Construction Of Aspergillus Oryzae Expression System And Heterologous Expression Of α-amylase From Penicillium Chrysogenum

Filamentous fungi have been used as powerful hosts because of their desirable growth characteristics and strong ability to produce and secrete exceptionally large amounts of proteins. They can correctly modify post-translational proteins by glycosylation and disulfide bridge formation, etc. Aspergillus oryzae has been used for producing tranditional soybean sauce and soy for over1000years in China, without any safety problems, and in that case, Aspergillus oryzae has been regarded as GRAS strains. Because of its strong ability to produce valuable industry enzymes and secondary metabolite, Aspergillus oryzae has been used to express heterologous gene in order to improve the yield by utilizing molecular biological technique.The method of preparation for Aspergillus oryzae protoplast was established. It is the first time in our lab to transform expression vectors by using this method. pBC-hygro was used as original vector and got improved. The bleomycin expression cassette was constructed as selection marker. With the help of chlorpromazine and TritonX-100, it is successful to improve the sensitivity of Aspergillus oryzae RIB40to bleomycin, decreasing the bleomycin concentrations from200μg/mL to100μg/mL. According to the expression of green fluorescent protein reporter gene, the strongest promoter from VamyB, PenoA, VsodM was screened. At last, VamyB was chosen as promoter to express homologous amyB gene from Aspergillus oryzae, and successfully obtain a transformant whose a-activity has increased106.27%compared with the host.A pyrG auxotrophic strain was obtained by UV mutagenesis as a transformed host, named as PF2.Expression vector pBCPaTP was constructed by inserting pryG expression cassette, promoter PamyB and terminator TamyB to motified pBC. Using pBCPaTP it is successful to express enhanced green fluorescent protein(EGFP) from eukaryotes and lipase from Proteus sp., which demonstrated that this transformation system is suitable for expression of heterologous proteins from both prokaryoticorganism and eukaryotic organism.An a-amylase-producing strain was identificated, which proved to be Penicillium chrysogenum, and named as3-5.The a-amylase gene named as PcAmy, was obtained by extracting the total mRNA and was cloned by reverse transcription PCR. amyB from Aspergillus oryzae was used as gene carrier and KEX2site as a linker to construct fusion protein expression vector pBCPaTPAKah.The PcAmy was purified by Ni-NTA affinity from fermentation supernatant. It is the first time for heterologous expression of a-amylase from Penicillium chrysogenum, and also the first time for Aspergillus oryzae expression system to be used to express a-amylase gene from Penicillium sp.