Development of a CRISPRi system in Fusarium fujikuroi and its application in gibberellic acid production

Gene knockdown is a pivotal genetic manipulation technique, particularly when targeting lethal genes or genes involved in product synthesis pathways, where complete gene knockout is not a viable option. This approach is particularly valuable in multinucleate species, such as Fusarium fujikuroi, where generating homogeneous gene knockouts is notoriously difficult. To address these limitations, we first screened a set of repression domains, and then leveraged the optimal candidates to construct a CRISPR/dCas9-mediated knockdown platform for F. fujikuroi. By targeting erg9, which encodes squalene synthase, the first committed enzyme in the mevalonate pathway for ergosterol biosynthesis, we successfully diverted a portion of the metabolic flux from sterol production to gibberellic acid (GA) biosynthesis. This strategy minimizes carbon loss to competing pathways while retaining phenotypically normal growth. Additionally, CRISPR/dCas9-mediated knockdown of the dehydrogenase gene des enhanced GA₄ production by 2.62-fold and eliminated the intermediate GA₇, generating a GA₃₊₄-producing strain and fine-tuning its metabolic profile. Using our CRISPRi system, we achieved a 70-89 % reduction in erg9 mRNA levels and a 67- 84 % reduction in des mRNA levels. Our findings establish a tailored CRISPRi platform for effective gene repression in F. fujikuroi.