Screening On Low-pigment Pullulans Strain And Cloning Of Pyrophosphorylase Gene

Pullulan is an extracellular microbial polysaccharide produced by strain of Aureobasidium pullulans. It is a natural water-soluble neutral glucan. Pullulan has excellent physical and chemical nature of colorless, tasteless, non-toxic, excellent film forming properties, water solubility, adhesion, easy biodegradation and so on. Therefore it can be widely used in food, medicine, daily chemicals, building materials, paper, printing, electronics, tobacco and other industries. It is a new multi-functional biological product with great economic value. It can be easily degradation and have not an impact on the environment. Pullulan has been widely used in many fields, but it is very expensive and no large-scale production in China, mainly because the melanin which was secreted during the fermentation process adheres firmly to the pullulan and is hard to remove.Breeding low-pigment and high-yield strains was a fundamental solution to the problem above. The subject was used of genetic engineering methods, which was based on the previous mutation (UV and NTG mutagenesis). It was a effective way to simplify the production process and reduce production costs.In this study, Aureobasidium pullulans was used as the original strain which was purchased from China General Microbiological Culture Collection Center(CGMCC NO.3.837). Firstly, a low pigment mutant strain was obtained by UV and NTG mutation. Secondly, the pyrophosphorylase (UDPGase) which controlled pullulan production was cloned in order to obtain low-pigment and high-yield strains.The main contents and results were as follows:(1) The original strain was mutated by UV and NTG. After many rounds, strain f61 which was a low-pigmented strain was obtained. The color of the fermentation liquor and polysaccharide was more slightly. The color of the fermentation liquor of original strain was black or dark green and the color of polysaccharide was dark. The color of the fermentation liquor of the strain f61 was light yellow and the color of polysaccharide was white or light yellow.(2) The pyrophosphorylase (UDPGase) gene which had a major impact on pullulan production was cloned by Degenerate PCR. The target gene was connected to pMD19-T vector and then transformed into E. coli DH5αcompetent cells. After incubation, the positive clones were screened. Finally the recombinant plasmid was detected by gel electrophoresis. The results showed that:the target gene and the vector were connected perfectly. Gene was cloned successfully.