Biology subjects

Haslinger, K.

Publications and source records attributed to Haslinger, K..

16 recordsLinked to original sources

bioRxiv2025

Integrating genomics and metabolomics to accelerate the discovery of anti-MRSA natural products from the endophytic fungus Neocucurbitaria sp. VM-36

Endophytic fungi in medicinal plants are a rich source of bioactive natural products. Herein, we performed a comprehensive genomic and metabolic analysis of an uncharacterized endophytic fungus Neocucurbitaria sp. VM-36. Whole-genome sequencing and comparative analysis of the encoded biosynthetic gene clusters with six Cucurbitariaceae strains predicted its potential to produce compounds related to griseofulvin, usnic acid, hypothemycin, and phomasetin. Untargeted metabolomics confirmed several of these predictions with the presence of phomasetin analogs and isousnic acid, and uncovered a diverse range of other secondary metabolites, including specialized lipids, amino acids, and peptides, such as cyclic hexapeptides. We successfully isolated the main compound (1), a phomasetin analog, and show that it has bactericidal activity against different methicillin-resistant and -sensitive Staphylococcus aureus strains comparable in strength to vancomycin and daptomycin. Checkerboard assays with these compounds revealed mostly indifferent interactions. These findings demonstrate the antibacterial potential of compound 1 and Neocucurbitaria sp. VM-36. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/681339v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@19326aeorg.highwire.dtl.DTLVardef@1d078adorg.highwire.dtl.DTLVardef@1a2ba46org.highwire.dtl.DTLVardef@737ed1_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

De novo biosynthesis of alpinetin enhanced by directed evolution of 5-O-methyltransferase

Alpinetin ((2S)-7-Hydroxy-5-methoxyflavan-4-one) is a natural flavonoid found in various medicinal herbs and is frequently used in Chinese patent medicines. It exhibits a wide range of bioactivities, including anti-inflammatory, cardiovascular protective, lung protective, antiviral, hepatoprotective, and antitumor effects. Alpinetin features a 5-methyl group on the A ring, a rare characteristic among methylated flavonoids. The limited abundance of Alpinetin in plant biomass, its laborious extraction and purification from this biomass, and the toxicity and lack of regio- and chemoselectivity in organic synthesis render its biosynthesis in engineered microbes an attractive alternative. In this study, we aimed to achieve the de novo biosynthesis of alpinetin in Escherichia coli. In a series of optimization steps, we varied the selection of pathway enzymes, plasmid configurations, medium composition, and fermentation strategies. Lastly, we applied a directed evolution campaign to the 5-O-methyltransferase to successfully enable the de novo biosynthesis of alpinetin in E. coli for the first time. Moreover, we demonstrated the ability of O-methyltransferase to methylate a broad range of flavonoid substrates, leading to the production of valuable O-methylated flavonoids. Our study represents the first example of alpinetin biosynthesis in a heterologous host and paves the way to produce other valuable O-methylated flavonoids enzymatically.

biochemistry↗

bioRxiv2025

De novo whole genome assembly of the globally invasive green shore crab Carcinus maenas (Linnaeus, 1758) via long-read Oxford Nanopore MinION sequencing

Invasive species are rapidly reshaping aquatic ecosystems worldwide at an accelerating pace, with profound ecological and economic impacts. Many crustacean species have demonstrated invasive potential or are already well-established invaders. The green shore crab, Carcinus maenas, native to Europe and North Africa, is one of the most successful global marine invaders and is now present on six continents. Although the role of genomics in invasion science is increasingly recognized, genomic resources for brachyuran crabs remain limited, including the notable absence of a reference genome for C. maenas. Here we report on a de novo whole genome assembly of C. maenas via long-read Oxford Nanopore Technology sequencing. The assembly spans 1.09 Gbp across 21,887 scaffolds (N50 = 15 Mbp) with a BUSCO completeness of 98.4%, providing a high-quality resource for future genomic analyses. Additionally, we provide a detailed protocol for obtaining high-quality DNA to successfully sequence brachyuran crabs using a long-read approach, including strategies to address nanopore blockage issues. This new resource expands available genomic data for the species-rich infraorder Brachyura, and provides a valuable foundation for understanding the genetic factors underlying the global invasion success of C. maenas, supporting future research in marine invasion genomics.

genomics↗

bioRxiv2024

The endophytic fungus Cosmosporella sp. VM-42 from Vinca minor is a source of bioactive compounds with potent activity against drug-resistant bacteria

Medicinal plants serve as valuable resources for the isolation of endophytic fungi. Vinca minor is a well-known producer of important vinca alkaloids and emerges as a promising source of endophytic fungi with antibacterial potential and biosynthetic capacity. In this study, we isolated an endophytic fungus from V. minor and identified it as Cosmosporella sp. VM-42. To date, relatively little is known about this fungal genus. The ethyl acetate extract of this isolate selectively inhibited Gram-positive bacteria, such as methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA). Therefore, we isolated the most abundant compound from the crude extract and identified it as nectriapyrone with MIC and MBC values ranging from 125 to 62.5 {micro}g/mL against MSSA and MRSA strains. We further sequenced and annotated the 39.07 Mb genome of the isolate, revealing that it encodes 9,842 protein-coding genes, including 415 genes for carbohydrate-active enzymes and various biosynthetic gene clusters. Our untargeted metabolomic analysis shows that the fungus produces various secondary metabolites, including cyclodepsipeptides, dimeric naphtho-{gamma}-pyrones, and macrolactones, which are known to have antifungal and antibacterial activities. In addition, we used small-molecule epigenetic modulators to activate the expression of silent biosynthetic gene clusters to broaden the chemical profile of Cosmosporella sp. VM-42. Taken together, we provide a first systematic analysis of Cosmosporella sp. VM-42, and our results show that it is a promising source of compounds with pharmacological potential against drug resistant bacteria. HighlightsO_LIFirst comprehensive study of Cosmosporella sp. VM-42 by genomics and metabolomics C_LIO_LIThe fungus produces chemically-diverse secondary metabolites with antibacterial activity C_LIO_LINectriapyrone, the main compound, shows bactericidal activity against MSSA and MRSA C_LIO_LISmall-molecule epigenetic modulators trigger production of putatively new secondary metabolites. C_LIO_LIOther secondary metabolites of Cosmosporella may present novel bioactivities. C_LI

biochemistry↗

bioRxiv2024

A biocatalytic platform for the production of substituted 2-quinolones and (thio)coumarins

2-quinolones are privileged scaffolds for drug discovery that are relatively rare in nature. Here, we characterise two promiscuous fungal polyketide synthases AthePKS and FerePKS, which we had previously found to produce quinolones in vitro. We challenged the enzymes with several substituted anthranilic acid derivatives, revealing their ability to produce precursors of pharmaceutically relevant quinolones. We also discovered that AthePKS and FerePKS accept other 2-substituted benzoic acids, leading to the formation of coumarin and thiocoumarin scaffolds. We applied AthePKS in an artificial enzymatic cascade towards an antimicrobial 4-methoxy-1-methyl-2-quinolone and demonstrated its in vivo feasibility by successfully expressing the pathway in Escherichia coli. Lastly, we determined the crystal structure of AthePKS, suggesting hotspots for enhancing its catalytic efficiency by enzyme engineering. Our results provide a framework for further engineering of enzymatic routes towards privileged heteroaromatic scaffolds and derivatives thereof.

biochemistry↗

bioRxiv2024

Effective metabarcoding of endophytic fungi through host-exclusive primer design for nanopore long-read sequencing

Metabarcoding is a powerful tool to simultaneously identify multiple taxa within a habitat. However, its application to host-associated microbiomes is challenged by substantial co-amplification of host DNA. Here we developed a host-exclusive primer design workflow, to selectively generate amplicons from target taxa while excluding the host. This workflow is centered around a new computational tool, mbc-prime, that can generate a list of discriminative candidate primers and score them. We showcase the use of this tool in the design of primers for long-read metabarcoding of endophytic fungi in Vinca minor. Mbc-prime streamlines the design of fungus-specific primers, enabling efficient and plant-free amplification of fungal rDNA from mixed DNA samples. Our workflow can be used to study the composition of complex host-associated microbiomes. It should be universally applicable for the design of discriminative primers in a user-friendly and practical manner and thus be of use for various researchers in microbiome research.

molecular biology↗

bioRxiv2024

Unravelling the functional diversity of type III polyketide synthases in fungi

Type III polyketide synthases (T3PKSs) are enzymes that produce diverse compounds of ecological and clinical importance. While well-studied in plants and bacteria, only a handful of T3PKSs from fungi have been characterised to date. Here, we developed a comprehensive workflow for kingdom-wide characterisation of T3PKSs. Using publicly available genomes, we mined more than 1000 putative enzymes and analysed their active site architecture and genomic neighbourhood. From there, we selected 37 representative PKS candidates for cell-free expression and prototyping with a diverse set of Coenzyme A activated substrates, revealing unique patterns in substrate and cyclisation specificity, as well as the preferred number of malonyl-Coenzyme A extensions. Using the 341 enzyme-substrate pairs generated in this study, we trained a machine learning model to predict T3PKS substrate specificity and experimentally validated it with an extended panel of non-natural substrates. We anticipate that the model will be useful for in silico screening of T3PKSs, while the insight into the product scope of these enzymes offers interesting starting points for further exploration.

biochemistry↗

bioRxiv2024

Identification and partial reconstitution of the biosynthetic pathway of bioactive meroterpenoids from Hericium erinaceus (Lion's Mane mushroom).

Hericium erinaceus (Lions Mane mushroom) is widely consumed for its numerous reported benefits for brain health. A growing body of evidence suggests that these benefits are likely attributable to aromatics contained in its fruiting bodies, including the meroterpenoids hericenones. Here, we report the identification and reconstitution of the first two steps of the biosynthetic pathway of hericenones via heterologous expression of the PKS HerA and the carboxylic acid reductase HerB in Aspergillus oryzae. Furthermore, we investigate a putative prenyltransferase that might be responsible for the following biosynthetic step. Ongoing efforts to reconstitute the full pathway will enable large scale production of hericenones and other meroterpenoids in heterologous hosts.

microbiology↗

bioRxiv2024

Discovery and heterologous expression of functional 4-O-dimethylallyl-L-tyrosine synthases from lichen-forming fungi.

Fungal DMATS-type aromatic prenyltransferases are a family of biosynthetic enzymes that catalyze the prenylation of a range of aromatic substrates during the biosynthesis of bioactive indole alkaloids, diketopiperazines, and meroterpenoids. Together with their broad substrate scope and soluble nature, this makes DMATS-type prenyltransferases particularly adept for applications in biocatalysis, for example to derivatize aromatic drug leads and improve their bioactivity. Here, we investigated four putative DMATS-type prenyltransferases from lichen-forming fungi, an underexplored group of organisms that produce more than 1,000 unique metabolites. We were able to successfully express two functional lichen prenyltransferases in the heterologous host A. oryzae, which allowed us to identify them as 4-O-dimethylallyltyrosine synthases. Our extensive bioinformatic analysis shows that related lichen prenyltransferases are likely not active on indoles but rather on aromatic polyketides and phenylpropanoids, common metabolites in these organisms. Overall, our work not only provides new insights into fungal DMATS-type prenyltransferases at the family level, but it also enables future efforts aimed at identifying new candidates for biocatalytic transformations of aromatic compounds.

microbiology↗

bioRxiv2023

Genome sequencing and molecular networking analysis of the wild fungus Anthostomella pinea reveal its ability to produce a diverse range of secondary metabolites

BackgroundFilamentous fungi are prolific producers of bioactive molecules and enzymes with important applications in industry. Yet, the vast majority of fungal species remain undiscovered or uncharacterized. Here we focus our attention to a wild fungal isolate that we identified as Anthostomella pinea. The fungus belongs to a complex polyphyletic genus in the family of Xylariaceae, which is known to comprise endophytic and pathogenic fungi that produce a plethora of interesting secondary metabolites. Despite that, Anthostomella is largely understudied and only two species have been fully sequenced and characterized at a genomic level. ResultsIn this work, we used long-read sequencing to obtain the complete 53.7 Mb genome sequence including the full mitochondrial DNA. We performed extensive structural and functional annotation of coding sequences, including genes encoding enzymes with potential applications in biotechnology. Among others, we found that the genome of A. pinea encodes 91 biosynthetic gene clusters, more than 600 CAZymes, and 164 P450s. Furthermore, untargeted metabolomics and molecular networking analysis of the cultivation extracts revealed a rich secondary metabolism, and in particular an abundance of sesquiterpenoids and sesquiterpene lactones. We also identified the polyketide antibiotic xanthoepocin, to which we attribute the anti-Gram-positive effect of the extracts that we observed in antibacterial plate assays. ConclusionsTaken together, our results provide a first glimpse into the potential of Anthstomella pinea to provide new bioactive molecules and biocatalysts and will facilitate future research into these valuable metabolites.

microbiology↗

bioRxiv2023

Heterologous naringenin production in the filamentous fungus Penicillium rubens

Naringenin is a natural product with several reported bioactivities and is the key intermediate for the entire class of plant flavonoids. The translation of flavonoids into modern medicine as pure compounds is often hampered by their low abundance in nature and difficult chemical synthesis. Here, we investigated new avenues toward producing high levels of naringenin in microbial hosts. Penicillium rubens is a well characterized and highly engineered traditional "workhorse" for the production of {beta}-lactam antibiotics and cholesterol-lowering statins. We explored a secondary metabolite deficient P. rubens strain, P. rubens 4xKO, that was derived from an earlier industrial production strain as a promising microbial host for a recombinant flavonoid pathway. By integrating two plant genes encoding for enzymes in the naringenin biosynthesis pathway into the genome of this strain, we achieved a high naringenin titer in flask fermentations 36 h after feeding the precursor p-coumaric acid. Along with the rapid product accumulation of up to an 88% molar yield, we also observed rapid degradation of naringenin. Based on high-resolution mass spectrometric analysis, we identified the degradation products and proposed a naringenin degradation pathway in P. rubens 4xKO, which is distinct from other flavonoid-converting pathways reported in fungi. Our approach combines fundamental research with application-oriented microbial engineering, and our findings will pave the way to the more sustainable and economically feasible production of flavonoids for pharmaceutical and nutraceutical applications.

biochemistry↗

bioRxiv2023

Structural characterization and extended substrate scope analysis of two Mg2+-dependent O-methyltransferases from bacteria

Oxygen-directed methylation is a ubiquitous tailoring reaction in natural product pathways catalysed by O-methyltransferases (OMTs). Promiscuous OMT biocatalysts are thus a valuable asset in the toolkit for sustainable synthesis and optimization of known bioactive scaffolds for drug development. Here, we characterized two bacterial OMTs from Desulforomonas acetoxidans and Streptomyces avermitilis in terms of their enzymatic properties and substrate scope and determined their crystal structures. Both OMTs methylated a wide range of catechol-like substrates, including flavonoids, coumarins, hydroxybenzoic acids and their respective aldehydes, an anthraquinone and an indole. One enzyme also accepted a steroid. The product range included pharmaceutically relevant compounds such as (iso)fraxidin, iso(scopoletin), chrysoeriol, alizarin 1-methyl ether and 2-methoxyestradiol. Interestingly, certain non-catechol flavonoids and hydroxybenzoic acids were also methylated. This study expands the knowledge on substrate preference and structural diversity of bacterial catechol OMTs and paves the way for their use in (combinatorial) pathway engineering. Table of contentsTwo promiscuous O-methyltransferases from bacteria were found to methylate a panel of catechol substrates towards high-value medicinal compounds. Surprisingly, the non-catechol substrates 5-hydroxyflavonoids and o-hydroxybenzoic acids/aldehydes were also methylated at low conversion rates. The crystal structures reveal potential target sites for enzyme engineering for biocatalytic applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/526015v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@4d9028org.highwire.dtl.DTLVardef@334ceborg.highwire.dtl.DTLVardef@1ff016forg.highwire.dtl.DTLVardef@b74b8b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Engineering a plant polyketide synthase for the biosynthesis of methylated flavonoids

Homoeriodictyol and hesperetin are naturally occurring O-methylated flavonoids with many health-promoting properties. They are produced in plants in low abundance and as complex mixtures of similar compounds that are difficult to separate. Synthetic biology offers the opportunity to produce various flavonoids in a targeted, bottom-up approach in engineered microbes with high product titers. However, the production of O-methylated flavonoids is currently still highly inefficient. In this study, we investigated and engineered a combination of enzymes that had previously been shown to support homoeriodictyol and hesperetin production in Escherichia coli from fed O-methylated cinnamic acids. We determined the crystal structures of the enzyme catalyzing the first committed step of the pathway, chalcone synthase from Hordeum vulgare, in three ligand-bound states. Based on these structures and a multiple sequence alignment with other chalcone synthases, we constructed mutant variants and assessed their performance in E. coli towards producing methylated flavonoids. With our best mutant variant, HvCHS (Q232P, D234V), we were able to produce homoeriodictyol and hesperetin at 2 times and 10 times higher titers than previously reported. Our findings will facilitate the further engineering of this enzyme towards higher production of methylated flavonoids.

biochemistry↗

bioRxiv2020

Rapid in vitro prototyping of O-methyltransferases for pathway applications in Escherichia coli

O-methyltransferases are ubiquitous enzymes involved in biosynthetic pathways for secondary metabolites such as bacterial antibiotics, human catecholamine neurotransmitters, and plant phenylpropanoids. While thousands of putative O-methyltransferases are found in sequence databases, few examples are functionally characterized. From a pathway engineering perspective, however, it is crucial to know the substrate and product ranges of the respective enzymes to fully exploit their catalytic power. In this study, we developed an in vitro prototyping workflow that allowed us to screen ~30 enzymes against five substrates in three days with high reproducibility. We combined in vitro transcription/translation of the genes of interest with a microliter-scale enzymatic assay in 96-well plates. The substrate conversion was indirectly measured by quantifying the consumption of the S-adenosyl-L-methionine co-factor by time-resolved fluorescence resonance energy transfer rather than time-consuming product analysis by chromatography. This workflow allowed us to rapidly prototype thus-far uncharacterized O-methyltransferases for future use as biocatalysts.

biochemistry↗

bioRxiv2020

Natural combinatorial genetics and prolific polyamine production enable siderophore diversification in Serratia plymuthica

Siderophores are small molecules with unmatched capacity to scavenge iron from proteins and the extracellular milieu, where it mostly occurs as insoluble Fe3+. Siderophores chelate Fe3+ for uptake into the cell, where it is reduced to soluble Fe2+. As iron is essential for bacterial survival, siderophores are key molecules in low soluble iron conditions. Bacteria have devised many strategies to synthesize proprietary siderophores to avoid siderophore piracy by competing organisms, e.g., by incorporating different polyamine backbones into siderophores, while maintaining the catechol moieties. We report that Serratia plymuthica V4 produces a variety of siderophores, which we term the siderome, and which are assembled by the concerted action of enzymes encoded in two independent gene clusters. Besides assembling serratiochelin with diaminopropane, S. plymuthica utilizes putrescine and the same set of enzymes to assemble photobactin, a siderophore described for Photorhabdus luminescens. The enzymes encoded by one of the gene clusters can independently assemble enterobactin. A third, independent operon is responsible for biosynthesis of the hydroxamate siderophore aerobactin, initially described in Enterobacter aerogenes. Mutant strains not synthesizing polyamine-siderophores significantly increased enterobactin production levels, though lack of enterobactin did not impact serratiochelin production. Knocking out SchF0, an enzyme involved in the assembly of enterobactin alone, significantly reduced bacterial fitness. This study illuminates the interplay between siderophore biosynthetic pathways and polyamine production superpathways, indicating routes of molecular diversification. Given its natural yields of diaminopropane (97.75 μmol/g DW) and putrescine (30.83 μmol/g DW), S. plymuthica can be exploited for the industrial production of these compounds.Significance Statement Siderophores are molecules crucial for bacterial survival in low iron environments. Bacteria have evolved the capacity to pirate siderophores made by other bacterial strains and to diversify the structure of their own siderophores, to prevent piracy. We found that Serratia plymuthica V4 produces five different siderophores using three gene clusters and a polyamine production superpathway. The most well studied siderophore, enterobactin, rather than the strain’s proprietary and by far most abundant siderophore, serratiochelin, displayed a crucial role in the fitness of S. plymuthica. Our results also indicate that this strain is a good candidate for engineering the large-scale production of diaminopropane (DAP), as without any optimization it produced the highest amounts of DAP reported for wild-type strains.Competing Interest StatementT.K.L. is a co-founder of Senti Biosciences, Synlogic, Engine Biosciences, Tango Therapeutics, Corvium, BiomX, and Eligo Biosciences. T.K.L. also holds financial interests in nest.bio, Ampliphi, IndieBio, MedicusTek, Quark Biosciences, and Personal Genomics.View Full Text

molecular biology↗

bioRxiv2019

Heterologous caffeic acid biosynthesis in Escherichia coli is affected by choice of tyrosine ammonia lyase and redox partners for bacterial Cytochrome P450

BackgroundCaffeic acid is industrially recognized for its antioxidant activity and therefore its potential to be used as an anti-inflammatory, anticancer, antiviral, antidiabetic and antidepressive agent. It is traditionally isolated from lignified plant material under energy-intensive and harsh chemical extraction conditions. However, over the last decade bottom-up biosynthesis approaches in microbial cell factories have been established, that have the potential to allow for a more tailored and sustainable production. One of these approaches has been implemented in Escherichia coli and only requires a two-step conversion of supplemented L-tyrosine by the actions of a tyrosine ammonia lyase and a bacterial Cytochrome P450 monooxygenase. Although the feeding of intermediates demonstrated the great potential of this combination of heterologous enzymes compared to others, no de novo synthesis of caffeic acid from glucose has been achieved utilizing the bacterial Cytochrome P450 thus far.\n\nResultsThe herein described work aimed at improving the efficiency of this two-step conversion in order to establish de novo caffeic acid formation from glucose. We implemented alternative tyrosine ammonia lyases that were reported to display superior substrate binding affinity and selectivity, and increased the efficiency of the Cytochrome P450 by altering the electron-donating redox system. With this strategy we were able to achieve final titers of more than 300 M or 47 mg/L caffeic acid over 96 h in an otherwise wild type E. coli MG1655(DE3) strain with glucose as the only carbon source. We observed that the choice and gene dose of the redox system strongly influenced the Cytochrome P450 catalysis. In addition, we were successful in applying a tethering strategy that rendered even an initially unproductive Cytochrome P450/ redox system combination productive.\n\nConclusionsThe caffeic acid titer achieved in this study is about 25% higher than titers reported for other heterologous caffeic acid pathways in wildtype E. coli without L-tyrosine supplementation. The tethering strategy applied to the Cytochrome P450 appears to be particularly useful for non-natural Cytochrome P450/redox partner combinations and could be useful for other recombinant pathways utilizing bacterial Cytochromes P450.

biochemistry↗