Key Laboratory of Marine Drugs and Key Laboratory of Evolution and Marine Biodiversity (the Ministry of Education of China), Institute of Evolution & Marine Biodiversity, School of Medicine and Pharmacy, Ocean University of China, Qingdao, 266003, China.
Fujian Key Laboratory on Conservation and Sustainable Utilization of Marine Biodiversity, Institute of Oceanography, Minjiang University, Fuzhou, 350108, China.
Microb Cell Fact. 2024 May 9;23(1):134. doi: 10.1186/s12934-024-02396-z.
Lovastatin has widespread applications thanks to its multiple pharmacological effects. Fermentation by filamentous fungi represents the major way of lovastatin production. However, the current lovastatin productivity by fungal fermentation is limited and needs to be improved.
In this study, the lovastatin-producing strains of Aspergillus terreus from marine environment were screened, and their lovastatin productions were further improved by genetic engineering. Five strains of A. terreus were isolated from various marine environments. Their secondary metabolites were profiled by metabolomics analysis using Ultra Performance Liquid Chromatography-Mass spectrometry (UPLC-MS) with Global Natural Products Social Molecular Networking (GNPS), revealing that the production of secondary metabolites was variable among different strains. Remarkably, the strain of A. terreus MJ106 could principally biosynthesize the target drug lovastatin, which was confirmed by High Performance Liquid Chromatography (HPLC) and gene expression analysis. By one-factor experiment, lactose was found to be the best carbon source for A. terreus MJ106 to produce lovastatin. To improve the lovastatin titer in A. terreus MJ106, genetic engineering was applied to this strain. Firstly, a series of strong promoters was identified by transcriptomic and green fluorescent protein reporter analysis. Then, three selected strong promoters were used to overexpress the transcription factor gene lovE encoding the major transactivator for lov gene cluster expression. The results revealed that compared to A. terreus MJ106, all lovE over-expression mutants exhibited significantly more production of lovastatin and higher gene expression. One of them, LovE-b19, showed the highest lovastatin productivity at a titer of 1512 mg/L, which represents the highest production level reported in A. terreus.
Our data suggested that combination of strain screen and genetic engineering represents a powerful tool for improving the productivity of fungal secondary metabolites, which could be adopted for large-scale production of lovastatin in marine-derived A. terreus.
洛伐他汀具有多种药理作用，因此应用广泛。丝状真菌发酵是洛伐他汀生产的主要方式。然而，真菌发酵生产洛伐他汀的当前效率有限，需要加以改进。
本研究从海洋环境中筛选出产洛伐他汀的土曲霉菌株，并通过基因工程进一步提高其洛伐他汀的产量。从各种海洋环境中分离出 5 株土曲霉。采用超高效液相色谱-质谱联用技术（UPLC-MS）结合全球天然产物社会分子网络（GNPS）对其代谢组学进行分析，结果表明不同菌株的次生代谢产物产量存在差异。值得注意的是，菌株 A. terreus MJ106 主要合成目标药物洛伐他汀，这一点通过高效液相色谱（HPLC）和基因表达分析得到了证实。通过单因素实验发现，乳糖是 A. terreus MJ106 生产洛伐他汀的最佳碳源。为提高 A. terreus MJ106 洛伐他汀的产量，对该菌株进行了基因工程改造。首先，通过转录组和绿色荧光蛋白报告分析鉴定了一系列强启动子。然后，使用三个选定的强启动子过表达转录因子基因 lovE，该基因编码 lov 基因簇表达的主要转录激活因子。结果表明，与 A. terreus MJ106 相比，所有 lovE 过表达突变体的洛伐他汀产量均显著增加，基因表达水平也更高。其中，LovE-b19 的洛伐他汀产量最高，达到 1512mg/L，这是 A. terreus 中报道的最高产量。
本研究结果表明，通过菌株筛选和遗传工程相结合，为提高真菌次生代谢产物的产量提供了一种有力工具，可用于从海洋来源的土曲霉中大规模生产洛伐他汀。
