Enhancing the Production of Xenocoumacin 1 in Xenorhabdus nematophila CB6 by a Combinatorial Engineering Strategy

Abstract

Xenocoumacin 1 (Xcn1) is an excellent antimicrobial natural product against Phytophthora capsici. However, the commercial development of Xcn1 is hindered by the low yield, which results in high application costs. In this study, multiple metabolic strategies, including blocking the degradation pathway, promoter engineering, and deletion of competing biosynthetic gene clusters, were employed to improve the production of Xcn1, which was increased from 0.07 to 0.91 g/L. The formation of Xcn1 reached 1.94 g/L in the TB medium with the final strain T3 in a shake flask and further reached 3.52 g/L in a 5 L bioreactor, which is the highest yield ever reported. The engineered strain provides a valuable platform for production of Xcn1, and the possible commercial development of the biofungicide. We anticipate that the metabolic engineering strategies utilized in this study and the constructed constitutive promoter library can be widely applied to other bacteria of the genera Xenorhabdus and Photorhabdus.

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

“…budapestensis XBD8. Thereafter, a recombinant plasmid library containing 30 previously identified constitutive promoters with different strengths was constructed . The plasmid library was then simultaneously transformed into the recombinant host strain to add the constitutive promoters in front of fclC to enhance the production of Fcl-7 and Fcl-8.…”

“…Construction of a Promoter-Replacement Mutant Library in X. budapestensis XBD8. Using the previously constructed constitutive promoters as templates, 50 we amplified promoter fragments with universal primers Pqs-F and Pqs-R. We then successfully created the MX14-pR6K plasmid library, which can be used to replace the native promoter of fclC. Finally, we used a twostep selection/counterselection method to generate seamless fclC promoter substitution mutants.…”

“…Thereafter, a recombinant plasmid library containing 30 previously identified constitutive promoters with different strengths was constructed. 50 The plasmid library was then simultaneously transformed into the recombinant host strain to add the constitutive promoters in front of fclC to enhance the production of Fcl-7 and Fcl-8. We picked 240 colonies and confirmed them by PCR and sequencing and successfully obtained 111 correct recombinants, resulting in a positive rate of 46.3% (Figure S6).…”