Screening And Identification Of Aureobasidium Pullulans HIT-LCY3~T And Recycling Of Potato Dregs And Waste Water

Aureobasidium pullulans is an amorphous fungus of the genus A.pullulans.Pullulan is a colorless and odorless extracellular polysaccharide produced by Aureobasidium pullulans fermentation.Because of its excellent properties such as solubility,stability,stickiness,plasticity,film-forming and safety,pullulan has been widely used in food industry,biomedicine,environmental protection,material packaging and disease treatment.Based on the analysis of the research on Aureobasidium pullulans and the production of pullulan polysaccharides at home and abroad,this paper aims to use potato starch industrial wastes as cheap raw materials to produce pullulan polysaccharides.The synthesis and resource utilization of pullulan from Aureobasidium pullulans were studied by strain screening and identification,product fermentation and genetic engineering.At the same time,through the adaptive evolution of bacteria,optimization of fermentation conditions and expanded production,it laid an industrial foundation for the production of high value-added pullulan products from waste produced by resource potato starch industry,that is,potato dregs and waste water.This not only reduces the production cost of pullulan,but also solves the environmental problems caused by large-scale food processing,which is in line with the carbon cycle policy advocated by the state.A microbial strain HIT-LCY3~T producing extracellular polysaccharides was successfully screened from humus soil,and it was identified as Aureobasidium pullulans by morphological observation and molecular biological identification,and the related components and functions of the gene of the strain were understood by whole genome sequencing.After the extracellular polysaccharides were separated and purified,the extracellular polysaccharides were identified by high performance liquid chromatography,nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy.The polysaccharides were determined as the target product-pullulan,and the fermentation conditions were optimized when the seed age was 48 h,the rotation was 160 rpm,the initial pH was 6.0,1.5%inoculation,26℃and 5 days fermentation time.The yield of pullulan was 23.47 g/L.The mutagenic strain with high yield of pullulan was obtained by ultraviolet mutagenesis of Aureobasidium pullulans HIT-LCY3~T.Some differentially expressed genes were obtained by transcriptome high-throughput sequencing analysis,and the typical genes ghf3 and udpg with large difference multiple were overexpressed.The results showed that the yield of pullulan was increased by 2.12 fold after overexpression of ghf3 gene,reaching 49.70 g/L.The gene expression of ghf3increased by 2.38 fold.After overexpression of udpg gene,the yield of pullulan increased by 2.50 fold,reaching 58.70 g/L.The gene expression of udpg increased by 2.87 fold,and the biomass of both of them had no significant change after overexpression.The engineering strain UDPG-GHF3 was constructed.Through verification,it was found that the yield of pullulan increased by 3.14 fold,reached 74.00 g/L,the biomass almost did not change,and the gene expression also increased significantly.After that,the adaptive evolution of the engineering strain was studied.With the gradual addition of potato dregs and waste water,the efficiency of producing pullulan increased from 7.19 g/L to 23.48 g/L,which increased by 3.27 fold.At the same time,the expression of pgm,ugp and fks,the key genes of pullulan synthesis,increased significantly.The conditions for the production of pullulan from potato starch industrial waste by over-expressed engineering strain after adaptive evolution were explored.The results showed that the yield of pullulan reached 29.10 g/L when fermentation temperature was 28℃,pH was 5.0,aeration rate was 0.25 vvm and fermentation time was 5 days.Using this technical parameter,the 50 L expansion experiment was carried out,and the yield of pullulan polysaccharide was32.55 g/L.To sum up,the differentially expressed genes were analyzed by transcriptome high-throughput sequencing technique,and the regulation of pullulan production by Aureobasidium pullulans hydrolase 3 gene ghf3 and UDP glycosyltransferase gene udpg was revealed.It was clear that the combination of sugar family hydrolase 3gene ghf3 and UDP glycosyltransferase gene udpg could significantly improve the production of pullulan polysaccharide.It was clarified that the essence of strain adaptive evolution was the improvement of key gene expression of target product synthesis,and it was confirmed that Aureobasidium pullulans could be produced from potato starch industrial waste,which could reduce production cost and benefit ecological environment.