Presentation Information
[P01-077]Metabolic engineering unlocks cryptic specialised metabolites in the human pathogen Nocardia terpenica
○Napawit Ham Nonthakaew1, Jiaqi Li1,2, Liam K.R. Sharkey1, Weiguang Zeng1, Marion Herisse1, Jordan Staiger-Creed1, Mark Rizzacasa2, Sacha J. Pidot1 (1. Department of Microbiology and Immunology at the Doherty Institute, University of Melbourne, Australia (Australia), 2. School of Chemistry, Bio21 Institute, University of Melbourne, Australia (Australia))
Keywords:
Actinomycetes,Nocardia,Specialized metabolites,Biosynthetic gene cluster
Nocardia species are opportunistic bacterial pathogens capable of causing severe infections in humans. Despite their close relationship to the prolific metabolite-producing genus Streptomyces, relatively few specialised metabolites have been characterized from Nocardia. Unlocking the hidden chemical diversity within this genus requires strategies to activate silent specialised metabolite biosynthetic gene clusters (BGCs).
In this study, we employed a metabolic engineering strategy to activate cryptic metabolites in Nocardia terpenica SP0012. We selected a Nocardia terpenica transposon mutant, in which insertion into the first gene of terpenomycin, terA, abolished terpenomycin production. Under identical growth conditions, the terA mutant produced several metabolites that were not detected in the wild-type, indicating activation of cryptic metabolite production. However, initial yields were low, limiting further characterization.
To enhance production, label-free quantitative proteomics identified an upregulated BGC similar to the piericidin A cluster. Previous studies have shown that piericidin biosynthesis is influenced by phosphate availability and a global regulatory system, the phosphate-responsive regulator PhoP. Moreover, PhoP shares overlapping DNA-binding motifs with GlnR, a global nitrogen regulator responsive to amino acid levels. Based on this regulatory interplay, we hypothesized that modulation of phosphate and nitrogen sources could influence metabolite production. A new cultivation medium, termed ARLP, was developed by varying phosphate concentrations and amino acid supplementation. Increased phosphate significantly enhanced production, while amino acid supplementation suppressed it. Optimization resulted in a maximum yield of 269 mg/L, a 143-fold increase. Under optimised conditions, a new piericidin analogue along with its hydrolysis products were isolated and structurally characterized.
Overall, this study demonstrates that combining pathway inactivation and media optimization effectively activates cryptic pathways and enhances metabolite production.
In this study, we employed a metabolic engineering strategy to activate cryptic metabolites in Nocardia terpenica SP0012. We selected a Nocardia terpenica transposon mutant, in which insertion into the first gene of terpenomycin, terA, abolished terpenomycin production. Under identical growth conditions, the terA mutant produced several metabolites that were not detected in the wild-type, indicating activation of cryptic metabolite production. However, initial yields were low, limiting further characterization.
To enhance production, label-free quantitative proteomics identified an upregulated BGC similar to the piericidin A cluster. Previous studies have shown that piericidin biosynthesis is influenced by phosphate availability and a global regulatory system, the phosphate-responsive regulator PhoP. Moreover, PhoP shares overlapping DNA-binding motifs with GlnR, a global nitrogen regulator responsive to amino acid levels. Based on this regulatory interplay, we hypothesized that modulation of phosphate and nitrogen sources could influence metabolite production. A new cultivation medium, termed ARLP, was developed by varying phosphate concentrations and amino acid supplementation. Increased phosphate significantly enhanced production, while amino acid supplementation suppressed it. Optimization resulted in a maximum yield of 269 mg/L, a 143-fold increase. Under optimised conditions, a new piericidin analogue along with its hydrolysis products were isolated and structurally characterized.
Overall, this study demonstrates that combining pathway inactivation and media optimization effectively activates cryptic pathways and enhances metabolite production.
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