Qin Lina, Jiang Xianzhang, Dong Zhiyang, Huang Jianzhong, Chen Xiuzhen
1National and Local Joint Engineering Research Center of Industrial Microbiology and Fermentation Technology, College of Life Sciences, Fujian Normal University, Qishan Campus, No.1 Keji Road, Shangjie, Minhou, Fuzhou, 350117 Fujian China.
3Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, College of Life Sciences, Fujian Normal University, Fuzhou, 350117 Fujian China.
Biotechnol Biofuels. 2018 May 17;11:142. doi: 10.1186/s13068-018-1139-3. eCollection 2018.
The ascomycete fungus was widely used as a biotechnological workhorse for production of cellulases and recombinant proteins due to its large capacity of protein secretion. Transgenesis by random integration of a gene of interest (GOI) into the genome of can generate series of strains that express different levels of the indicated transgene. The insertion site of the GOI plays an important role in the ultimate production of the targeted proteins. However, so far no systematic studies have been made to identify transgene integration loci for optimal expression of the GOI in . Currently, only the locus of exocellobiohydrolases I encoding gene ( is widely used as a promising integration site to lead to high expression level of the GOI. No additional sites associated with efficient gene expression have been characterized.
To search for gene integration sites that benefit for the secreted expression of GOI, the food-and-mouth disease virus 2A protein was applied for co-expression of an gene and sp. gene in by random integration of the expression cassette into the genome. We demonstrated that the fluorescent intensity of RFP (red fluorescent protein) inside of the cell was well correlated with the secreted lipase yields, based on which, we successfully developed a high-throughput screening method to screen strains with relatively higher secreted expression of the GOI (in this study, lipase). The copy number and the insertion sites of the transgene were investigated among the selected highly expressed strains. Eventually, in addition to gene locus, two other genome insertion loci that efficiently facilitate gene expression in were identified.
We have successfully developed a high-throughput screening method to screen strains with optimal expression of the indicated secreted proteins in . Moreover, we identified two optimal genome loci for transgene expression, which could provide new approach to modulate gene expression levels while retaining the indicated promoter and culture conditions.
由于其强大的蛋白质分泌能力，子囊菌被广泛用作生产纤维素酶和重组蛋白的生物技术主力菌株。通过将感兴趣的基因（GOI）随机整合到该菌的基因组中进行转基因操作，可以产生一系列表达不同水平目标转基因的菌株。GOI的插入位点对目标蛋白的最终产量起着重要作用。然而，到目前为止，尚未进行系统研究来确定在该菌中实现GOI最佳表达的转基因整合位点。目前，只有外切葡聚糖酶I编码基因（ ）的位点被广泛用作有望实现GOI高表达水平的整合位点。尚未鉴定出与高效基因表达相关的其他位点。
为了寻找有利于GOI分泌表达的基因整合位点，通过将表达盒随机整合到该菌基因组中，应用口蹄疫病毒2A蛋白在该菌中共同表达一个基因和该菌的基因。我们证明细胞内红色荧光蛋白（RFP）的荧光强度与分泌的脂肪酶产量密切相关，基于此，我们成功开发了一种高通量筛选方法，以筛选具有相对较高GOI分泌表达水平（在本研究中为脂肪酶）的菌株。在选定的高表达菌株中研究了转基因的拷贝数和插入位点。最终，除了基因位点外，还鉴定出另外两个能有效促进该菌中基因表达的基因组插入位点。
我们成功开发了一种高通量筛选方法，以筛选在该菌中具有目标分泌蛋白最佳表达的菌株。此外，我们鉴定出两个转基因表达的最佳基因组位点，这可以在保留特定启动子和培养条件的同时，为调节基因表达水平提供新方法。
