posted on 2023-12-18, 18:38authored byJiaqi Duan, Baoming Yuan, Fenglian Jia, Xiaohui Li, Chang Chen, Guangyue Li
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.