National and Local Joint Engineering Research Center for Biomanufacturing of Choral Chemicals, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
Appl Microbiol Biotechnol. 2023 May;107(9):3071-3084. doi: 10.1007/s00253-023-12498-0. Epub 2023 Apr 4.
Gibberellic acid (GA), one of the natural diterpenoids produced by Fusarium fujikuroi, serves as an important phytohormone in agriculture for promoting plant growth. Presently, the metabolic engineering strategies for increasing the production of GA are progressing slowly, which seriously restricted the advancing of the cost-effective industrial production of GA. In this study, an industrial strain with high-yield GA of F. fujikuroi was constructed by metabolic modification, coupling with transcriptome analysis and promoter engineering. The over-expression of AreA and Lae1, two positive factors in the regulatory network, generated an initial producing strain with GA production of 2.78 g L. Compared with a large abundance of transcript enrichments in the GA synthetic gene cluster discovered by the comparative transcriptome analysis, geranylgeranyl pyrophosphate synthase 2 (Ggs2), and cytochrome P450-3 genes, two key genes that respectively participated in the initial and final step of biosynthesis, were identified to be downregulated when the highest GA productivity was obtained. Employing with a nitrogen-responsive bidirectional promoter, the two rate-limiting genes were dynamically upregulated, and therefore, the production of GA was increased to 3.02 g L. Furthermore, the top 20 upregulated genes were characterized in GA over-production, and their distributions in chromosomes suggested potential genomic regions with a high transcriptional level for further strain development. The construction of a GA high-yield-producing strain was successfully achieved, and insights into the enriched functional transcripts provided novel strain development targets of F. fujikuroi, offering an efficient microbial development platform for industrial GA production. KEY POINTS: • Global regulatory modification was achieved in F. fujikuroi for GA overproduction. • Comparative transcriptome analysis revealed bottlenecks in GA specific-pathway. • A dynamically nitrogen-regulated bidirectional promoter was cloned and employed.
赤霉素（GA）是一种由藤仓镰刀菌产生的天然二萜类化合物，作为农业中促进植物生长的重要植物激素。目前，提高 GA 产量的代谢工程策略进展缓慢，严重限制了 GA 具有成本效益的工业生产的推进。在这项研究中，通过代谢修饰，结合转录组分析和启动子工程，构建了一种赤霉菌高产 GA 的工业菌株。两个正调控因子 AreA 和 Lae1 的过表达产生了一个初始生产菌株，GA 产量为 2.78 g/L。与比较转录组分析发现的 GA 合成基因簇中大量转录本的丰度相比，香叶基香叶基焦磷酸合酶 2（Ggs2）和细胞色素 P450-3 基因，这两个分别参与生物合成初始和最终步骤的关键基因，在获得最高 GA 生产力时被下调。采用氮响应双向启动子，两个限速基因被动态上调，因此，GA 的产量增加到 3.02 g/L。此外，对 GA 过量产生的 20 个上调基因进行了特征描述，它们在染色体上的分布表明了进一步菌株开发的具有高转录水平的潜在基因组区域。成功构建了 GA 高产生产菌株，并深入了解了丰富的功能转录本，为藤仓镰刀菌提供了新的菌株开发目标，为工业 GA 生产提供了高效的微生物开发平台。关键点： • 在藤仓镰刀菌中实现了全局调控修饰以实现 GA 过量生产。 • 比较转录组分析揭示了 GA 特定途径中的瓶颈。 • 克隆并采用了一个动态氮调节的双向启动子。
