Development of an Efficient and Seamless Genetic Manipulation Method for Xenorhabdus and Its Application for Enhancing the Production of Fabclavines.

Xenorhabdus can produce numerous natural products, but their development has been hampered by the lack of a seamless genetic manipulation method. In this study, we compared several lethal genes and determined the sacB gene as the most effective counter-selection marker and then established a dual selection/counter-selection system by integrating neo and sacB genes into one cassette. This provides an efficient and seamless genetic manipulation method for Xenorhabdus . Using this method, DNA fragments ranging from 205 to 47,788 bp in length were seamlessly knocked out or replaced with impressively high positive rates of 80 to 100% in Xenorhabdus budapestensis XBD8. In addition, the method was successfully applied with good efficiency (45-100%) in Xenorhabdus nematophila CB6. To further validate the method, different constitutive promoters were used to replace the native fclC promoter in a batch experiment. The positivity rate remained consistently high, at 46.3%. In comparison to WT XBD8, the recombinant strain MX14 demonstrated a significant increase in the production of fabclavine 7 and fabclavine 8 by 4.97-fold and 3.22-fold, respectively, while the overall production of fabclavines was enhanced by 3.52-fold.