黑曲霉转化系统的优化及重组菌株高效筛选  

Optimization of Aspergillus niger transformation system and efficient screening of recombinant strains

作　　者：李岑 刘松 堵国成[2] LI Cen;LIU Song;DU Guocheng(National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]粮食发酵工艺与技术国家工程实验室(江南大学),江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与发酵工业》2022年第3期1-8,共8页Food and Fermentation Industries

基　　金：国家重点研发计划项目(2019YFA0905300);国家自然科学基金项目(31771913);国家自然科学基金项目(32071474)。

摘　　要：黑曲霉(Aspergillus niger)是食品工业中重要的生产菌株,广泛应用于酶和有机酸生产。为提高黑曲霉遗传操作效率,对黑曲霉转化及重组菌株筛选策略进行优化。基于已报道的最佳转化条件对黑曲霉AG11进行电转化、农杆菌介导和聚乙二醇(polyethylene glycol,PEG)介导转化(Agrobacterium-mediated and PEG-mediated transformation,PMT),单个潮霉素转化板分别得到0、30和6个转化子。其中,PMT的转化子阳性率最高。基于优化后的原生质体制备条件:0.8~1 g松散的菌丝球于10 mL Yatalase溶液(0.8 mg/mL,pH 5.5)反应2.5 h,获得原生质体并进行PMT,转化效率较优化前提高14倍。为提高转化子筛选效率,将融合绿色荧光蛋白(green fluorescent protein,GFP)的天冬酰胺酶(asparaginase,ASN)基因整合至黑曲霉AG11糖化酶位点,并通过流式细胞仪分选表达ASN的孢子。分选结果显示,0.3%的孢子能表达GFP(共筛选30万个孢子)。酶活力分析表明,65%表达GFP的孢子能表达ASN。研究结果将为黑曲霉基因编辑提供重要方法学基础。Aspergillus niger is an important production strain in food industry,widely used in production of enzymes and organic acids.To improve the genetic efficiency of A.niger,the transformation and recombinant strain screening strategies were optimized.According to the reported optimal transformation conditions,A.niger AG11 was transformed via electroporation,Agrobacterium-mediated and PEG-mediated transformation(PMT),and a hygromycin transformation plate yield 0,30,and 6 transformants,respectively.PMT achieved the highest positive rate of transformants.Based on the optimized protoplast preparation condition:0.8-1 g loose mycelial pellets were incubated in 10 mL Yatalase solution(0.8 mg/mL,pH 5.5)for 2.5 h to obtain protoplasts and used to PMT,the transformation efficiency was increased by 14-fold than that before optimization.To improve the screening efficiency of transformants,the asparaginase(ASN)gene fused with green fluorescent protein(GFP)was integrated into the glycosylase locus of A.niger AG11,and those spores expressing ASN were sorted by flow cytometry.The results showed that 0.3%of the spores could express the GFP(a total of 300000 spores were screened).Enzyme activity analysis indicated that 65%of the spores expressing GFP could produce ASN.These results provide an important methodological basis for gene editing of A.niger.

关 键 词：黑曲霉 PEG介导转化 原生质体 流式细胞仪 绿色荧光蛋白 天冬酰胺酶 

分 类 号：TS201.3[轻工技术与工程—食品科学]