Tilo Lübken

Terpenes make up a structurally and functionally highly diverse class of natural products found across all living organisms. Pseudomonas species possess significant potential for the biosynthetic assembly of farnesyl pyrophosphate (FPP)-derived terpenes with unusual carbon skeletons. However, their structural and biosynthetic diversity has been largely unexplored. Here, we report the discovery of grimophan, a C₁₇ terpene featuring a rare deltacyclane skeleton. The compound was accessed by heterologous expression of the pgr biosynthetic gene cluster from Pseudomonas grimontii DSM 17515 in E. coli. The roles of the enzymes involved in grimophan biosynthesis were elucidated through in vitro reconstitution of the entire biosynthetic pathway. In-depth functional studies on the involved SAM-dependent methyltransferases led to the discovery of methyltransferase-like enzymes that do not perform functional group transfer, but rather significantly enhance the production titer of noncanonical terpenes. These enzymes thus constitute valuable tools for enhancing biotechnological production levels of noncanonical C₁₆ and C₁₇ terpenes. The function of the cognate terpene synthase PgrE was evaluated by targeted point mutations within the observed Asp-rich motif D₉₂DMPLG₉₇ to map the effects of active-site residues on product formation. These investigations facilitated redirecting product selectivity, leading to alternative products. Overall, our work sheds light on the general biosynthetic logic leading to noncanonical C₁₇ terpenes and provides a basis for their targeted discovery by genome mining and heterologous expression and for engineering C₁₇ terpene structural frameworks.

Silver antimony sulfide (AgSbS₂) has recently emerged as a promising semiconducting material for application in optoelectronics. Its nontoxicity, earth-abundant composition, high absorption coefficient, phase, and environmental stability make it particularly interesting for photovoltaic applications. This study presents a facile room-temperature synthesis of colloidal AgSbS₂ nanocrystals via cation exchange. The obtained AgSbS₂ nanocrystals show a unique postsynthetic size tunability with tunable optoelectronic properties while maintaining an ideal elemental composition. The great potential of these ternary nanocrystals is demonstrated by their integration into planar nanocrystal solar cells for the first time, in which the AgSbS₂ nanocrystals form a compact layer, reaching a promising power conversion efficiency of up to 1.99%, approaching the performance of bulk AgSbS₂ photovoltaic cells.

Terpenes are a structurally and functionally highly diverse class of natural products found across all living organisms. Pseudomonas species possess significant potential for the biosynthetic assembly of farnesyl pyrophosphate (FPP)-derived terpenes with unusual carbon skeletons. However, their structural and biosynthetic diversity is largely unexplored. Here, we report the discovery of grimophan, an unprecedented C17 terpene featuring a deltacyclane skeleton. The compound was accessed by heterologous expression of the pgr biosynthetic gene cluster from Pseudomonas grimontii DSM 17515 in E. coli. The roles of the enzymes involved in grimophan biosynthesis were elucidated through in vitro reconstitution of the entire biosynthetic pathway. In-depth functional studies on the involved SAM-dependent methyltransferases led to the discovery of novel methyltransferase-like enzymes that do not perform functional group transfer, but rather significantly enhance the production titer of non-canonical terpenes. These enzymes thus constitute valuable tools for increasing biotechnological production levels of terpenes. The function of the cognate terpene synthase PgrE was evaluated by targeted point mutations within the conserved Asp-rich motif D92DMPLG97 to map effects of active-site residues on product formation. These investigations facilitated redirecting product selectivity, leading to alternative products. Overall, our work sheds light on the general biosynthetic logic leading to non-canonical C17 terpenes, provides a basis for their targeted discovery by genome mining and heterologous expression, and for engineering C17 terpene structural frameworks.

Baeyer-Villiger monooxygenases (BVMOs) play crucial roles in the core-structure modification of natural products. They catalyze lactone formation by selective oxygen insertion into a carbon-carbon bond adjacent to a carbonyl group (Baeyer-Villiger oxidation, BVO). The homologous bacterial BVMOs, BraC and PxaB, thereby process bicyclic dihydroindolizinone substrates originating from a bimodular nonribosomal peptide synthetase (BraB or PxaA). While both enzymes initially catalyze the formation of oxazepine-dione intermediates following the identical mechanism, the final natural product spectrum diverges. For the pathway involving BraC, the exclusive formation of lipocyclocarbamates, the brabantamides, was reported. The pathway utilizing PxaB solely produces pyrrolizidine alkaloids, the pyrrolizixenamides. Surprisingly, replacing pxaB within the pyrrolizixenamide biosynthetic pathway by braC does not change the product spectrum to brabantamides. Factors controlling this product selectivity have remained elusive. In this study, we set out to solve this puzzle by combining the total synthesis of crucial pathway intermediates and anticipated products with in-depth functional in vitro studies on both recombinant BVMOs. This work shows that the joint oxazepine-dione intermediate initially formed by both BVMOs leads to pyrrolizixenamides upon nonenzymatic hydrolysis, decarboxylative ring contraction, and dehydration. Brabantamide biosynthesis is enzyme-controlled, with BraC efficiently transforming all the accepted substrates into its cognate final product scaffold. PxaB, in contrast, shows only considerable activity toward brabantamide formation for the substrate analog with a natural brabantamide-type side chain structure, revealing substrate-controlled product selectivity.

Daylight fluorescent paints are luminous colors that are increasingly used in contemporary art. The pigments consist of a synthetic resin in which fluorescent dyes and optical brighteners are embedded. In the recent years, several research articles have been published on the composition of daylight fluorescent pigments. Despite the growing research on the aging behavior of daylight fluorescent paints, little is known to date about the chemical processes involved in aging. In the research presented here, we used dialysis to separate the colorants from the resin. HPLC–HR-ESI–MS/MS was used to extend the elucidation of the dye composition. A variety of rhodamines and coumarins, an aminonaphthalimide dye and another optical brightener were determined. NMR was used to elucidate the structure of an additional hemicyanine dye not listed in the Colour Index. Furthermore, reference substances were artificially aged under visible light and UV radiation and the degradation products were analyzed accordingly. N-deethylation, hydroxylation and higher oxidation processes were found to be the main degradation pathways for all colorants. For most dyes and optical brighteners, there was no difference between aging under visible light and aging under UV radiation. When the results were checked on samples of aged paint mock-ups, it was found that only a few of the degradation products can still be detected in the case of very advanced aging even with the smallest sample quantities.

Parts of Salvia species such as its flowers and leaves are currently used as a culinary herb and for some medicinal applications. To distinguish the different sage extracts it is necessary to analyze their individual chemical compositions. Their characteristic compounds might be established as markers to differentiate between sage flowers and leaf extracts or to determine the manufacturing technology and storage conditions. Tri-p-coumaroylspermidine can be detected only in flowers and has been described here for Salvia and Lavandula species for the first time. Markers for oxidation processes are the novel compounds salviquinone A and B, which were generated from carnosol by contact with oxygen. Caffeic acid ethyl ester was established as an indirect marker for the usage of ethanol as extraction solvent. The compounds were identified by LC-QTOF-HRESIMS, LC-MS, NMR, IR, and single-crystal X-ray diffraction after isolation by semipreparative HPLC. Furthermore, sage flower resin showed interesting antibacterial in vitro activities against Gram-positive and Gram-negative bacteria.

The nematode Caenorhabditis elegans requires exogenous cholesterol to survive and its depletion leads to early developmental arrest. Thus, tight regulation of cholesterol storage and distribution within the organism is indispensable. Here, we present a novel class of C. elegans phosphorylated glycosphingolipids, phosphoethanolamine glucosylceramides (PEGCs), capable of rescuing larval arrest induced by sterol starvation. We describe the total synthesis of a major PEGC species and demonstrate that the PEGC synthetic counterpart suppresses the dauer-constitutive phenotype of Niemann-Pick C1 (NPC1) and DAF-7/TGF-β mutant worms caused by impaired intracellular sterol trafficking. PEGC biosynthesis depends on functional NPC1 and TGF-β, indicating that these proteins control larval development at least partly through PEGC. Furthermore, glucosylceramide deficiency dramatically reduced PEGC amounts. However, the resulting developmental arrest could be rescued by oversaturation of food with cholesterol. Taken together, these data show that PEGC is essential for C. elegans development through its regulation of sterol mobilization.

We investigated the three soft corals Sarcophyton stellatum, Capnella fungiformis and Lobophytum crassum and the sponge Pseudoceratina arabica, which have been collected at the coast of Madagascar. In addition to previously known marine natural products, S. stellatum provided the new (+)-enantiomer of the cembranoid (1E,3E,11E)-7,8-epoxycembra-1,3,11,15-tetraene (2). Capnella fungiformis afforded three new natural products, ethyl 5-[(1E,5Z)-2,6-dimethylocta-1,5,7-trienyl]furan-3-carboxylate (6), ethyl 5-[(1E,5E)-2,6-dimethylocta-1,5,7-trienyl]furan-3-carboxylate (7) and the diepoxyguaiane sesquiterpene oxyfungiformin (9a). The extracts of all three soft corals exhibited moderate activities against the malarial parasite Plasmodium falciparum. Extracts of the sponge Pseudoceratina arabica proved to be very active against a series of Gram-positive and Gram-negative bacteria.

New Zealand manuka honey is well-known for its unique antibacterial activity. Due to its high price and limited availability, this honey is often subject to honey fraud. Two pteridine derivatives, 3,6,7-trimethyl-2,4(1H,3H)-pteridinedione and 6,7-dimethyl-2,4(1H,3H)-pteridinedione, have now been identified in New Zealand manuka honey. Their structures were elucidated by LC-QTOF-HRMS, NMR, and single-crystal X-ray diffraction after isolation via semi-preparative HPLC. Their marker potential for authentic manuka honey was proved as both substances were neither detectable in the pollen-identical kanuka honey nor in the nine other kinds of monofloral New Zealand honey analyzed (clover, forest, kamahi, pohutukawa, rata, rewarewa, tawari, thyme, and vipers bugloss). The fluorescence property of the pteridine derivatives can be used as an easy and fast TLC screening method for the authentication of genuine manuka honey. 6,7-dimethyl-2,4(1H,3H)-pteridinedione has been described for the first time.