黑曲霉尿苷/尿嘧啶营养缺陷型转化系统的构建及应用  

Construction and application of Aspergillus niger uridine/uracilauxotroph transformation system

作　　者：王小平 宋问 张霏 刘燕 王升帆 邵东[3] 梁玲玲[3] 许新德[3] 郑建永 WANG Xiaoping;SONG Wen;ZHANG Fei;LIU Yan;WANG Shengfan;SHAO Dong;LIANG Lingling;XU Xinde;ZHENG Jianyong(Zhejiang Keming Biopharmaceuticals Co.,Ltd.,Xinchang 312500,China;College of Biotechnology and Bioengineering,Zhejiang University of Technology,Hangzhou 310014,China;Xinchang Pharmaceutical Factory,Zhejiang Medicine Co.,Ltd.,Xinchang 312500,China)

机构地区：[1]浙江可明生物医药有限公司,浙江新昌312500 [2]浙江工业大学生物工程学院,浙江杭州310014 [3]浙江医药股份有限公司新昌制药厂,浙江新昌312500

出　　处：《浙江工业大学学报》2024年第1期105-111,共7页Journal of Zhejiang University of Technology

基　　金：国家自然科学基金资助项目(31600639);重庆市技术创新与应用发展专项重点项目(cstc2021jscx-jbgsX0002)。

摘　　要：黑曲霉(Aspergillus niger)是一种重要的工业发酵菌株,它具有强大的蛋白分泌表达能力。为了提高黑曲霉遗传操作效率及优化重组菌株的筛选策略,构建以尿苷/尿嘧啶营养缺陷型为筛选标记的转化系统。利用CRISPR/Cas9技术实现pyrG基因的敲除,在含有尿嘧啶核苷和5-氟乳清酸(5-FOA)的抗性培养基中筛选表型正确的转化子。经基因组PCR验证,黑曲霉营养缺陷型菌株可稳定遗传。利用该转化系统可成功实现增强型绿色荧光蛋白在黑曲霉中的表达。通过结合增强型绿色荧光蛋白和流式细胞仪建立了黑曲霉转化子的高通量筛选模型。Aspergillus niger is an industrially important fermentation strain known for its robust protein secretion and expression capabilities.To enhance the efficiency of genetic manipulation in A.niger andoptimize the screening strategy for recombinant strains,we developed a transformation system employing uridine/uracil nutrient deficiency as a screening marker.This system involved the complete knockout ofthe pyr G gene through CRISPR/Cas9 technology,with subsequently screening of correct transformants in a medium containing uridine and 5-FOA.Following PCR validation,we successfully obtained a stable genetic strain of A.niger uridine/uracil auxotroph.Using this transformation system,we also achieved the successful expression of enhanced green fluorescent protein in A.niger.Furthermore,we established a high-throughput screening model for A.niger transformants by combining enhanced fluorescent protein with flow cytometry.

关 键 词：黑曲霉 CRISPR/Cas9 基因敲除 尿嘧啶营养缺陷型 增强型绿色荧光蛋白(EGFP) 

分 类 号：Q939.9[生物学—微生物学]