Linda Ahonen

Laetiporus sulphureus is an edible wood-decaying basidiomycete whose fruiting body is valued for its characteristic chicken-like texture and flavor. Although the species has been investigated for various biotechnological applications, information on the submerged fermentation physiology remains limited. In this study, two newly isolated Danish L. sulphureus strains (NFICC9002 and NFICC9004) were evaluated with respect to cultivation conditions and potential mycotoxin production. The effect of incubation temperature, initial pH, carbon source, nitrogen sources, and agitation was investigated in liquid cultivations. Both strains exhibited greater biomass production at 25–30 °C than at 35–37 °C and at low pH. Carbon and nitrogen sources exerted strain-specific influence on biomass production. HPLC analysis demonstrated that substrate consumption was not always proportional to biomass formation, with glycerol supporting high biomass production despite only moderate substrate utilization. Whole-genome sequencing and untargeted metabolomics were used to support strain characterization and to screen for evidence of known mycotoxins. Whole-genome sequencing confirmed the taxonomic identity of the two strains as L. sulphureus and revealed no evidence of known toxin- or mycotoxin-associated genes. Untargeted metabolomics detected no known mycotoxins under the cultivation, extraction and detection methods used. These findings provide physiological and genomic information that may support the future development of L. sulphureus for submerged cultivation and food-related applications.

Pests cause up to 40% of global crops losses. Pesticide overuse drives resistance and poses notable risks to public health and the environment. Many hypocrealean fungi form symbiotic relationships with plants while antagonizing pests, making them valuable sources of biocontrol agents and biopesticides. However, little is known about their biosynthetic capabilities. Here we use phylogenomics, metabolomics and heterologous expression to catalog the biosynthetic repertoire of 82 plant-associated and insect-associated Hypocreales species. Annotation of 5,221 biosynthetic gene clusters reveals that ~80% of them encode unknown products. By linking biosynthetic gene clusters to molecules, we investigate the biosynthesis of several natural products, including pyridones, dethiosecoemestrin and efrapeptin. Additionally, by combining our metabologenomics workflow with synthetic biology, we characterize four nonribosomal peptide synthetase-like synthetases involved in the biosynthesis of hitherto unknown products. We believe that this work lays the groundwork for future efforts toward sustainable pest control in agriculture. The alt text for this image may have been generated using AI.

Pests are responsible for the loss of up to 40% of global crops. Widespread resistance to pesticides has emerged due to the overuse of a limited set of compounds and targets. This overreliance has driven the rapid adaptation of pest populations, making pesticides increasingly ineffective while posing significant risks to public health and the environment. Many fungal species form symbiotic relationships with plants while antagonizing insects, nematodes, and bacteria, making them valuable sources of sustainable biocontrol agents and biopesticides for agriculture. However, despite their potential, few of these species are used in biocontrol. Furthermore, little is known about their biosynthetic capabilities. Here we used phylogenomics, metabolomics, and heterologous expression to catalog and characterize the biosynthetic repertoire of biocontrol species within the Hypocreales order. Our workflow, applied to 82 plant- and insect-associated Hypocreales species, led to the annotation of 5221 biosynthetic gene clusters and putative products hitherto for 1034 of them. By linking biosynthetic gene clusters to molecules, we investigated the biosynthesis of several natural products. Additionally, by combining our metabologenomics workflow with synthetic biology we characterized 4 NRPS-like synthetases involved in the biosynthesis of hitherto unknow products. We believe this biosynthetic survey will provide a valuable foundation for developing sustainable pest control strategies in agriculture.

Fungal natural products, including polyketides, are a rich source of bioactive molecules. Their biosynthetic enzymes are encoded within biosynthetic gene clusters, which are often activated by specific environmental conditions. As a result, many natural products are not produced under standard laboratory conditions. Heterologous expression bypasses native regulation, enabling a systematic approach for polyketide discovery. The most widely used fungal hosts for natural product production are Saccharomycetales yeasts, and filamentous Eurotiomycetes. Yeasts are highly tractable but have a narrow product scope due to their limited secondary metabolism, while filamentous Eurotiomycetes have a richer secondary metabolism but are more difficult to engineer. In this work, we established two yeasts of the genera Exophiala and Knufia as novel heterologous hosts for a broad range of polyketides. These hosts combine the genetic tractability of yeast with the metabolic robustness of filamentous fungi. We developed genetic engineering tools for precise gene integration and genome editing, allowing us to heterologously express five fungal polyketide synthases with different domain architectures, including one involved in the biosynthesis of a previously undescribed 2-pyridone. Our findings demonstrate that these novel yeast hosts can efficiently produce complex polyketides, paving the way for systematic polyketide synthase expression and engineering.

Monoterpene indole alkaloids (MIAs) from Mitragyna speciosa (“kratom”), such as mitragynine and speciogynine, are promising novel scaffolds for opioid receptor ligands for treatment of pain, addiction, and depression. While kratom leaves have been used for centuries in South-East Asia as stimulant and pain management substance, the biosynthetic pathway of these psychoactives have only recently been partially elucidated. Here, we demonstrate the de novo production of mitragynine and speciogynine in Saccharomyces cerevisiae through the reconstruction of a five-step synthetic pathway from common MIA precursor strictosidine comprising fungal tryptamine 4-monooxygenase to bypass an unknown kratom hydroxylase. Upon optimizing cultivation conditions, a titer of ∼290 µg/L kratom MIAs from glucose was achieved. Untargeted metabolomics analysis of lead production strains led to the identification of numerous shunt products derived from the activity of strictosidine synthase (STR) and dihydrocorynantheine synthase (DCS), highlighting them as candidates for enzyme engineering to further improve kratom MIAs production in yeast. Finally, by feeding fluorinated tryptamine and expressing a human tailoring enzyme, we further demonstrate production of fluorinated and hydroxylated mitragynine derivatives with potential applications in drug discovery campaigns. Altogether, this study introduces a yeast cell factory platform for the biomanufacturing of complex natural and new-to-nature kratom MIAs derivatives with therapeutic potential.

Background and aim: Genetic markers are established as predictive of type 1 diabetes, but unknown early life environment is believed to be involved. Umbilical cord blood may reflect perinatal metabolism and exposures. We studied whether selected polar metabolites in cord blood contribute to prediction of type 1 diabetes.Methods: Using a targeted UHPLC-QQQ-MS platform, we quantified 27 low-molecular-weight metabolites (including amino acids, small organic acids, and bile acids) in 166 children, who later developed type 1 diabetes, and 177 random control children in the Norwegian Mother, Father, and Child cohort. We analyzed the data using logistic regression (estimating odds ratios per SD [adjusted odds ratio (aOR)]), area under the receiver operating characteristic curve (AUC), and k-means clustering. Metabolites were compared to a genetic risk score based on 51 established non-HLA single-nucleotide polymorphisms, and a 4-category HLA risk group.Results: The strongest associations for metabolites were aminoadipic acid (aOR = 1.23; 95% CI, 0.97-1.55), indoxyl sulfate (aOR = 1.15; 95% CI, 0.87-1.51), and tryptophan (aOR = 0.84; 95% CI, 0.65-1.10), with other aORs close to 1.0, and none significantly associated with type 1 diabetes. K-means clustering identified 6 clusters, none of which were associated with type 1 diabetes. Cross-validated AUC showed no predictive value of metabolites (AUC 0.49), whereas the non-HLA genetic risk score AUC was 0.56 and the HLA risk group AUC was 0.78.Conclusions: In this large study, we found no support of a predictive role of cord blood concentrations of selected bile acids and other small polar metabolites in the development of type 1 diabetes.

Aims/hypothesis Abnormal gut microbiota and blood metabolome profiles have been reported both in children and adults with uncomplicated type 1 diabetes as well as in adults with type 1 diabetes and advanced stages of diabetic nephropathy. In this study we aimed to investigate the gut microbiota and a panel of targeted plasma metabolites in individuals with type 1 diabetes of long duration without and with different levels of albuminuria. Methods In a cross-sectional study we included 161 individuals with type 1 diabetes and 50 healthy control individuals. Individuals with type 1 diabetes were categorised into three groups according to historically measured albuminuria: (1) normoalbuminuria (−1 (1.73 m)⁻². Measures of the gut microbial beta diversity differed significantly between healthy controls and individuals with type 1 diabetes, either with micro- or macroalbuminuria. Taxonomic analyses showed that 79 of 324 genera differed in relative abundance between individuals with type 1 diabetes and healthy controls and ten genera differed significantly among the three albuminuria groups with type 1 diabetes. For the measured plasma metabolites, 11 of 31 metabolites differed significantly between individuals with type 1 diabetes and healthy controls. When individuals with type 1 diabetes were stratified by the level of albuminuria, individuals with macroalbuminuria had higher plasma concentrations of indoxyl sulphate and l-citrulline than those with normo- or microalbuminuria and higher plasma levels of homocitrulline and l-kynurenine compared with individuals with normoalbuminuria. Whereas plasma concentrations of tryptophan were lower in individuals with macroalbuminuria compared with those with normoalbuminuria. Conclusions/interpretation We demonstrate that individuals with type 1 diabetes of long duration are characterised by aberrant profiles of gut microbiota and plasma metabolites. Moreover, individuals with type 1 diabetes with initial stages of diabetic nephropathy show different gut microbiota and plasma metabolite profiles depending on the level of albuminuria. Graphical abstract

Adipose tissue plays a pivotal role in storing excess fat and its composition reflects the history of person’s lifestyle and metabolic health. Broad profiling of lipids with mass spectrometry has potential for uncovering new knowledge on the pathology of obesity, metabolic syndrome, diabetes and other related conditions. Here, we developed a lipidomic method for analyzing human subcutaneous adipose biopsies. We applied the method to four body areas to understand the differences in lipid composition between these areas. Adipose tissue biopsies from 10 participants were analyzed using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. The sample preparation optimization included the optimization of the lipid extraction, the sample amount and the sample dilution factor to detect lipids in an appropriate concentration range. Lipidomic analyses were performed for adipose tissue collected from the abdomen, breast, thigh and lower back. Differences in lipid levels between tissues were visualized with heatmaps. Lipidomic analysis on human adipose biopsies lead to the identification of 186lipids in 2 mg of sample. Technical variation of the lipid-class specific internal standards were below 5%, thus indicating acceptable repeatability. Triacylglycerols were highly represented in the adipose tissue samples, and lipids from 13 lipid classes were identified. Long polyunsaturated triacylglycerols in higher levels in thigh (q<0.05), when compared with the abdomen, breast and lower back, indicating that the lipidome was area-specific. The method presented here is suitable for the analysis of lipid profiles in 2 mg of adipose tissue. The amount of fat across the body is important for health but we argue that also the distribution and the particular profile of the lipidome may be relevant for metabolic outcomes. We suggest that the method presented in this paper could be useful for detecting such aberrations.

Background and aims Adipose tissue plays a pivotal role in storing excess fat and its composition reflects the history of person’s lifestyle and metabolic health. Broad profiling of lipids with mass spectrometry has potential for uncovering new knowledge on the pathology of obesity, metabolic syndrome, diabetes and other related conditions. Here, we developed a lipidomic method for analyzing human subcutaneous adipose biopsies. We applied the method to four body areas to understand the differences in lipid composition between these areas. Materials and methods Adipose tissue biopsies from 10 participants were analyzed using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. The method development included the optimization of the lipid extraction, the sample amount and the sample dilution factor to detect lipids in an appropriate concentration range. Lipidomic analyses were performed for adipose tissue collected from the abdomen, breast, thigh and lower back. Differences in lipid levels between tissues were visualized with heatmaps. Results Lipidomic analysis on human adipose biopsies lead to the identification of 187 lipids in 2 mg of sample. Technical variation of the lipid-class specific internal standards were below 5 %, thus indicating acceptable repeatability. Triacylglycerols were highly represented in the adipose tissue samples, and lipids from 13 lipid classes were identified. Long polyunsaturated triacylglycerols in higher levels in thigh (qConclusion The method presented here is suitable for the analysis of lipid profiles in 2 mg of adipose tissue. The amount of fat across the body is important for health but we argue that also the distribution and the particular profile of the lipidome may be relevant for metabolic outcomes. We suggest that the method presented in this paper could be useful for detecting such aberrations.