructed.Design primers based on the predicted sequence,13 gene fragments were amplified into the pMD19T vector,and then sequenced the intron-containing coding sequences to find that all the exon parts completely match the predicted sequences.Next,a functional expression vector was constructed in A.pullulans,including a promoter,a terminator,and a hygromycin selection marker gene,namely,the effective element PtrpC-hyg-TtrpC.Finally,the effective element was inserted into the constructed cloning vectors of 13 sugar transporter candidate genes through enzymatic digestion,and the corresponding knockout vectors for homologous recombination were completed.3.Gene engineering was used to knock out the sugar transporter coding genes of A.pullulans CCTCC M 2012259.First of all,the growth status of A.pullulans was investigated.A.pullulans was cultured in YPD medium,and the corresponding growth curve was drawn.As a result,it was found that the cells were in the logarithmic growth phase after being cultured for about 25 hours and could be used for subsequent electrotransformation experiments.Secondly,the appropriate hygromycin resistance screening concentration was determined.As a result,no trace of colony growth was found on the 100 ?g/mL hygromycin screening plate.Therefore,a hygromycin concentration of 100 ?g/mL was selected as a condition for screening positive transformants.Then,the corresponding knockout fragments were used to knock out sugar transporter genes in A.pullulans.And mutant strains were successfully obtained.Finally,under the conditions of shake flask culture,the starting strain and mutant strains were fermented to produce pullulan.It was found that compared with the original strain,cell dry weight of mutant strains was increased,and the yield of pullulan was significantly decreased.The above results indicate that sugar transporters investigated in this paper may play a role in the extracellular secretion of pullulan,which provides favorable evidence for further transformation of the strain from the molecular level to improve the efficiency of pullulan synthesis.