A kind of Aspergillus terreus strain producing aconitic acid and its construction method and application
A technology of aconitic acid and Aspergillus terreus, applied in the field of microorganisms, can solve the problems of many by-products, high cost and complex process
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Embodiment 1
[0042] Example 1, Construction of cadA gene targeting element cadA-KO1
[0043] According to the genome information of Aspergillus terreus, the following primers were designed and synthesized:
[0044] U-cadA-F:5'-agccgctaccagccgaagcaatag-3';
[0045] U-cadA-R: 5'-GTTCAATCACCATCTCCCTTAatcgtcagtatactttgtagac-3';
[0046] D-cadA-F:5'-CGTATTTCTCGCCTGTGTG aaggttactccatctcatagc-3';
[0047] D-cadA-R: 5'-tcgactatagctggattgatcac-3'.
[0048] Aspergillus terreus (Aspergillus terreus) CICC 40205 genomic DNA was used as a template, and pfu DNA polymerase (Fermentas, catalog number: EP0501) was used for PCR amplification, and the primer pair U-cadA-F / U-cadA-R could amplify The upstream homology arm U-cadA of the cadA gene with a size of about 1.2kb is obtained, and the downstream homology arm D-cadA of the cadA gene with a size of 1.2kb can be amplified with the primer pair D-cadA-F / D1-cadA-R . Using the plasmid pXH-106 as a template, the primer pair pyrGAn-F (5'-taagggagatggtgattga...
Embodiment 2
[0050] Example 2, construction of cadA gene deletion Aspergillus terreus strain At-ΔcadA
[0051] Aspergillus terreus At-Δku80 is an engineering strain for knocking out the ku80 gene of Aspergillus terreus CICC40205, and At-Δku80-ΔpyrG is a uracil auxotrophic engineering strain obtained after knocking out the pyrG gene on the basis of the At-Δku80 strain (Lv Xuefeng et al. , a method and application of improving the application efficiency of gene targeting technology in Aspergillus terreus, ZL201510275491.5).
[0052] Inoculate the spores of the engineering strain At-Δku80-ΔpyrG into 50mL IPM liquid medium, so that the spore concentration is about 10 7 cells / mL, cultured at 200rpm, 32°C for 12-18h. Collect the growing mycelium by filtering with a sterile single layer of 500-mesh nylon cloth, and filter with sterile 0.6M MgSO 4 The solution was rinsed three times, pressed dry and placed in a sterile 50ml Erlenmeyer flask, adding an appropriate amount of enzymatic solution acc...
Embodiment 3
[0054] Example 3, Shake Flask Analysis of Aconitic Acid Produced by Aspergillus terreus Strain At-ΔcadA Fermentation
[0055] Select genetically engineered strain At-ΔcadA transformants and control strain At-Δku80 to inoculate into Aspergillus terreus sporulation slant medium (10g L -1 Glucose, 2gL -1 NaNO 3 , 0.2g L -1 ,KH 2 PO 4 , 20 mg L -1 FeSO 4 , 5g L -1 MgSO 4 , 0.5g L -1 NaCl, 40 mg L -1 ZnSO 4 , 40 mg L -1 CuSO 4 , 0.5% bran, 1.5% agar, sterilized at 115°C for 15 minutes, then distributed to test tubes, and then sterilized at 115°C for 25 minutes to prepare slant), cultivated at 32°C for 7 days to obtain mature spores. Then respectively inoculate the spores on one slant into a bottle of itaconic acid fermentation medium (55ml medium is housed in the 500ml Erlenmeyer flask), the inoculation amount is about final concentration 2×10 5 spores / mL, 12 bottles were fermented in shake flasks for each strain, 37°C, 220rpm for 96h, and samples were taken 5 times i...
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