Transcriptome-Based Mining of the Strong Promoters for Hyperproduction of Gibberellin GA3 in <i>Fusarium fujikuroi</i>

Gibberellin GA3 is a plant growth regulator with significant applications in agriculture, and <i>Fusarium fujikuroi</i> has gained attention as an excellent host for the industrial production of GA3. Although numerous gene-editing tools have been developed, the precise metabolic flux regulation in <i>F. fujikuroi</i> was significantly hindered because the endogenous promoters were rarely identified. In this study, a library containing 20 potential promoters was mined and constructed for the first time through transcriptome sequencing. Using β-glucuronidase as a reporter gene, promoter <i>P</i>_<i>10594</i> showed the highest relative activity and had a stable expression in different media, which was identified as a strong constitutive promoter. Subsequently, <i>P</i>_<i>10594</i> was used to regulate the metabolic flux for GA3 overproduction. The yield reached 0.89 g/L in the shake flask, 17.1% higher than the control strain. Finally, 2.38 g/L GA3 can be obtained in a 5 L bioreactor using the engineered strain FF-2. In general, the work enriched the promoter library in <i>F. fujikuroi</i> and provided help for reshaping the complex metabolic flow.