Regioselective Oxidative Phenol Coupling by a Mushroom Unspecific Peroxygenase.

Bioactive dimeric (pre-)anthraquinones are ubiquitous in nature. Their biosynthesis via an oxidative phenol coupling (OPC) step is catalyzed by either cytochrome P450 enzymes, peroxidases, or laccases. While the biocatalysis of OPC in molds (Ascomycota) is well-known, the respective enzymes of mushroom-forming fungi (Basidiomycota) are still unknown. Here, we report on the biosynthesis of the atropisomers phlegmacin A1 and B1, unsymmetrical 7,10'-homo-coupled dihydroanthracenones of the mushroom Cortinarius odorifer. The biosynthesis was heterologously reconstituted in the mold Aspergillus niger. We show that methylation of the dimeric (pre-)anthraquinone building block atrochrysone to its 6-O-methyl ether torosachrysone by the O-methyltransferase (CoOMT1) precedes the regioselective homo-coupling to phlegmacin, catalyzed by an unspecific peroxygenase (CoUPO1). Our results revealed an unprecedented UPO-mediated unsymmetric OPC reaction, thereby expanding the biocatalytic portfolio of OPC-type reactions beyond the commonly reported enzymes. The findings highlight the pivotal role of OPC in natural processes, demonstrating that Basidiomycota employed peroxygenases to develop the ability to selectively couple aryls, distinct and convergent to any other group of organisms.

The Ketosynthase Domain Controls Chain Length in Mushroom Oligocyclic Polyketide Synthases.

The nonreducing iterative type I polyketide synthases (NR-PKSs) CoPKS1 and CoPKS4 of the webcap mushroom Cortinarius odorifer share 88 % identical amino acids. CoPKS1 almost exclusively produces a tricyclic octaketide product, atrochrysone carboxylic acid, whereas CoPKS4 shows simultaneous hepta- and octaketide synthase activity and also produces the bicyclic heptaketide 6-hydroxymusizin. To identify the region(s) controlling chain length, four chimeric enzyme variants were constructed and assayed for activity in Aspergillus niger as heterologous expression platform. We provide evidence that the ß-ketoacyl synthase (KS) domain determines chain length in these mushroom NR-PKSs, even though their KS domains differ in only ten amino acids. A unique proline-rich linker connecting the acyl carrier protein with the thioesterase domain varies most between these two enzymes but is not involved in chain length control.

Fast histological assessment of adipose tissue inflammation by label-free mid-infrared optoacoustic microscopy.

Conventional histology, as well as immunohistochemistry or immunofluorescence, enables the study of morphological and phenotypical changes during tissue inflammation with single-cell accuracy. However, although highly specific, such techniques require multiple time-consuming steps to apply exogenous labels, which might result in morphological deviations from native tissue structures. Unlike these techniques, mid-infrared (mid-IR) microspectroscopy is a label-free optical imaging method that retrieves endogenous biomolecular contrast without altering the native composition of the samples. Nevertheless, due to the strong optical absorption of water in biological tissues, conventional mid-IR microspectroscopy has been limited to dried thin (5-10 µm) tissue preparations and, thus, it also requires time-consuming steps-comparable to conventional imaging techniques. Here, as a step towards label-free analytical histology of unprocessed tissues, we applied mid-IR optoacoustic microscopy (MiROM) to retrieve intrinsic molecular contrast by vibrational excitation and, simultaneously, to overcome water-tissue opacity of conventional mid-IR imaging in thick (mm range) tissues. In this proof-of-concept study, we demonstrated application of MiROM for the fast, label-free, non-destructive assessment of the hallmarks of inflammation in excised white adipose tissue; i.e., formation of crown-like structures and changes in adipocyte morphology.

Unprecedented Mushroom Polyketide Synthases Produce the Universal Anthraquinone Precursor.

(Pre-)anthraquinones are widely distributed natural compounds and occur in plants, fungi, microorganisms, and animals, with atrochrysone (1) as the key biosynthetic precursor. Chemical analyses established mushrooms of the genus Cortinarius-the webcaps-as producers of atrochrysone-derived octaketide pigments. However, more recent genomic data did not provide any evidence for known atrochrysone carboxylic acid (4) synthases nor any other polyketide synthase (PKS) producing oligocyclic metabolites. Here, we describe an unprecedented class of non-reducing (NR-)PKS. In vitro assays with recombinant enzyme in combination with in vivo product formation in the heterologous host Aspergillus niger established CoPKS1 and CoPKS4 of C. odorifer as members of a new class of atrochrysone carboxylic acid synthases. CoPKS4 catalyzed both hepta- and octaketide synthesis and yielded 6-hydroxymusizin (6), along with 4. These first mushroom PKSs for oligocyclic products illustrate how the biosynthesis of bioactive natural metabolites evolved independently in various groups of life.

Directed evolution of biofuel-responsive biosensors for automated optimization of branched-chain alcohol biosynthesis.

The biosynthesis of short-chain alcohols is a carbon-neutral alternative to petroleum-derived production, but strain screening operations are encumbered by laborious analytics. Here, we built, characterized and applied whole cell biosensors by directed evolution of the transcription factor AlkS for screening microbial strain libraries producing industrially relevant alcohols. A selected AlkS variant was applied for in situ product detection in two screening applications concerning key steps in alcohol production. Further, the biosensor strains enabled the implementation of an automated, robotic platform-based workflow with data clustering, which readily allowed the identification of significantly improved strain variants for isopentanol production.