米曲霉异源表达合成虫草素

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

摘要/Abstract

摘要：

【目的】 通过米曲霉中异源表达虫草素合成关键基因，构建米曲霉工程菌株合成虫草素。【方法】 以米曲霉尿苷/尿嘧啶和组氨酸双营养缺陷型菌株AoΔpyrGΔHisB为背景菌株，利用农杆菌介导的转化方法对虫草素合成关键基因CmCns1-CmCns3进行过表达；通过荧光显微镜观察CmCns1和CmCns2在米曲霉中的亚细胞定位；利用高效液相色谱法（HPLC）测定转基因米曲霉虫草素含量，同时对米曲霉发酵液添加合成虫草素的前体甘氨酸和腺嘌呤，探究其在米曲霉中对合成虫草素的影响。【结果】 蛹虫草CmCns1和CmCns2在米曲霉中定位于脂滴；并且米曲霉中单独过表达CmCns1、共表达CmCns1和CmCns2以及共表达CmCns1-CmCns3均能合成虫草素，发酵48 h虫草素胞外产量最高达到37.74 μg/mL；向米曲霉发酵液添加甘氨酸和腺嘌呤，不能有效提升虫草素的含量。【结论】 成功在米曲霉异源表达合成虫草素。

关键词: 虫草素, 米曲霉, 异源表达, 亚细胞定位, 腺嘌呤, 甘氨酸

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

闫欢欢, 尚怡彤, 王丽红, 田学琴, 廖海艳, 曾斌, 胡志宏. 米曲霉异源表达合成虫草素[J]. 生物技术通报, 2024, 40(6): 290-298.

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.

图/表 5

表1 构建载体所用引物

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

图1 CmCns1和CmCns2在米曲霉中亚细胞定位 A：Cns1-pEX1-GFP、Cns2-pEX2D-DsRed在米曲霉中过表达载体构建示意图；B：野生型AoRIB40、营养缺陷型菌株AoΔpyrGΔHisB、转化子CmCns1/AoΔpyrGΔHisB、CmCns2/AoΔpyrGΔHisB和CmCns1 CmCns2/AoΔpyrGΔHisB在CD+Uri+Ura+His培养3-5 d的表型；C：转化子荧光观察，从上到下依次是AoRIB40、转化子CmCns1/AoΔpyrGΔHisB、CmCns2/AoΔpyrGΔHisB和CmCns1 CmCns2/AoΔpyrGΔHisB在20倍物镜菌丝体的显微镜观察；D：CmCns1和CmCns2在米曲霉中的亚细胞定位，从上到下依次为AoRIB40、AoRIB40与尼罗红染料染色、转化子CmCns1/AoΔpyrGΔHisB与尼罗红染料染色、CmCns1 CmCns2/AoΔpyrGΔHisB在63倍镜下菌丝体的显微镜观察。C和D中从左至右：DIC，GFP绿色荧光，DsRed红色荧光，GFP、DsRed与DIC组合图像

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

图2 米曲霉异源表达CmCns1和CmCns2虫草素含量 A：米曲霉转化子CmCns1/AoΔpyrGΔHisB和CmCns1 CmCns2/AoΔpyrGΔHisB虫草素的液相色谱峰及CmCns1 CmCns2/AoΔpyrGΔHisB的LC-MS/MS分析，黑色和红色峰形分别代表CmCns1/AoΔpyrGΔHisB和CmCns1 CmCns2/AoΔpyrGΔHisB；B：米曲霉对照AoRIB40、转化子CmCns1/AoΔpyrGΔHisB、CmCns2/AoΔpyrGΔHisB和CmCns1 CmCns2/AoΔpyrGΔHisB液体发酵培养基中虫草素的含量

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

图3 米曲霉异源表达CmCns1、CmCns2和CmCns3虫草素含量 A：Cns1-GFP/Cns2-DsRed-pEX2D、Cns3-pEX1-BFP载体构建示意图；B：野生型米曲霉AoRIB40、背景菌株AoΔpyrGΔHisB、米曲霉转化子CmCns1 CmCns2-pEX2D/AoΔpyrGΔHisB、CmCns3/AoΔpyrGΔHisB和CmCns1 CmCns2 CmCns3/AoΔpyrGΔHisB在30℃使用CD+Uri+Ura+His培养3-5 d的表型；C：从上到下依次是米曲霉转化子CmCns1 CmCns2-pEX2D/AoΔpyrGΔHisB、CmCns3/AoΔpyrGΔHisB和CmCns1 CmCns2 CmCns3/AoΔpyrGΔHisB在40倍镜下菌丝体显微镜观察，从左至右依次是DIC，GFP绿色荧光，DsRed红色荧光，BFP蓝色荧光，GFP、DsRed、BFP三种荧光与DIC组合图像；D：米曲霉AoRIB40、异源表达转化子CmCns1/AoΔpyrGΔHisB、CmCns1 CmCns2/AoΔpyrGΔHisB和CmCns1 CmCns2 CmCns3/AoΔpyrGΔHisB液体发酵培养基中虫草素的含量；E：米曲霉转化子CmCns1/AoΔpyrGΔHisB、CmCns1 CmCns2/AoΔpyrGΔHisB和CmCns1 CmCns2 CmCns3/AoΔpyrGΔHisB虫草素的液相色谱峰，红色、蓝色和黑色峰形分别对应CmCns1/AoΔpyrGΔHisB、CmCns1 CmCns2/AoΔpyrGΔHisB和CmCns1 CmCns2 CmCns3/AoΔpyrGΔHisB。**表示与对照组相比有显著差异（P<0.01），下同

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

图4 米曲霉异源表达CmCns1、CmCns2和CmCns3液体发酵液添加腺嘌呤和甘氨酸虫草素含量 A：米曲霉AoRIB40、米曲霉转化子CmCns1/AoΔpyrGΔHisB、CmCns1 CmCns2/AoΔpyrGΔHisB和CmCns1 CmCns2 CmCns3/AoΔpyrGΔHisB在DPY培养基以及在培养基中分别添加甘氨酸和腺嘌呤的虫草素含量，AoRIB40+A代表向培养基中添加腺嘌呤，AoRIB40+G代表在培养基中添加甘氨酸，其余以此类推；B：米曲霉转化子CmCns1/AoΔpyrGΔHisB、CmCns1 CmCns2/AoΔpyrGΔHisB和CmCns1 CmCns2 CmCns3/AoΔpyrGΔHisB及添加甘氨酸后虫草素的液相色谱图，青色、红色、黑色、紫色、绿色和蓝色分别是CmCns1/AoΔpyrGΔHisB、CmCns1 CmCns2/AoΔpyrGΔHisB和CmCns1 CmCns2 CmCns3/AoΔpyrGΔHisB及添加甘氨酸后的峰形；C：米曲霉AoRIB40和转化子CmCns1/AoΔpyrGΔHisB添加腺嘌呤前以及添加后虫草素的液相色谱峰，黑色、蓝色和绿色峰形分别对应AoRIB40、CmCns1/AoΔpyrGΔHisB添加腺嘌呤后和CmCns1/AoΔpyrGΔHisB添加腺嘌呤前

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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