Publications

• Production of alpha-keto carboxylic acid dimers in yeast by overexpression of NRPS-like genes from Aspergillus terreus.

  Huehner E., Backhaus K., Kraut R., Li S.M.

  Non-ribosomal peptide synthetases (NRPSs) are key enzymes in microorganisms for the assembly of peptide backbones of biologically and pharmacologically active natural products. The monomodular NRPS-like enzymes comprise often an adenylation (A), a thiolation (T), and a thioesterase (TE) domain. In ... >> More

  Non-ribosomal peptide synthetases (NRPSs) are key enzymes in microorganisms for the assembly of peptide backbones of biologically and pharmacologically active natural products. The monomodular NRPS-like enzymes comprise often an adenylation (A), a thiolation (T), and a thioesterase (TE) domain. In contrast to the NRPSs, they do not contain any condensation domain and usually catalyze a dimerization of α-keto carboxylic acids and thereby provide diverse scaffolds for further modifications. In this study, we established an expression system for NRPS-like genes in Saccharomyces cerevisiae. By expression of four known genes from Aspergillus terreus, their predicted function was confirmed and product yields of up to 35 mg per liter culture were achieved. Furthermore, expression of ATEG_03090 from the same fungus, encoding for the last uncharacterized NRPS-like enzyme with an A-T-TE domain structure, led to the formation of the benzoquinone derivative atromentin. All the accumulated products were isolated and their structures were elucidated by NMR and MS analyses. This study provides a convenient system for proof of gene function as well as a basis for synthetic biology, since additional genes encoding modification enzymes can be introduced. << Less

  Appl. Microbiol. Biotechnol. 102:1663-1672(2018) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Three redundant synthetases secure redox-active pigment production in the basidiomycete Paxillus involutus.

  Braesel J., Gotze S., Shah F., Heine D., Tauber J., Hertweck C., Tunlid A., Stallforth P., Hoffmeister D.

  The symbiotic fungus Paxillus involutus serves a critical role in maintaining forest ecosystems, which are carbon sinks of global importance. P. involutus produces involutin and other 2,5-diarylcyclopentenone pigments that presumably assist in the oxidative degradation of lignocellulose via Fenton ... >> More

  The symbiotic fungus Paxillus involutus serves a critical role in maintaining forest ecosystems, which are carbon sinks of global importance. P. involutus produces involutin and other 2,5-diarylcyclopentenone pigments that presumably assist in the oxidative degradation of lignocellulose via Fenton chemistry. Their precise biosynthetic pathways, however, remain obscure. Using a combination of biochemical, genetic, and transcriptomic analyses, in addition to stable-isotope labeling with synthetic precursors, we show that atromentin is the key intermediate. Atromentin is made by tridomain synthetases of high similarity: InvA1, InvA2, and InvA5. An inactive atromentin synthetase, InvA3, gained activity after a domain swap that replaced its native thioesterase domain with that of InvA5. The found degree of multiplex biosynthetic capacity is unprecedented with fungi, and highlights the great importance of the metabolite for the producer. << Less

  Chem. Biol. 22:1325-1334(2015) [PubMed] [EuropePMC]

• Characterization of the Suillus grevillei quinone synthetase GreA supports a nonribosomal code for aromatic alpha-keto acids.

  Wackler B., Lackner G., Chooi Y.H., Hoffmeister D.

  The gene greA was cloned from the genome of the basidiomycete Suillus grevillei. It encodes a monomodular natural product biosynthesis protein composed of three domains for adenylation, thiolation, and thioesterase and, hence, is reminiscent of a nonribosomal peptide synthetase (NRPS). GreA was bi ... >> More

  The gene greA was cloned from the genome of the basidiomycete Suillus grevillei. It encodes a monomodular natural product biosynthesis protein composed of three domains for adenylation, thiolation, and thioesterase and, hence, is reminiscent of a nonribosomal peptide synthetase (NRPS). GreA was biochemically characterized in vitro. It was identified as atromentin synthetase and therefore represents one of only a limited number of biochemically characterized NRPS-like enzymes which accept an aromatic α-keto acid. Specificity-conferring amino acid residues--collectively referred to as the nonribosomal code--were predicted for the primary sequence of the GreA adenylation domain and were an unprecedented combination for aromatic α-keto acids. Plausible support for this new code came from in silico simulation of the adenylation domain structure. According to the model, the predicted residues line the active site and, therefore, very likely contribute to substrate specificity. << Less

  ChemBioChem 13:1798-1804(2012) [PubMed] [EuropePMC]

• Characterization of the atromentin biosynthesis genes and enzymes in the homobasidiomycete Tapinella panuoides.

  Schneider P., Bouhired S., Hoffmeister D.

  This report highlights the first biochemical characterization of a multi-domain biosynthetic enzyme for basidiomycete secondary metabolism: the tri-domain enzyme atromentin synthetase AtrA, from Tapinella panuoides, which adenylates and dimerizes 4-hydroxyphenylpyruvic acid into atromentin. Also, ... >> More

  This report highlights the first biochemical characterization of a multi-domain biosynthetic enzyme for basidiomycete secondary metabolism: the tri-domain enzyme atromentin synthetase AtrA, from Tapinella panuoides, which adenylates and dimerizes 4-hydroxyphenylpyruvic acid into atromentin. Also, the l-tyrosine:2-oxoglutarate aminotransferase AtrD, which provides the substrate for this dimerization step, has been characterized. AtrA and AtrD expand the shikimic acid pathway from l-tyrosine to atromentin, the central terphenylquinone intermediate for a prominent and widely occurring class of basidiomycete pigments, among them various pharmaceutically relevant compounds. The genes atrA and atrD were cloned and found to be clustered within one genetic locus. Given the broad distribution of atromentin-derived compounds among homobasidiomycetes we expect our system represents a widely applicable model. << Less

  Fungal Genet. Biol. 45:1487-1496(2008) [PubMed] [EuropePMC]