Eric J. N. Helfrich’s research while affiliated with Goethe University Frankfurt and other places

This review explores recent insights into the roles and enzymatic mechanisms of canonical and non-canonical tailoring enzymes in shaping and modifying terpene hydrocarbon scaffolds during bacterial terpene biosynthesis.

Specialized or secondary metabolites are small molecules of biological origin, often showing potent biological activities with applications in agriculture, engineering and medicine. Usually, the biosynthesis of these natural products is governed by sets of co-regulated and physically clustered genes known as biosynthetic gene clusters (BGCs). To share information about BGCs in a standardized and machine-readable way, the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard and repository was initiated in 2015. Since its conception, MIBiG has been regularly updated to expand data coverage and remain up to date with innovations in natural product research. Here, we describe MIBiG version 4.0, an extensive update to the data repository and the underlying data standard. In a massive community annotation effort, 267 contributors performed 8304 edits, creating 557 new entries and modifying 590 existing entries, resulting in a new total of 3059 curated entries in MIBiG. Particular attention was paid to ensuring high data quality, with automated data validation using a newly developed custom submission portal prototype, paired with a novel peer-reviewing model. MIBiG 4.0 also takes steps towards a rolling release model and a broader involvement of the scientific community. MIBiG 4.0 is accessible online at https://mibig.secondarymetabolites.org/.

Many complex terpenoids, predominantly isolated from plants and fungi, show drug-like physicochemical properties. Recent advances in genome mining revealed actinobacteria as an almost untouched treasure trove of terpene biosynthetic gene clusters (BGCs). In this study, we characterized a terpene BGC with an unusual architecture. The selected BGC includes, among others, genes encoding a terpene cyclase fused to a truncated reductase domain and a cytochrome P450 monooxygenase (P450) that is split over three gene fragments. Functional characterization of the BGC in a heterologous host led to the identification of several new members of the trans-eunicellane family of diterpenoids, the euthailols, that feature unique oxidation patterns. A combination of bioinformatic analyses, structural modeling studies, and heterologous expression revealed a dual function of the pathway-encoded hypothetical protein that acts as an isomerase and an oxygenase. Moreover, in the absence of other tailoring enzymes, a P450 hydroxylates the eunicellane scaffold at a position that is not modified in other eunicellanes. Surprisingly, both the modifications installed by the hypothetical protein and one of the P450s exhibit partial redundancy. Bioactivity assays revealed that some of the euthailols show growth inhibitory properties against Gram-negative ESKAPE pathogens. The characterization of the euthailol BGC in this study provides unprecedented insights into the partial functional redundancy of tailoring enzymes in complex diterpenoid biosynthesis and highlights hypothetical proteins as an important and largely overlooked family of tailoring enzymes involved in the maturation of complex terpenoids.

Background Sequencing and annotating genomes of non-model organisms helps to understand genome architecture, the genetic processes underlying species traits, and how these genes have evolved in closely-related taxa, among many other biological processes. However, many metazoan groups, such as the extremely diverse molluscs, are still underrepresented in the number of sequenced and annotated genomes. Although sequencing techniques have recently improved in quality and quantity, molluscs are still neglected due to difficulties in applying standardized protocols for obtaining genomic data. Results In this study, we present the chromosome-level genome assembly and annotation of the sacoglossan sea slug species Elysia timida, known for its ability to store the chloroplasts of its food algae. In particular, by optimizing the long-read and chromosome conformation capture library preparations, the genome assembly was performed using PacBio HiFi and Arima HiC data. The scaffold and contig N50s, at 41.8 Mb and 1.92 Mb, respectively, are approximately 30-fold and fourfold higher compared to other published sacoglossan genome assemblies. Structural annotation resulted in 19,904 protein-coding genes, which are more contiguous and complete compared to publicly available annotations of Sacoglossa with respect to metazoan BUSCOs. We found no evidence for horizontal gene transfer (HGT), i.e. no photosynthetic genes encoded in the sacoglossan nucleus genome. However, we detected genes encoding polyketide synthases in E. timida, indicating that polypropionates are produced. HPLC–MS/MS analysis confirmed the presence of a large number of polypropionates, including known and yet uncharacterised compounds. Conclusions We can show that our methodological approach helps to obtain a high-quality genome assembly even for a "difficult-to-sequence" organism, which may facilitate genome sequencing in molluscs. This will enable a better understanding of complex biological processes in molluscs, such as functional kleptoplasty in Sacoglossa, by significantly improving the quality of genome assemblies and annotations.

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) constitute a diverse class of natural products. Atropopeptides are a recent addition to the class. Here we developed AtropoFinder, a genome mining algorithm to chart the biosynthetic landscape of the atropopeptides. AtropoFinder identified more than 650 atropopeptide biosynthetic gene clusters (BGCs). We pinpointed crucial motifs and residues in leader and core peptide sequences, prompting a refined definition of the atropopeptide RiPP family. Our study revealed that a substantial subset of atropopeptide BGCs harbors multiple tailoring genes, thus suggesting a broader structural diversity than previously anticipated. To verify AtropoFinder, we heterologously expressed four atropopeptide BGCs, which resulted in the identification of novel atropopeptides with varying peptide lengths, number and types of modifications. Atropopeptides serve as a proof-of-principle for the versatile genome mining approach developed in this study that can be repurposed for the identification of RiPP and other BGCs that currently evade detection.

Natural products are molecules that fulfil a range of important ecological functions. Many natural products have been exploited for pharmaceutical and agricultural applications. In contrast to many other specialised metabolites, the products of modular nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) systems can often (partially) be predicted from the DNA sequence of the biosynthetic gene clusters. This is because the biosynthetic pathways of NRPS and PKS systems adhere to consistent rulesets. These universal biosynthetic rules can be leveraged to generate biosynthetic models of biosynthetic pathways. While these principles have been largely deciphered, software that leverages these rules to automatically generate visualisations of biosynthetic models has not yet been developed. To enable high-quality automated visualisations of natural product biosynthetic pathways, we developed RAIChU (Reaction Analysis through Illustrating Chemical Units), which produces depictions of biosynthetic transformations of PKS, NRPS, and hybrid PKS/NRPS systems from predicted or experimentally verified module architectures and domain substrate specificities. RAIChU also boasts a library of functions to perform and visualise reactions and pathways whose specifics (e.g., regioselectivity, stereoselectivity) are still difficult to predict, including terpenes, ribosomally synthesised and posttranslationally modified peptides and alkaloids. Additionally, RAIChU includes 34 prevalent tailoring reactions to enable the visualisation of biosynthetic pathways of fully maturated natural products. RAIChU can be integrated into Python pipelines, allowing users to upload and edit results from antiSMASH, a widely used BGC detection and annotation tool, or to build biosynthetic PKS/NRPS systems from scratch. RAIChU’s cluster drawing correctness (100%) and drawing readability (97.66%) were validated on 5000 randomly generated PKS/NRPS systems, and on the MIBiG database. The automated visualisation of these pathways accelerates the generation of biosynthetic models, facilitates the analysis of large (meta-) genomic datasets and reduces human error. RAIChU is available at https://github.com/BTheDragonMaster/RAIChU and https://pypi.org/project/raichu. Scientific contribution RAIChU is the first software package capable of automating high-quality visualisations of natural product biosynthetic pathways. By leveraging universal biosynthetic rules, RAIChU enables the depiction of complex biosynthetic transformations for PKS, NRPS, ribosomally synthesised and posttranslationally modified peptide (RiPP), terpene and alkaloid systems, enhancing predictive and analytical capabilities. This innovation not only streamlines the creation of biosynthetic models, making the analysis of large genomic datasets more efficient and accurate, but also bridges a crucial gap in predicting and visualising the complexities of natural product biosynthesis.

Species of the genus Blautia are not only abundant in the human gut but also contribute to human well‐being. Our study demonstrates that the gut acetogen Blautia schinkii can grow on myo ‐inositol. We identified the pathway of myo ‐inositol degradation through a combination of physiological and biochemical studies, genome‐wide expression profiling and homology searches. Initially, myo ‐inositol is oxidized to 2‐keto‐ myo ‐inositol. This compound is then metabolized by a series of enzymes – a dehydratase, hydrolase, isomerase and kinase – to form 2‐deoxy‐5‐keto‐ d ‐gluconic acid 6‐phosphate. This intermediate is split by an aldolase into malonate semialdehyde and dihydroxyacetone phosphate, which is an intermediate of the Embden–Meyerhof–Parnas pathway. This pathway leads to the production of pyruvate and, subsequently, acetate. Concurrently, malonate semialdehyde is reduced to 3‐hydroxypropionate (3‐HP). The genes responsible for myo ‐inositol degradation are clustered on the genome, except for the gene encoding the aldolase. We identified the putative aldolase Fba_3 and 3‐HP dehydrogenase Adh1 encoding genes bioinformatically and verified them biochemically using enzyme assays with heterologously produced and purified protein. The major fermentation end products were 3‐HP and acetate, produced in similar amounts. The production of the unusual fermentation end product 3‐HP is significant not only for human health but also for the potential bioindustrial production of this highly desired compound.

Background: Sequencing and annotating genomes of non-model organisms helps to understand genome architecture, the genetic processes underlying species traits, and how these genes have evolved in closely-related taxa, among many other biological processes. However, many metazoan groups, such as the extremely diverse molluscs, are still underrepresented in the number of sequenced and annotated genomes. Although sequencing techniques have recently improved in quality and quantity, molluscs are still neglected due to difficulties in applying standardized protocols for obtaining genomic data. Results: In this study, we present the chromosome-level genome assembly and annotation of the marine sacoglossan species Elysia timida, known for its ability to store the chloroplasts of its food algae. In particular, by optimizing the Long-read and chromosome conformation capture library preparations, the genome assembly was performed using PacBio HiFi and Arima HiC data. The scaffold and contig N50s, at 41.8 Mb and 1.92 Mb, respectively, are 100-fold and 4-fold higher compared to other published sacoglossan genome assemblies. Structural annotation resulted in 19,904 protein-coding genes, which are more contiguous and complete compared to publicly available annotations of Sacoglossa. We detected genes encoding polyketide synthases in E. timida, indicating that polypropionates are produced. HPLC-MS/MS analysis confirmed the presence of a large number of polypropionates, including known and yet uncharacterised compounds. Conclusions: We can show that our methodological approach helps to obtain a high-quality genome assembly even for a "difficult-to-sequence" organism, which may facilitate genome sequencing in molluscs. This will enable a better understanding of complex biological processes in molluscs, such as functional kleptoplasty in Sacoglossa, by significantly improving the quality of genome assemblies and annotations.

Bacteria produce natural products to interact with their environment. These structurally diverse small molecules exhibit various bioactivities and have been exploited for the treatment of many diseases. The discovery pace of truly novel natural products using traditional methods has markedly declined. As an alternative, sophisticated bioinformatic tools have been developed that leverage genome sequence information for the targeted discovery of bioactive compounds. To chart microbial biosynthetic dark matter and identify non-canonical natural product biosynthetic pathways that escape unrecognised by current bioinformatic tools, artificial intelligence has recently been introduced into the genome mining process, holding promise to expand natural product chemical and biosynthetic space.

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) constitute a diverse class of natural products. Atropopeptides are a recent addition to the fast-growing number of RiPP families. Characterized members of the peptide family feature a particular intricate three-dimensional shape. Here we developed AtropoFinder, a machine learning-based algorithm to chart the biosynthetic landscape of the atropopeptides. AtropoFinder identified more than 650 atropopeptide biosynthetic gene clusters (BGCs). Through bioinformatics and modeling analyses, we pinpointed crucial motifs and residues in leader and core peptide sequences, prompting a refined definition of the atropopeptide RiPP family. Our study revealed that a substantial subset of atropopeptide BGCs harbors multiple tailoring genes, thus suggesting a broader structural diversity than previously anticipated. To verify AtropoFinder, we heterologously expressed four atropopeptide BGCs, which resulted in the identification of novel atropopeptides with varying peptide lengths, number and type of modifications. Most notably, our study resulted in the characterization of an atropopeptide that is more extensively modified than previously identified members, resulting in an even more rigid 3-dimensional shape. Moreover, one characterized atropopeptide BGC encoding a single P450 is involved in the biosynthesis of two peptides with the same sequence but distinct and non-overlapping modification patterns. This work expands the atropopeptide chemical space, advances our understanding of atropopeptide biosynthesis and underscores the potential of machine learning in uncovering the uncharted biosynthetic diversity encoded in RiPP biosynthetic blueprints.