Establishing a CRISPR/Cas9 Genome Editing System Combined with <i>URA3</i> -Blaster in <i>Botrytis cinerea</i> for Enhanced Abscisic Acid Production

Abscisic acid (ABA) is a key plant growth regulator widely used in agriculture and ecological restoration. Although metabolic engineering of the fungus <i>Botrytis cinerea</i> can enhance ABA production, it has been hindered by inefficient genetic tools. In this study, we first established a recyclable selection marker system in <i>B. cinerea</i> 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 <i>Bcpks12</i> and <i>Bcphs1</i> all significantly promoted ABA accumulation. Notably, co-overexpression of <i>Bcacly1</i> and <i>Bcacly2</i> 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 <i>B. cinerea</i>.