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The new farnesyl pyrophosphate (FPP) derivative with a shifted olefinic double bond from C6‐C7 to C7‐C8 is accepted and converted by the sesquiterpene cyclases protoilludene synthase (Omp7) as well as viridiflorene synthase (Tps32). In both cases, a so far unknown germacrene derivative was found to be formed, which we name “germacrene F”. Both cases are examples in which a modification around the central olefinic double bond in FPP leads to a change in the mode of initial cyclization (from 1→11 to 1→10). For Omp7 a rationale for this behaviour was found by carrying out molecular docking studies. Temperature‐dependent NMR experiments, accompanied by NOE studies, show that germacrene F adopts a preferred mirror‐symmetric conformation with both methyl groups oriented in the same directions in the cyclodecane ring.

Silyl ether protecting groups are important tools in organic synthesis, ensuring selective reactions of hydroxyl functional groups. Enantiospecific formation or cleavage could simultaneously enable the resolution of racemic mixtures and thus significantly increase the efficiency of complex synthetic pathways. Based on reports that lipases, which today are already particularly important tools in chemical synthesis, can catalyze the enantiospecific turnover of trimethylsilanol (TMS)-protected alcohols, the goal of this study was to determine the conditions under which such a catalysis occurs. Through detailed experimental and mechanistic investigation, we demonstrated that although lipases mediate the turnover of TMS-protected alcohols, this occurs independently of the known catalytic triad, as this is unable to stabilize a tetrahedral intermediate. The reaction is essentially non-specific and therefore most likely completely independent of the active site. This rules out lipases as catalysts for the resolution of racemic mixtures alcohols through protection or deprotection with silyl groups.

Macrocyclization of peptides is typically used to fix specific bioactive conformations and improve their pharmacological properties. Recently, macrobicyclic peptides have received special attention owing to their capacity to mimic protein structures or be key components of peptide-drug conjugates. Here, we describe the development of novel synthetic strategies for two distinctive types of peptide macrobicycles. A multicomponent macrocyclo-dimerization approach is introduced for the production of interconnected β-turns, allowing two macrocyclic rings to be formed and dimerized in one pot. Also, an on-resin double stapling strategy is described for the assembly of lactam-bridged macrobicycles with stable tertiary folds.

The preprophase band (PPB) is a transient cytokinetic structure that marks the future division plane at the onset of mitosis. The PPB forms a dense cortical ring of mainly microtubules, actin filaments, endoplasmic reticulum, and associated proteins that encircles the nucleus of mitotic cells. After PPB disassembly, the positional information is preserved by the cortical division zone (CDZ). The formation of the PPB and its contribution to timely CDZ set-up involves activities of functionally distinct microtubule-associated proteins (MAPs) that interact physically and genetically to support robust division plane orientation in plants. Recent studies identified two types of plant-specific MAPs as key regulators of PPB formation, the TON1 RECRUITMENT MOTIF (TRM) and IQ67 DOMAIN (IQD) families. Both families share hallmarks of disordered scaffold proteins. Interactions of IQDs and TRMs with multiple binding partners, including the microtubule severing KATANIN1, may provide a molecular framework to coordinate PPB formation, maturation, and disassembly.

Terpene synthase-mediated biotransformation of eleven synthetic sulfur- or oxygen-containing non-natural prenyl diphosphates resulted in the formation of five novel terpenoids and analogues. Uniquely, they trap intermediate steps and form heterocycles or compounds with alkyne side chains. Computational modelling differentiates convertible from inconvertible substrates and thereby provides an understanding of the detailed molecular mechanism of terpene cyclases. Two terpene cyclases were used as biocatalytic tool, namely, limonene synthase from Cannabis sativa (CLS) and 5-epi-aristolochene synthase (TEAS) from Nicotiana tabacum. They showed significant substrate flexibility towards non-natural prenyl diphosphates to form novel terpenoids, including core oxa- and thia-heterocycles and alkyne-modified terpenoids. We elucidated the structures of five novel monoterpene-analogues and a known sesquiterpene-analogue. These results reflected the terpene synthases′ ability and promiscuity to broaden the pool of terpenoids with structurally complex analogues. Docking studies highlight an on-off conversion of the unnatural substrates.

4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H), a flavin-dependent monooxygenase from E. coli that catalyzes the hydroxylation of monophenols to catechols, was modified by rational re-design to convert also more bulky substrates, especially phenolic natural products like phenylpropanoids, flavones or coumarins. Selected amino acid positions in the binding pocket of 4HPA3H were exchanged by residues from the homologous protein from Pseudomonas aeruginosa, yielding variants with improved conversion of spacious substrates such as the flavonoid naringenin or the alkaloid mimetic 2-hydroxycarbazole. Reactions were followed by an adapted Fe(III)-catechol chromogenic assay selective for the products. Especially substitution of the residue Y301 facilitated modulation of substrate specificity: introduction of non-aromatic but hydrophobic (iso)leucine resulted in the preference of the substrate ferulic acid (having a guaiacyl (guajacyl) moiety, part of the vanilloid motif) over unsubstituted monophenols. The in vivo (whole-cell biocatalysts) and in vitro (three-enzyme cascade) transformations of substrates by 4HPA3H and its optimized variants was strictly regiospecific and proceeded without generation of by-products.

Plant specialized metabolites are often synthesized and stored in dedicated morphological structures such as glandular trichomes, resin ducts, or laticifers where they accumulate in large concentrations. How this high productivity is achieved is still elusive, in particular, with respect to the interface between primary and specialized metabolism. Here, we focus on glandular trichomes to survey recent progress in understanding how plant metabolic cell factories manage to balance homeostasis of essential central metabolites while producing large quantities of compounds that constitute a metabolic sink. In particular, we review the role of gene duplications, transcription factors and photosynthesis.

Type 1 secretion systems (T1SS) have a relatively simple architecture compared to other classes of secretion systems and therefore, are attractive to be optimized by protein engineering. Here, we report a KnowVolution campaign for the hemolysin (Hly) enhancer fragment, an untranslated region upstream of the hlyA gene, of the hemolysin T1SS of Escherichia coli to enhance its secretion efficiency. The best performing variant of the Hly enhancer fragment contained five nucleotide mutations at  five positions (A30U, A36U, A54G, A81U, and A116U) resulted in a 2-fold increase in the secretion level of a model lipase fused to the secretion carrier HlyA1. Computational analysis suggested that altered affinity to the generated enhancer fragment towards the S1 ribosomal protein contributes to the enhanced secretion levels. Furthermore, we demonstrate that involving a native terminator region along with the generated Hly enhancer fragment increased the secretion levels of the Hly system up to 5-fold.

Human drug‐metabolizing cytochrome P450 monooxygenases (CYPs) have enormous substrate promiscuity; this makes them promising tools for the expansion of natural product diversity. Here, we used CYP3A4 for the targeted diversification of a plant biosynthetic route leading to monoterpenoid indole alkaloids. In silico, in vitro and in planta studies proved that CYP3A4 was able to convert the indole alkaloid vinorine into vomilenine, the former being one of the central intermediates in the ajmaline pathway in the medicinal plant Rauvolfia serpentina (L.) Benth. ex Kurz. However, to a much larger extent, the investigated conversion yielded vinorine (19R ,20R)‐epoxide, a new metabolite with an epoxide functional group that is rare for indole alkaloids. The described work represents a successful example of combinatorial biosynthesis towards an increase in biodiversity of natural metabolites. Moreover, characterisation of the products of the in vitro and in planta transformation of potential pharmaceuticals with human CYPs might be indicative of the route of their conversion in the human organism.