Method for producing recombinant acremonium chrysogenum strain producing 7-aminocephalosporanic acid at high concentration, and strain produced using method
A technology of Cephalosporium acremonium and Cephalosporium acremonium, applied in biochemical equipment and methods, recombinant DNA technology, using vectors to introduce foreign genetic material, etc., can solve problems such as low conversion rate, low CPC productivity, and productivity that has not yet been reached
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Embodiment 1
[0111] Production of Cephalosporin acremonium strain with improved CPC (cephalosporin C) productivity by NTG treatment
[0112] N-methyl-N'-nitro-N-nitrosoguanidine (NTG, N-methyl-N'-nitro-N-nitrosoguanidine) solution was dissolved in acetone to NTG 10mg / mL concentration, thereby making a stock solution ( stock solution), store it at 70°C and wash it with citrate buffer (22.32g / L sodium citrate, 4.63g / L citric acid, pH 5.5) before use Dilute for use.
[0113] Place a colony of Acremonium chrysogenum in LB medium (10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride (NaCl)) Inoculate in 200rpm at 30°C for 3 to 7 days with shaking, collect by centrifugation, wash twice with citrate buffer, add NGT solution, and place at 30°C for 30 minutes. After placing it on ice to cool it down, the NTG solution was discarded by centrifugation, and then washed with potassium phosphate buffer (107.13g / L K 2 HPO 4 ,52.39g / L KH 2 PO 4 ) after washing twice, the same buffer (buffer) w...
Embodiment 2
[0116] 7-ACA production by introducing CPC acyltransferase into E1 strain
[0117] Design of CPC Acyltransferase Expression Vector (design)
[0118]In this step, as a gene having a conversion rate of 7-ACA (7-aminocephalosporanicacid) in CPC (cephalosporin C), the gene (sequence No. 18). The promoter for expressing this gene generally uses the well-known gpdA promoter and trpC terminator of Aspergillus nidulans (Punt PJ et al., Gene. 1990 Sep 1; 93(1):101-109, Functional elements in the promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase).
[0119] The plasmid vector used to introduce the CPC acyltransferase gene will be introduced into the pBluescript II SK+ cloning vector with antibiotic hygromycin B (hygromycin B), geneticin (geneticin), phleomycin (phleomycin) resistant marker gene cassette (cassette) and the Flp cassette for demarking for the purpose of multiple import as the basic framework.
[0120] The gpdA pro...
Embodiment 3
[0135] Improvement of 7-ACA productivity through repeated transformation
[0136] Promoter tuning and 7-ACA productivity improvement
[0137] The CPC acyltransferase of the E2 strain is expressed as a gpdA promoter, and the expression level is not high so that promoter tuning is performed, thereby increasing the strength of the promoter.
[0138] Promoter tuning is a technique that seeks to increase the strength of the promoter by randomly and abruptly mutating the working part of the promoter through various methods, and is currently being actively studied due to the emergence of synthetic biology ((Alper H, Fischer C, Nevoigt E, Stephanopoulos G., Proc Natl Acad Sci U S A. 2005 Sep6; 102(36): 12678-83. Epub 2005 Aug 25, Tuning genetic control through promoter engineering; Santos CN, Stephanopoulos G., Curr Opin Chem Biol. 2008 Apr; 12 (2):168-76., Combinatorial engineering of microbes for optimizing cellularphenotype,; Dehli T, Solem C, Jensen PR., Subcell Biochem.2012; ...
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