Genetic Engineering Activates Biosynthesis of Aromatic Fumaric Acid Amides in the Human Pathogen Aspergillus fumigatus

Abstract

The Aspergillus fumigatus nonribosomal peptide synthetase FtpA is among the few of this species whose natural product has remained unknown. Both FtpA adenylation domains were characterized in vitro. Fumaric acid was identified as preferred substrate of the first and both L-tyrosine and L-phenylalanine as preferred substrates of the second adenylation domain. Genetically engineered A. fumigatus strains expressed either ftpA or the regulator gene ftpR, encoded in the same cluster of genes, under the control of the doxycycline-inducible tetracycline-induced transcriptional activation (tet-on) cassette. These strains produced fumaryl-L-tyrosine and fumaryl-L-phenylalanine which were identified by liquid chromatography and high-resolution mass spectrometry. Modeling of the first adenylation domain in silico provided insight into the structural requirements to bind fumaric acid as peptide synthetase substrate. This work adds aromatic fumaric acid amides to the secondary metabolome of the important human pathogen A. fumigatus which was previously not known as a producer of these compounds.

An Iterative O‐Methyltransferase Catalyzes 1,11‐Dimethylation of Aspergillus fumigatus Fumaric Acid Amides

“…[6] Isolation of genomic A. fumigatus DNA and Southern blot analysis were as previously described. Fumaryl-l-tyrosine and fumaryl-l-phenylalanine were synthesized as described previously.…”

“…Fumaryl-l-tyrosine and fumaryl-l-phenylalanine were synthesized as described previously. [6] Isolation of genomic A. fumigatus DNA and Southern blot analysis were as previously described. [14] Synthetic DNA was produced by Life Technologies.…”

“…[1] Since the publication of the genomic sequence, [2] research into its capacity to produce secondary metabolitesh as gained momentum:t he A. fumigatus genome encodes as many as 15 polyketide synthases (PKSs) and 16 nonribosomal peptides ynthetases (NRPSs), including those for toxins such as gliotoxin, [3] fumitremorgin, [4] and fumigaclavine. [6] The gene cluster also encodes ap utative Sadenosyl-l-methionine (SAM)-dependentm ethyltransferase (FtpM, AFUA_3G15280;2 82 aa, calculated mass 31.5 kDa). However, the function of ac luster of five genes around the gene AFUA_3G15270 (Scheme 1), which encodes at wo-module NRPS, hasr emained elusive, as the cluster was not expressed under laboratory conditions.…”

“…By placing either AFUA_3G15270 (here referred to as ftpA)o rAFUA_ 3G15290 (regulatorg ene ftpR)u nder the control of at etracycline-inducible promoter,wer ecently showedt hat FtpA produces both fumaryl-l-tyrosine (1,S cheme 1B)a nd fumaryl-l-phenylalanine (2). [6] The gene cluster also encodes ap utative Sadenosyl-l-methionine (SAM)-dependentm ethyltransferase (FtpM, AFUA_3G15280;2 82 aa, calculated mass 31.5 kDa). However,p robably because of insufficient activation of ftpM in as train that overexpresses ftpR,m ethylated derivatives were found in only minor amounts, and their structures remained undetermined.…”

The cyclochlorotine mycotoxin is produced by the nonribosomal peptide synthetase CctN in Talaromyces islandicus (‘Penicillium islandicum’)

“…The incidence of homologous NRPS biosynthetic genes of which one is silent has been found earlier in work on Aspergillus fumigatus (Steinchen et al ., ). Genetic engineering and metabolite analysis revealed that both NRPSs are functional and produce a similar set of small metabolites (Kalb et al ., ). Apparently, the effort of harbouring redundant NRPS pathways is prevalent in filamentous fungi and might emphasize the significance of the corresponding SMs.…”