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

Abstract

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.

Keywords

CRISPR/Cas12a / fabclavines / genome editing / LuxR/LuxI / Xenorhabdus budapestensis

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