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Appl Environ Microbiol. 2018 Sep 14. pii: AEM.01789-18. doi: 10.1128/AEM.01789-18. [Epub ahead of print]

Engineering pseudochelin production in Myxococcus xanthus.

Author information

1
Department of Biochemical and Chemical Engineering, Laboratory of Technical Biology, TU Dortmund University, Emil-Figge-Strasse 66, 44227 Dortmund, Germany.
2
Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, Beutenbergstr. 11a, 07745 Jena, Germany.
3
Department of Biochemical and Chemical Engineering, Laboratory of Technical Biology, TU Dortmund University, Emil-Figge-Strasse 66, 44227 Dortmund, Germany markus.nett@tu-dortmund.de.

Abstract

Myxobacteria utilize the catechol natural products myxochelin A and B in order to maintain their iron homeostasis. Recently, the production of these siderophores was reported from the marine bacterium Pseudoalteromonas piscicida S2040, along with a new myxochelin derivative named pseudochelin A. The latter features a characteristic imidazoline moiety, which was proposed to originate from an intramolecular condensation reaction of the β-aminoethyl amide group in myxochelin B. To identify the enzyme catalyzing this conversion, we compared the myxochelin regulons of two myxobacterial strains, which solely produce myxochelin A and B, with P. piscicida S2040. This approach revealed a gene exclusive to the myxochelin regulon in P. piscicida S2040, coding for an enzyme of the amidohydrolase superfamily. To prove that this enzyme is indeed responsible for the postulated conversion, the reaction was reconstituted in vitro using a hexahistidyl-tagged recombinant protein made in Escherichia coli and myxochelin B as substrate. To test the production of pseudochelin A under in vivo conditions, the amidohydrolase gene was cloned into the myxobacterial plasmid pZJY156 and placed under the control of a copper-inducible promoter. The resulting vector was introduced into the myxobacterium Myxococcus xanthus DSM16526, a native producer of myxochelin A and B. Following the induction with copper, the myxobacterial expression strain was found to synthesize small quantities of pseudochelin A. Replacement of the copper-inducible promoter with the constitutive pilA promoter led to increased production levels in M. xanthus, which facilitated the isolation and subsequent structural verification of the heterologously produced compound.Importance In this study, an enzyme for imidazoline formation in pseudochelin biosynthesis was identified. Evidence for the involvement of this enzyme in the postulated reaction was obtained after in vitro reconstitution. Furthermore, the function of this enzyme was also demonstrated in vivo by transferring the corresponding gene into the bacterium Myxococcus xanthus, which thereby became a producer of pseudochelin A. Aside from clarifying the molecular basis of imidazoline formation in siderophore biosynthesis, we describe the heterologous expression of a gene in a myxobacterium without its chromosomal integration. Due to its metabolic proficiency, M. xanthus represents an interesting alternative to established host systems for the reconstitution and manipulation of biosynthetic pathways. Since the plasmid used in this study is easily adaptable for the expression of other enzymes as well, we expand the conventional expression strategy for myxobacteria, which is based on the integration of biosynthetic genes into the host genome.

PMID:
30217842
PMCID:
PMC6210110
[Available on 2019-04-30]
DOI:
10.1128/AEM.01789-18

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