Genomic Locus of a Penicillium crustosum Pigment as an Integration Site for Secondary Metabolite Gene Expression.

Heterologous expression of secondary metabolite genes and gene clusters has been proven to be a successful strategy for identification of new natural products of cryptic or silent genes hidden in the genome sequences. It is also a useful tool to produce designed compounds by synthetic biology approaches. In this study, we demonstrate the potential usage of the gene locus pcr4401 in the fast-growing filamentous fungus Penicillium crustosum as an integration site for heterologous gene expression. The deduced polyketide synthase (PKS) Pcr4401 is involved in the dihydroxynaphthalene (DHN)-melanin pigment formation, and its deletion in P. crustosum PRB-2 led to an albino phenotype. Heterologous expression of pcr4401 in Aspergillus nidulans proved its function as the melanin precursor YWA1 synthase. To ensure gene expression after genomic integration and to easily identify the potential transformants by visualization, the gene locus of pcr4401 was chosen as an integration site. For heterologous expression in P. crustosum, the expression constructs were created by ligation-independent homologous recombination in Escherichia coli or Saccharomyces cerevisiae. A pyrG deficient strain was also created, so that both the pyrG and hph resistance gene can be used as selection markers. Successful expression in P. crustosum was demonstrated by using one uncharacterized PKS gene from Aspergillus and two from Penicillium strains. All three genes were successfully introduced, heterologously expressed, and their biosynthetic products elucidated. The results presented in this study demonstrated that P. crustosum can be used as a suitable host for heterologous expression of secondary metabolite genes.

Peniphenone and Penilactone Formation in Penicillium crustosum via 1,4-Michael Additions of ortho-Quinone Methide from Hydroxyclavatol to γ-Butyrolactones from Crustosic Acid.

Penilactones A and B consist of a γ-butyrolactone and two clavatol moieties. We identified two separate gene clusters for the biosynthesis of these key building blocks in Penicillium crustosum. Gene deletion, feeding experiments, and biochemical investigations proved that a nonreducing PKS ClaF is responsible for the formation of clavatol and the PKS-NRPS hybrid TraA is involved in the formation of crustosic acid, which undergoes decarboxylation and isomerization to the predominant terrestric acid. Both acids are proposed to be converted to γ-butyrolactones with involvement of a cytochrome P450 ClaJ. Oxidation of clavatol to hydroxyclavatol by a nonheme FeII/2-oxoglutarate-dependent oxygenase ClaD and its spontaneous dehydration to an ortho-quinone methide initiate the two nonenzymatic 1,4-Michael addition steps. Spontaneous addition of the methide to the γ-butyrolactones led to peniphenone D and penilactone D, which undergo again stereospecific attacking by methide to give penilactones A/B.

Convenient synthetic approach for tri- and tetraprenylated cyclodipeptides by consecutive enzymatic prenylations.

The prenyltransferases EchPT1 and EchPT2 from Aspergillus ruber are responsible for the consecutive prenylations of cyclo-L-Trp-L-Ala, leading to the formation of the triprenylated echinulin as the predominant product. In this study, we demonstrate that EchPT1 also accepts all stereoisomers of cyclo-Trp-Ala and cyclo-Trp-Pro and catalyses regiospecific reverse C2-prenylation at the indole nucleus. EchPT1 products were well accepted by EchPT2 for multiple consecutive prenylations, with conversion yields of 84 to 98% for six of the eight substrates. C2-, C5- and C7-triprenylated derivatives are identified as major enzyme products, with product yields of 40 to 86% in seven cases. High product yields of 25-36%, i.e. approximate 30% of the total enzyme products, were observed for tetraprenylated derivatives in the four reaction mixtures with one D- and one L-configured amino acid residues. To the best of our knowledge, enzymatic preparation of tetraprenylated cyclodipeptides with such high efficacy has not been reported prior to this study.

Two Prenyltransferases Govern a Consecutive Prenylation Cascade in the Biosynthesis of Echinulin and Neoechinulin.

Two prenyltransferases from Aspergillus ruber control the echinulin biosynthesis via exceptional sequential prenylations. EchPT1 catalyzes the first prenylation step, leading to preechinulin. The unique EchPT2 attaches, in a consecutive prenylation cascade, up to three dimethylallyl moieties to preechinulin and its dehydro forms neoechinulins A and B, resulting in the formation of at least 23 2- to 4-fold prenylated derivatives. Confirming these products in fungal extracts unravels the unprecedented catalytic relevance of EchPT2 for structural diversity.

PrenDB, a Substrate Prediction Database to Enable Biocatalytic Use of Prenyltransferases.

Prenyltransferases of the dimethylallyltryptophan synthase (DMATS) superfamily catalyze the attachment of prenyl or prenyl-like moieties to diverse acceptor compounds. These acceptor molecules are generally aromatic in nature and mostly indole or indole-like. Their catalytic transformation represents a major skeletal diversification step in the biosynthesis of secondary metabolites, including the indole alkaloids. DMATS enzymes thus contribute significantly to the biological and pharmacological diversity of small molecule metabolites. Understanding the substrate specificity of these enzymes could create opportunities for their biocatalytic use in preparing complex synthetic scaffolds. However, there has been no framework to achieve this in a rational way. Here, we report a chemoinformatic pipeline to enable prenyltransferase substrate prediction. We systematically catalogued 32 unique prenyltransferases and 167 unique substrates to create possible reaction matrices and compiled these data into a browsable database named PrenDB. We then used a newly developed algorithm based on molecular fragmentation to automatically extract reactive chemical epitopes. The analysis of the collected data sheds light on the thus far explored substrate space of DMATS enzymes. To assess the predictive performance of our virtual reaction extraction tool, 38 potential substrates were tested as prenyl acceptors in assays with three prenyltransferases, and we were able to detect turnover in >55% of the cases. The database, PrenDB (www.kolblab.org/prendb.php), enables the prediction of potential substrates for chemoenzymatic synthesis through substructure similarity and virtual chemical transformation techniques. It aims at making prenyltransferases and their highly regio- and stereoselective reactions accessible to the research community for integration in synthetic work flows.

The sulfur oxygenase reductase from the mesophilic bacterium Halothiobacillus neapolitanus is a highly active thermozyme.

A biochemical, biophysical, and phylogenetic study of the sulfur oxygenase reductase (SOR) from the mesophilic gammaproteobacterium Halothiobacillus neapolitanus (HnSOR) was performed in order to determine the structural and biochemical properties of the enzyme. SOR proteins from 14 predominantly chemolithoautotrophic bacterial and archaeal species are currently available in public databases. Sequence alignment and phylogenetic analysis showed that they form a coherent protein family. The HnSOR purified from Escherichia coli after heterologous gene expression had a temperature range of activity of 10 to 99°C with an optimum at 80°C (42 U/mg protein). Sulfite, thiosulfate, and hydrogen sulfide were formed at various stoichiometries in a range between pH 5.4 and 11 (optimum pH 8.4). Circular dichroism (CD) spectroscopy and dynamic light scattering showed that the HnSOR adopts secondary and quaternary structures similar to those of the 24-subunit enzyme from the hyperthermophile Acidianus ambivalens (AaSOR). The melting point of the HnSOR was ≈20°C lower than that of the AaSOR, when analyzed with CD-monitored thermal unfolding. Homology modeling showed that the secondary structure elements of single subunits are conserved. Subtle changes in the pores of the outer shell and increased flexibility might contribute to activity at low temperature. We concluded that the thermostability was the result of a rigid protein core together with the stabilizing effect of the 24-subunit hollow sphere.