Biotechnology Bulletin ›› 2024, Vol. 40 ›› Issue (6): 290-298.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0006
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YAN Huan-huan1,2(), SHANG Yi-tong1,2, WANG Li-hong1,2, TIAN Xue-qin1,2, LIAO Hai-yan1,2, ZENG Bin4, HU Zhi-hong1,2,3()
Received:
2024-01-04
Online:
2024-06-26
Published:
2024-05-14
Contact:
HU Zhi-hong
E-mail:huan89215@163.com;huzhihong426@163.com
YAN Huan-huan, SHANG Yi-tong, WANG Li-hong, TIAN Xue-qin, LIAO Hai-yan, ZENG Bin, HU Zhi-hong. Heterologous Biosynthesis of Cordycepin in Aspergillus oryzae[J]. Biotechnology Bulletin, 2024, 40(6): 290-298.
Primer name | Forward primer(5'-3') | Reverse primer(5'-3') |
---|---|---|
Cns1-pEX1-GFP | GAGCAGACATCACCCTCGAGATGGCCATGAACGAGAACGC | ATGGTACCTACGTACTCGAGGGCTATGCCCACCTTGGATC |
Cns2-pEX2D-DsRed | CACAGAAGGCATTTCACGTGATGTCTTGTCCTACCAGCGC | TCCTTAAGCACGGGCACGTGTCGATGCTGCGTGCGGCTC |
Cns3-pEX1 | GAGCAGACATCACCCTCGAGATGTCCGAGTCAACCGCCTA | ATGGTACCTACGTACTCGAGCACACGCTGATAAAGGCCGA |
Cns3-pEX1-BFP | CCCTCGAGTACGTAGGTACCATGGTGTCTAAGGGCGAAGA | CCCTTGCTCACCATGGTACCATTAAGCTTGTGCCCCAGTT |
Cns1-GFP/Cns2-DsRed-pEX2D | TATGACATGATTACGAATTCTCAGAGCCTAGCCAACTAGT | CTCCAGATCGCAGCGAATTCCGACGGCCAGTGCCAAGCTT |
Table 1 Primers used for vector construction
Primer name | Forward primer(5'-3') | Reverse primer(5'-3') |
---|---|---|
Cns1-pEX1-GFP | GAGCAGACATCACCCTCGAGATGGCCATGAACGAGAACGC | ATGGTACCTACGTACTCGAGGGCTATGCCCACCTTGGATC |
Cns2-pEX2D-DsRed | CACAGAAGGCATTTCACGTGATGTCTTGTCCTACCAGCGC | TCCTTAAGCACGGGCACGTGTCGATGCTGCGTGCGGCTC |
Cns3-pEX1 | GAGCAGACATCACCCTCGAGATGTCCGAGTCAACCGCCTA | ATGGTACCTACGTACTCGAGCACACGCTGATAAAGGCCGA |
Cns3-pEX1-BFP | CCCTCGAGTACGTAGGTACCATGGTGTCTAAGGGCGAAGA | CCCTTGCTCACCATGGTACCATTAAGCTTGTGCCCCAGTT |
Cns1-GFP/Cns2-DsRed-pEX2D | TATGACATGATTACGAATTCTCAGAGCCTAGCCAACTAGT | CTCCAGATCGCAGCGAATTCCGACGGCCAGTGCCAAGCTT |
Fig. 1 Subcellular localization of CmCns1 and CmCns2 in A. oryzae A: The scheme of Cns1-pEX1-GFP and Cns2-pEX2D-DsRed overexpression vectors in A. oryzae. B: Wild-type AoRIB40, nutrient deficient strain AoΔpyrGΔHisB, transformant CmCns1/AoΔpyrGΔHisB, CmCns2/AoΔpyrGΔHisB and CmCns1 CmCns2/AoΔpyrGΔHisB were phenotyped in CD+Uri+Ura+His medium at 30℃ for 3-5 d. C: Fluorescence observation of transformants, from top to bottom: microscopic observation of mycelium of AoRIB40, transformant CmCns1/AoΔpyrGΔHisB, CmCns2/AoΔpyrGΔHisB and CmCns1 CmCns2/AoΔpyrGΔHisB in a 20-fold objective. D: Subcellular localization of CmCns1 and CmCns2 in A. oryzae, from top to bottom: microscopic observation of mycelium with AoRIB40, AoRIB40 stained with Nile red dye, transformant of CmCns1/AoΔpyrGΔHisB stained with Nile red dye, and CmCns1 CmCns2/AoΔpyrGΔHisB under 63-fold microscope. C and D: From left to right: DIC, green fluorescence of GFP, red fluorescence of DsRed, and combined images of GFP, DsRed and DIC
Fig. 2 Cordycepin contents of CmCns1 and CmCns2 heterologously expressed in A. oryzae A: The chromatogram peaks of cordycepin in A. oryzae transformant CmCns1/AoΔpyrGΔHisB and CmCns1 CmCns2/AoΔpyrGΔHisB by HPLC, and LC-MS/MS analysis of CmCns1 CmCns2/AoΔpyrGΔHisB. The black and red peaks respectively indicate CmCns1/AoΔpyrGΔHisB and CmCns1 CmCns2/AoΔpyrGΔHisB.B: Cordycepin contents in A. oryzae AoRIB40, heterologous expressing transformant CmCns1/AoΔpyrGΔHisB, CmCns2/AoΔpyrGΔHisB and CmCns1 CmCns2/AoΔpyrGΔHisB in liquid fermentation medium
Fig. 3 Cordycepin contents of CmCns1, CmCns2 and CmCns3 heterologously expressed in A. oryzae A: The scheme of Cns1-GFP/Cns2-DsRed-pEX2D and Cns3-pEX1-BFP vectors. B: Wild-type A. oryzae AoRIB40, background strain AoΔpyrGΔHisB, transformant CmCns1 CmCns2-pEX2D/AoΔpyrGΔHisB, CmCns3/AoΔpyrGΔHisB and CmCns1 CmCns2 CmCns3/AoΔpyrGΔHisB were phenotyped in CD+Uri+Ura+His medium at 30℃ for 3-5 d.C: From top to bottom : Microscopic observation of mycelium of transformant CmCns1CmCns2-pEX2D/AoΔpyrGΔHisB, CmCns3/AoΔpyrGΔHisB and CmCns1 CmCns2 CmCns3/AoΔpyrGΔHisB in a 40-fold objective; from left to right: DIC, green fluorescence of GFP, red fluorescence of DsRed, blue fluorescence of BFP, combined images of GFP, DsRed, BFP and DIC. D: The contents of cordycepin in A. oryzae AoRIB40 and heterologous expression transformant CmCns1/AoΔpyrGΔHisB, CmCns1 CmCns2/AoΔpyrGΔHisB and CmCns1 CmCns2 CmCns3/AoΔpyrGΔHisB liquid fermentation medium. E: The chromatogram peaks of cordycepin contents in A. oryzae transformants CmCns1/AoΔpyrGΔHisB, CmCns1 CmCns2/AoΔpyrGΔHisB and CmCns1 CmCns2 CmCns3/AoΔpyrGΔHisB by HPLC, the red, blue, and black peaks respectively correspond to CmCns1/AoΔpyrGΔHisB, CmCns1 CmCns2/AoΔpyrGΔHisB and CmCns1 CmCns2 CmCns3/AoΔpyrGΔHisB. ** indicate a significant difference compared to the control(P<0.01), the same below
Fig. 4 Cordycepin contents of CmCns1, CmCns2 and CmCns3 heterologously expressed in A. oryzae in the liquid fermentation broth with adenine and glycine A: Cordycepin contents of A. oryzae AoRIB40, transformant CmCns1/AoΔpyrGΔHisB, CmCns1 CmCns2/AoΔpyrGΔHisB and CmCns1CmCns2 CmCns3/AoΔpyrGΔHisB in DPY medium and with glycine and adenine in the medium, respectively, AoRIB40+A indicates the addition of adenine to the medium, AoRIB40+G indicates the addition of glycine to the medium, and so on. B: Cordycepin content plots of A. oryzae transformant CmCns1/AoΔpyrGΔHisB, CmCns1 CmCns2/AoΔpyrGΔHisB, CmCns1 CmCns2 CmCns3/AoΔpyrGΔHisB and addition of glycine to the medium by HPLC, cyan, red, black, purple, green, and blue are the peaks of CmCns1/AoΔpyrGΔHisB, CmCns1 CmCns2/AoΔpyrGΔHisB, CmCns1 CmCns2 CmCns3/AoΔpyrGΔHisB and the addition of glycine, respectively. C: The chromatogram peaks of cordycepin in AoRIB40 and transformant CmCns1/AoΔpyrGΔHisB by HPLC before and after addition of adenine, the black, blue and green peaks correspond to AoRIB40, CmCns1/AoΔpyrGΔHisB after adding adenine and CmCns1/AoΔpyrGΔHisB before adding adenine, respectively
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