Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing, 210023, P. R. China.
J Microbiol. 2019 Feb;57(2):154-162. doi: 10.1007/s12275-019-8259-8. Epub 2019 Jan 31.
Hypocrellin A (HA) is a perylenequinone (PQ) isolated from Shiraia bambusicola that shows antiviral and antitumor activities, but its application is limited by the low production from wild fruiting body. A gene overexpressing method was expected to augment the production rate of HA in S. bambusicola. However, the application of this molecular biology technology in S. bambusicola was impeded by a low genetic transformation efficiency and little genomic information. To enhance the plasmid transformant ratio, the Polyethylene Glycol-mediated transformation system was established and optimized. The following green fluorescent protein (GFP) analysis showed that the gene fusion expression system we constructed with a GAPDH promoter Pgpd1 and a rapid 2A peptide was successfully expressed in the S. bambusicola S4201 strain. We successfully obtained the HA high-producing strains by overexpressing O-methyltransferase/FAD-dependent monooxygenase gene (mono) and the hydroxylase gene (hyd), which were the essential genes involved in our putative HA biosynthetic pathway. The overexpression of these two genes increased the production of HA by about 200% and 100%, respectively. In general, this study will provide a basis to identify the genes involved in the hypocrellin A biosynthesis. This improved transformation method can also be used in genetic transformation studies of other fungi.
竹红菌素 A（HA）是从竹黄中分离得到的一种二萘醌类化合物，具有抗病毒和抗肿瘤活性，但由于其野生子实体产量低，其应用受到限制。预计通过基因过表达方法可以提高竹黄中 HA 的产量。然而，由于遗传转化效率低和基因组信息少，该分子生物学技术在竹黄中的应用受到阻碍。为了提高质粒转化体的比例，建立并优化了聚乙二醇介导的转化系统。以下绿色荧光蛋白（GFP）分析表明，我们构建的带有 GAPDH 启动子 Pgpd1 和快速 2A 肽的基因融合表达系统在 S. bambusicola S4201 菌株中成功表达。通过过表达参与我们假定的 HA 生物合成途径的 O-甲基转移酶/FAD 依赖性单加氧酶基因（mono）和羟化酶基因（hyd），我们成功获得了 HA 高产菌株。这两个基因的过表达分别使 HA 的产量增加了约 200%和 100%。总的来说，这项研究将为鉴定参与竹红菌素 A 生物合成的基因提供基础。这种改进的转化方法也可用于其他真菌的遗传转化研究。
