Heterologous Biosynthesis of Cordycepin in Aspergillus oryzae

doi:10.13560/j.cnki.biotech.bull.1985.2024-0006

Abstract

Abstract:

【Objective】 This study aims to synthesize cordycepin by constructing engineered Aspergillus oryzae strains that heterologously express key genes for cordycepin synthesis. 【Method】 The uidine/uracil and histidine diauxotrophic strain of Aspergillus oryzae AoΔpyrGΔHisB served as the background, and overexpressed the key gene CmCns1-CmCns3 for cordycepin synthesis using the Agrobacterium-mediated transformation technique. The subcellular localization of CmCns1 and CmCns2 within A. oryzae was visualized through fluorescence microscopy. High-performance liquid chromatography（HPLC）was used to determine the content of cordycepin in transgenic A. oryzae. Additionally, glycine and adenine, both precursors in the synthesis of cordycepin, were introduced into the fermentation broth to investigate their impact on cordycepin synthesis in A. oryzae. 【Result】 The CmCns1 and CmCns2 from Cordyceps militaris were localized in lipid droplets in A. oryzae. Cordycepin synthesis achieved through the overexpression of CmCns1 alone, the co-expression of CmCns1 and CmCns2, and the co-expression of CmCns1-CmCns3 in A. oryzae, and the highest extracellular production of cordycepin reached 37.74 μg/mL after 48 h of fermentation. The addition of glycine and adenine to the fermentation broth of A. oryzae could not effectively increase the content of cordycepin.【Conclusion】 This study successfully achieved heterologous expression of synthetic cordycepin genes in A. oryzae.

Key words: cordycepin, Aspergillus oryzae, heterologous expression, subcellular localization, adenine, glycine

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.

Figures/Tables 5

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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