Martin Grininger

Atropopeptides are a family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) that share conserved cross-link-forming amino acids in their core peptides, yet adopt architectures ranging from simple monocyclic to complex polycyclic structures with intriguing three-dimensional shapes. These complex architectures arise from multiple C–C and C–N cross-links that are installed by a single cytochrome P450 enzyme, but the factors governing these divergent cross-linking patterns and resulting structural complexities have remained elusive. Recent studies on RiPP biosynthesis have revealed a form of substrate-controlled catalysis, in which the precursor sequence, rather than the modifying enzyme, dictates the reaction course. Here, we describe a distinct form of substrate-controlled catalysis in atropopeptide biosynthesis, demonstrating that core peptide length, rather than sequence, determines the cross-linking pattern installed by an atropopeptide-forming cytochrome P450. We further uncover an unusual proteolytic excision of an internal amino acid in mature atropopeptides, generating peptides that spontaneously convert into pyrroloindoline-containing diketopiperazines (DKPs) and thereby effecting a nonenzymatic scaffold-hop from atropopeptides to DKP-derived alkaloids, for which we provide mechanistic insight. Both the atropopeptide and the corresponding DKP exhibit antiviral activity, and their structural diversity can be further expanded through precursor engineering. Our work reveals substrate length as a key determinant of atropopeptide complexity, provides insights into the catalysis of a multicross-link-forming P450, and establishes an unprecedented route connecting atropopeptides to pyrroloindoline DKP alkaloids.

Polyketides constitute a large class of natural products with important biological activities and applications such as antibiotics, antitumor agents, pesticides, and pigments. Their biosynthesis is catalyzed by polyketide synthases (PKSs) which are multidomain enzymes evolutionarily related to fatty acid synthases (FASs). Despite their close homology in structure and the chemistry they perform, FASs and PKSs differ fundamentally in their catalytic programming: FASs run fully reducing elongation reactions to yield saturated fatty acids, while iterative PKSs execute reductions just in selected cycles, generating complex oxidized compounds. In this study, we aimed at engineering the metazoan FAS in its ketoreduction (KR) domain to switch from fully reducing to a nonreducing mode during chain elongation. Guided by recent insights into KR programming, we incorporated a helix into metazoan FAS, which is found in KRs from iterative PKSs and type II FASs with chain length programming. These FAS variants initially catalyze complete fatty acid cycles but lose the ability of β ‐keto reduction in later elongation rounds, producing intermediates that spontaneously cyclize to pyrone products. Finally, our study provides valuable insight into the mechanism of KR catalysis identifying another amino acid next to the active tyrosine which is capable for intermediate protonation.

Metazoan fatty acid (FA) synthases (mFASs) facilitate the de novo synthesis of C16- and C18-FAs through iterative extensions within the FA cycle and hydrolytic release. Here we re-engineer mFAS to fine-tune the interplay between FA extension and FA hydrolytic release for the targeted production of short- and medium-chain fatty acids. Single amino acid exchanges in the ketosynthase domain can redirect FA product profiles from predominantly C8 (G113W) to C8/C10 (G113F) and C12/C14 (G113M). Integration of a thioreductase domain enables the production of medium-chain fatty aldehydes and alcohols. We apply our approach for controlling chain length in FA biosynthesis to the microbial production of C10- and C12-FAs, translate it into a yeast cell factory and achieve C10/C12-FAs titers of 674 mg l ⁻¹ and 67% purity of total free FAs. Our work demonstrates a modular platform for programmable FA synthesis and paves the way toward sustainable bioproduction of valuable oleochemicals.

Kurzzusammenfassung: Die Kompartimentierung biosynthetischer Wege stellt ein zentrales Ziel des Proteinengineerings dar, insbesondere in der synthetischen Biologie und bei der Optimierung komplexer Stoffwechselwege. Hier kann die Kompartimentierung die katalytische Effizienz steigern, reaktive Zwischenprodukte stabilisieren, die Bildung von Nebenprodukten verringern und darüber hinaus durch die Koordination mehrstufiger Reaktionen eine erhöhte synthetische Komplexität ermöglichen. In dieser Studie wurde eine chemo‐enzymatische Plattform zur Biosynthese von 2‐Pyronen – insbesondere Styrylpyrone und Hispidin – in einem Multienzym etabliert, das auf einer nicht‐reduzierenden (nr) Variante der murinen Fettsäuresynthase (FAS) basiert, welche natürlicherweise Palmitinsäure produziert. Durch die Einführung von zwei gezielten Aminosäuresubstitutionen in der Ketosynthase‐Domäne (KS), wurde die nrFAS‐vermittelte Synthese von Styrylpyronen aus nicht‐kanonischen Substraten, einschließlich halogenierter Derivate, verbessert. Das angepasste Enzym zeigte eine 66‐fach höhere katalytische Aktivität im Vergleich zur nicht‐mutierten nrFAS und übertraf dabei die Styrylpyronsynthase des Kavalacton‐Biosynthesewegs aus Piper methysticum . Darüber hinaus wurde ein 4‐Cumarat‐Ligase (4CL1) Lademodul mithilfe des SpyTag/SpyCatcher‐Systems in das Kompartiment integriert, wodurch die Aktivierung und direkte Übertragung von Zimtsäurederivaten auf die nrFAS ermöglicht wurde. Die resultierenden Styrylpyrone dienen als direkte Vorläufer pharmakologisch aktiver Kavalactone, während Hispidin als Vorläufer der Pilz‐Biolumineszenz fungiert.

Compartmentalizing biosynthetic pathways is a key objective in protein engineering, particularly in synthetic biology and metabolic engineering. It can improve catalytic efficiency, stabilize reactive intermediates, reduce by‐product formation, and, beyond these advantages, enable synthetic complexity by coordinating multistep pathways. In this study, we established a chemoenzymatic platform for producing 2‐pyrones—specifically styrylpyrones and hispidin—within a multienzyme based on a non‐reducing (nr) variant of the murine fatty acid synthase (FAS), which naturally produces palmitic acid. By introducing two amino acid substitutions in the ketosynthase (KS) domain, we enhanced the nrFAS‐mediated synthesis of styrylpyrones from non‐native substrates, including halogenated derivatives. The engineered enzyme exhibited a 66‐fold increase in activity compared to the non‐mutated nrFAS, surpassing the styrylpyrone synthase of the kavalactone pathway in Piper methysticum . Additionally, we integrated a 4‐coumarate ligase (4CL1) loading module into the compartment using the SpyTag/SpyCatcher system, enabling the activation and direct transfer of cinnamic acid derivatives to the nrFAS. The resulting styrylpyrones are direct precursors of pharmaceutically active kavalactones, while hispidin serves as the precursor of fungal bioluminescence.

Polyketides constitute a large class of natural products with important biological activities and applications such as antibiotics, antitumor agents, pesticides, and pigments. Their biosynthesis is catalyzed by polyketide synthases (PKSs) which are multi-domain enzymes evolutionarily related to fatty acid synthases (FASs). Despite their close homology in structure and the chemistry they perform, FASs and PKSs differ fundamentally in their catalytic programming: FASs run fully reducing elongation reactions to yield saturated fatty acids, while iterative PKSs execute reductions just in selected cycles, generating complex oxidized compounds. In this study, we aimed at engineering the metazoan FAS in its KR domain to switch from fully reducing to a non-reducing mode during chain elongation. Guided by recent insights into KR programming, we incorporated a helix into metazoan FAS, which is found in KRs from iterative PKSs and type II FASs with chain length programming. These FAS variants initially catalyze complete fatty acid cycles but lose the ability of β-keto reduction in later elongation rounds, producing intermediates that spontaneously cyclize to pyrone products. Finally, our study provides valuable insight into the mechanism of KR catalysis identifying another amino acid next to the active tyrosine which is capable for intermediate protonation.

Branched‐chain fatty acids (BCFAs) exhibit enhanced oxidative stability, lower melting points, and reduced viscosity compared to straight‐chain fatty acids (StCFAs), making them valuable for biological systems and industrial applications. Previous studies have shown that metazoan fatty acid synthase (mFAS) can produce BCFAs through the incorporation of branched starter units and branched extender substrates such as methylmalonyl‐CoA (metmal‐CoA). However, the mechanistic and kinetic underpinnings of BCFA biosynthesis in metazoans remain poorly understood. Here, we address this by characterizing the kinetic parameters of mFAS‐catalyzed BCFA synthesis using NADPH consumption assays and analyzing the synthesized products via GC–MS. These experiments revealed a lower turnover number of mFAS with metmal‐CoA and a shift toward medium‐chain fatty acids compared to the native StCFA biosynthesis with malonyl‐CoA. The ketoacyl synthase (KS) kinetic measurements revealed low elongation rates, indicating that the KS domain dictates the substrate specificity and speed of BCFA production. To gain a better understanding of the reaction mechanism, we performed molecular dynamic simulations, which revealed key KS: substrate interactions by conserved threonine residues. These insights support a refined decarboxylative condensation mechanism and illuminate how substrate specificity arises within mFAS. These findings offer a foundation for future protein engineering and inhibitor design strategies.