Establishing a CRISPR/Cas9 Genome Editing System Combined with URA3-Blaster in Botrytis cinerea for Enhanced Abscisic...

Abscisic acid (ABA) is a key plant growth regulator widely used in agriculture and ecological restoration. Although metabolic engineering of the fungus Botrytis cinerea can enhance ABA production, it has been hindered by inefficient genetic tools. In this study, we first established a recyclable selection marker system in B. cinerea based on orotidine-5'-phosphate decarboxylase. Subsequently, the CRISPR/Cas9 system was optimized, achieving up to 100% editing efficiency, far surpassing traditional homologous recombination. Based on this platform, multiple metabolic engineering strategies were systematically explored to enhance ABA biosynthesis. Increasing acetyl-CoA supply, inhibiting squalene synthesis, and knocking out key secondary metabolism genes Bcpks12 and Bcphs1 all significantly promoted ABA accumulation. Notably, co-overexpression of Bcacly1 and Bcacly2 combined with 1 g/L citrate increased ABA production to 1.36 g/L, representing a 38.66% improvement. Overall, this study provides an efficient genetic toolkit and a solid foundation for the industrial-scale production of ABA via engineered B. cinerea.