A genome‐integrated CRISPR/Cas12a system enables efficient genetic engineering in Xenorhabdus budapestensis XBD8

Xenorhabdus budapestensis strain XBD8, isolated from soil in northeastern China, produces a diverse array of natural products with promising agricultural applications, including fabclavines and GameXPeptides. However, the development and application of this strain have been significantly constrained because conventional genome‐editing methods rely on homologous recombination technology, which is inefficient and time‐consuming. The CRISPR/Cas technology, derived from the adaptive immune system of prokaryotes, provides a powerful and groundbreaking tool for microbial genome editing. In this study, we developed a CRISPR/Cas12a‐based genome‐editing approach for XBD8 that enables precise and seamless deletion or replacement of DNA fragments ranging from 379 to 45,168 base pairs, with a success rate exceeding 90%. Using this system, the quorum‐sensing regulatory module LuxR/LuxI was introduced into XBD8 to enable dynamic regulation of key biosynthetic genes involved in fabclavines biosynthesis. As a result, the production of fabclavine‐8 reached 140.10 mg/L, representing a 1.90‐fold increase compared with the wild‐type strain.