Biology subjects

Otto, M.

Publications and source records attributed to Otto, M..

At least 19 recordsLinked to original sources

bioRxiv2026

C3a receptor signalling in ObRb-expressing neurons promotes leptin resistance and obesity-associated cardiac remodelling

Obesity is associated with hypothalamic inflammation, leptin resistance and increased cardiometabolic risk, but the neuroimmune signals linking nutrient excess to these outcomes remain incompletely defined. Here we show that diet-induced obesity is accompanied by local complement activation in the mouse hypothalamus, leading to C3a generation that signals through C3a receptor (C3aR) on leptin receptor (ObRb)-expressing neurons and blunts leptin signaling. Deletion of C3aR in ObRb-expressing neurons preserves leptin responsiveness, reduces food intake and weight gain, and improves glucose homeostasis during dietary challenge. Systemic pharmacological C3aR targeting induces weight loss and improves leptin responsiveness in established obesity. Genetic or pharmacological C3aR targeting also attenuates obesity-associated diastolic dysfunction and myocardial fibrosis; pair-feeding experiments reveal an additional component of cardiac protection beyond reduced weight gain, associated with lower cardiac sympathetic innervation. In humans, circulating complement activation markers are elevated in obesity and decline after bariatric surgery. Together, our findings identify C3aR signaling in ObRb-expressing neurons as a neuroimmune pathway contributing to hypothalamic leptin resistance and obesity-associated cardiac remodeling, highlighting complement signaling as a potential therapeutic target in cardiometabolic disease.

immunology↗

bioRxiv2026

PRIME: A modular plasmid-based platform for continuous in vivo evolution across bacteria

Directed evolution enables the engineering of proteins with novel or improved functions, yet existing approaches often require extensive manual intervention and are difficult to sustain over long evolutionary trajectories. Here we introduce PRIME (Protein-primed Replication for In vivo Mutagenesis and Evolution), a plasmid-based orthogonal DNA replication system for continuous directed evolution in Escherichia coli. PRIME harnesses protein-primed DNA replication to establish an autonomous replicon that operates independently of the host genome. Coupling this system to an error-prone DNA polymerase enables targeted diversification of constructs encoded on the orthogonal plasmid while preserving genomic integrity. Using only standard laboratory equipment, we demonstrate the evolution of a construct expressing msfGFP with an 11.8-fold increase in fluorescence. The platform is fully compatible with standard molecular biology workflows and requires no specialised instrumentation. We further demonstrate its portability across bacterial hosts by establishing PRIME in Pseudomonas putida. PRIME thus provides a broadly accessible framework for continuous in vivo evolution, with wide-ranging applications in protein engineering and synthetic biology.

synthetic biology↗

bioRxiv2026

Dimerization-dependent cis-autophosphorylation activates the UPR and ISR kinases

Maintaining cellular homeostasis requires the ability to detect and respond to various forms of stress. Protein kinases of the unfolded protein response (UPR) monitor protein folding in the endoplasmic reticulum, while the integrated stress response (ISR) kinases sense viral infection, nutrient deprivation, mitochondrial oxidative stress and proteotoxic stress. However, the precise molecular mechanism by which these kinases are activated is still unknown. In this study, we show that the ISR kinases PERK, HRI, and Gcn2, as well as the UPR kinase Ire1, undergo dimerization-dependent cis-autophosphorylation of their activation loops. The back-to-back dimerization of the kinase domain allosterically activates each kinase. We derive a simple mathematical model for dimerization-dependent cis-autophosphorylation, which we use to obtain kinetic parameters of PERK autophosphorylation. We show that dimerization promotes not only activation loop phosphorylation of PERK, but also phosphorylation of its substrate, eIF2. In cells, kinase domain dimerization is necessary and sufficient for the activation of PERK. In summary, we propose a model in which a dimer is the minimal functional unit of all UPR and ISR kinases.

biochemistry↗

bioRxiv2026

Machine-learning MRI stratification of genetic frontotemporal dementia for clinical trial enrichment

BACKGROUNDGenetic frontotemporal dementia (FTD) shows large differences in symptom profiles, brain atrophy patterns, and progression rate, making clinical trials difficult to design and power. There is a need for biomarkers that can model disease progression, identify biologically distinct groups, and support efficient trial enrichment. METHODSWe applied contrastive trajectory inference (cTI), a machine-learning method, to structural MRI, white matter hyperintensity, and demographic data from 736 participants in the GENFI cohort, including non-carriers and carriers of C9orf72, GRN, or MAPT mutations. cTI produced an individual "genetic FTD progression score" (0-1) and grouped mutation carriers into data-driven subtypes. We tested construct validity using correlations between progression score and cognitive/functional measures, examined subtype differences in brain-behavior coupling, plasma neurofilament light (NfL), and longitudinal decline, and compared cTI-based trial enrichment against age, cortical thickness and NfL using analytic and simulation-based power analyses. RESULTSGenetic FTD progression scores correlated strongly with global dementia severity and multiple cognitive domains (all p < 0.001), confirming robust clinical scoring. Two mutation-carrier subtypes emerged: a Progressive Track (Subtype 2) with strong associations between progression score and cognitive/functional impairment, rising NfL, and faster longitudinal decline; and a Dissociated Track (Subtype 3) with comparable levels of structural variation but weak or absent clinical and NfL changes, suggesting relative biological stability. Baseline subtype membership added prognostic value for future decline in processing speed and language beyond baseline severity. Notably, for C9orf72 and GRN, cTI-informed enrichment reduced required recruited sample size per arm by about 61-75% compared with unenriched designs, and outperformed enrichment using age, cortical thickness or NfL in both analytic and simulation-based power analyses. CONCLUSIONSMachine-learning stratification of genetic FTD reveals a progressive and a dissociated disease track and provides individualized progression scores that closely track clinical status. cTI progression scores offer a powerful tool for trial enrichment, enabling smaller, more efficient prevention and early-intervention trials than conventional MRI or NfL markers alone.

neuroscience↗

bioRxiv2026

Ketogenic diet synergistic reprogramming of both host and microbiome promotes tissue regeneration

Nutrition influences host physiological processes, yet how diets reshape host physiology, microbial functions, or host-microbe interactions to promote regeneration remains poorly explored. Here, we show that a ketogenic diet (KD), enriched in fats and low in carbohydrates, reprograms both skin microbial and immune functions to promote tissue repair. KD enhances IL-17A activity in {gamma}{delta} T cells and mucosal-associated invariant T (MAIT) cells, accelerating tissue repair, while KD-induced skin lipidomic alterations enhance both the abundance and metabolic output of Staphylococcus epidermidis. Metatranscriptomic and lipidomic analyses revealed increased riboflavin biosynthesis and sphingomyelinase (Sph)-dependent ceramide production in S. epidermidis under KD conditions. Genetic depletion of microbial ribD, a key enzyme for riboflavin biosynthesis, or of sph compromised the ability of the bacteria to promote tissue repair. Thus, host nutritional status drives tissue regeneration by synergistically rewiring host and microbial functions, providing new insights into how diet can be harnessed to regulate host physiology.

immunology↗

bioRxiv2026

Herpes simplex virus infection promotes ALS pathology through ICP0-mediated PML body disruption

Transactive response DNA binding protein 43 kDa (TDP-43) pathology, is a central molecular hallmark of amyotrophic lateral sclerosis (ALS). However, the underlying triggers are incompletely understood. Here, we show that infection with herpes simplex virus (HSV) induces molecular hallmarks of ALS in various in vitro and in vivo models and is associated with an increased risk of ALS in human population data. German healthcare provider data (n = 238,440) and herpesvirus serology of an ALS patient and control cohort (n = 1,100) showed that HSV infection elevated the ALS risk by 210% and odds by [~]65%, respectively. On a molecular level, HSV infection promoted TDP-43 pathology in neuronal cell models, human iPSC-derived motoneurons and cerebral organoids, mice, and human tissue sections. This effect was triggered by HSV-1 or 2, but not by several other related herpesviruses. Mechanistically, the infected cell protein 0 (ICP0) of HSV-1/2 drives TDP-43 pathology by disturbance of promyelocytic leukemia nuclear bodies (PML-NBs), thereby abrogating TDP-43 SUMO2/3ylation. Taken together, we reveal a previously unrecognized association between HSV infection and ALS and clarify the underlying molecular mechanism that drives TDP-43 pathology. Our data may guide future studies into therapeutic and prophylactic interventions against ALS.

neuroscience↗

bioRxiv2025

MRI-based classifier to identify close-to-onset cases in C9orf72 genetic frontotemporal dementia

Predicting symptom onset in genetic frontotemporal dementia (FTD) is crucial for advancing targeted interventions and clinical trial design. Brain changes begin years before clinical symptoms emerge, making neuroimaging a strong candidate for onset prediction. However, FTD is highly heterogeneous, encompassing diverse molecular pathologies, affected brain networks, and symptom trajectories. This variability limits the predictive power of any single imaging biomarker and underscores the need for an integrative, multimodal approach to improve prediction accuracy and generalizability. We used machine learning to integrate diverse neuroimaging features, identifying a robust signature for risk stratification. We analyzed T1-weighted and T2-weighted MRI scans from 71 symptomatic C9orf72 carriers, 90 presymptomatic carriers, and 69 healthy controls from the GENFI cohort. We used FreeSurfer to measure cortical thickness and subcortical volumes, and BISON to quantify white matter hyperintensities (WMH). We applied Principal Component Analysis for dimensionality reduction and trained a random forest classifier to distinguish symptomatic carriers from controls. The model was subsequently applied to the presymptomatic cohort to identify individuals whose brain patterns resembled those of symptomatic cases, under the hypothesis that greater similarity indicated a higher risk of conversion. We validated the model with neuropsychological data and a two-year longitudinal follow-up. The classifier distinguished symptomatic C9orf72 carriers from controls with 87.0% accuracy. When applied to presymptomatic carriers, the model identified 21.1% of the cohort as having brain features comparable to those of symptomatic cases. This "high-risk group" showed significant neuropsychological weaknesses in executive function, language and social cognition compared to the non high-risk group. The model accurately predicted clinical conversion within a two-year period with 84.5% accuracy, a 70% sensitivity and a 93.3% negative predictive value. Our findings demonstrate the utility of a machine learning approach using multi-modal MRI to identify presymptomatic C9orf72 carriers at high risk of disease onset within the next two years. By capturing subtle neuroanatomical patterns associated with disease processes, this approach offers a promising method for stratifying genetic FTD carriers prior to symptom onset. Such predictive models could optimize patient selection in future clinical trials.

neuroscience↗

bioRxiv2025

Inferring the history of gene copy number evolution

Gene duplication plays a crucial role in the adaptive evolution and diversification of organisms by creating extra copies of genes that can evolve new functions while preserving the original. Duplicated genes can become fixed in populations or appear as copy number variants. However, inferring and dating these duplication events from present-day data is challenging, as gene copy count distributions could result from either a few ancient duplication events or many recent ones. Sequence based phylogenetic reconstruction, an often seen practice, does not include the history of individuals and hence may result in inconsistencies, which may lead to misinterpretations. Here, we introduce a novel model for inferring gene copy number evolution, which describes gene duplication and their evolution over time through a random walk on a coalescent duplication network. This approach is solely based on copy number counts and hence independent of the inconsistencies of sequence based inferences. Backward in time we implement structured coalescent simulations, where we re-interprete structure as genealogical distance based on copy number counts. We apply this model to the NB-ARC domain counts of NLR genes in A. thaliana to infer the number and times of duplication events that have led to the present day copy number distribution.

evolutionary biology↗

bioRxiv2025

Monocyte Dysregulation Defines an MDD-Specific Transcriptional Signature Closely Linked to Clinical MDD Traits

ObjectiveMajor depressive disorder (MDD) is a highly heterogeneous psychiatric condition contributing significantly to the global disease burden. In a subset of MDD patients inflammatory processes accompany the disease and this has also been reported for other psychiatric conditions like schizophrenia (SZ). However, little is known regarding immune deregulation in relation to clinical symptoms. MethodsCellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq) of peripheral blood mononuclear cells (PBMCs) from patients with MDD (n=22), SZ (n=10), and healthy controls (HCs, n=26) was performed, yielding 633,284 high-quality single-cell profiles. Differential gene expression, cell composition, pathway enrichment, and weighted gene co-expression network analyses were conducted. MDD symptom severity was assessed using the Beck Depression Inventory-II (BDI-II), enabling the correlation of gene expression with individual disease symptoms. ResultsWe identified disease-specific transcriptomic signatures for MDD as well as for SZ and shared gene expression changes between the two conditions. For MDD, monocyte numbers were elevated concomitant with a strong enrichment of interferon signaling. The MDD-specific signature involving all peripheral cell types correlated strongly with key symptoms of depression like sadness or pessimism. Reactome analysis identified nuclear factor kappa-light-chain-enhancer of activated B cells (NF-{kappa}B)-associated pathways among the genes positively correlating with disease symptoms and traits. ConclusionOur findings might help to stratify psychiatric patients and provide a multidimensional framework for investigating immune contributions to psychiatric disorders and highlight potential targets for therapeutic intervention. Clinical symptoms might be reflected on a molecular level paving the way for more targeted and effective interventions.

genomics↗

bioRxiv2025

Tyrosine availability shapes Staphylococcus aureus nasal colonization and interactions with commensal communities

Only certain individuals are nasally colonized by Staphylococcus aureus, making them more susceptible to developing endogenous infections. The role of the resident microbiota in modulating S. aureus nasal colonization remains largely unclear. Using a newly assembled nasal strain collection, we demonstrate that nasal commensals can either promote or inhibit S. aureus proliferation through strain- and community-specific effects in vitro and within gnotobiotic animals. We identified nutrient-dependent interactions as a key driver of these dynamics and found that S. aureus strains auxotrophic to tyrosine rely on amino acid-providing nasal communities to thrive in the nutrient-limited environment of the nose. Large-scale screenings revealed tyrosine dependency to affect 8% of nasal S. aureus strains and to impair S. aureus colonization. Our findings uncover a crucial factor in S. aureus physiology and its interactions as a commensal nasal species, paving the way for new decolonization strategies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/651429v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@17b6289org.highwire.dtl.DTLVardef@182249forg.highwire.dtl.DTLVardef@1324dc1org.highwire.dtl.DTLVardef@18ec3bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Alzheimer's disease and its co-pathologies: implications for hippocampal degeneration, cognitive decline, and the role of APOE ϵ4

INTRODUCTIONIn neurodegenerative dementias, the co-occurrence and interaction of A{beta}, tau, and other pathological lesions confound their individual contributions to neurodegeneration and their modulation by risk factors. METHODSWe analyzed 480 post-mortem human brains (ages 50-99) using regression and structural equation models to assess the relationships among A{beta}, tau, LATE-NC, -synuclein, other age-related lesions, and APOE {varepsilon}4, as well as their effects on CA1 neuronal density, brain weight, and cognitive status. RESULTSA{beta}, tau, LATE-NC, and amygdala-predominant -synuclein pathology were highly interconnected. Tau was the strongest predictor of global neurodegeneration, while LATE-NC primarily, but not exclusively, affected hippocampal neuron loss. Small vessel disease correlated with both LATE-NC and -synuclein, while APOE {varepsilon}4 was mainly associated with extracellular and capillary A{beta}. DISCUSSIONAlthough Alzheimers pathology plays a central role in brain degeneration, coexisting pathologies can both exacerbate and independently contribute to it. These factors should be considered in patient stratification.

neuroscience↗

bioRxiv2025

White Matter Hyperintensities Precede other Biomarkers in GRN Frontotemporal Dementia

INTRODUCTIONIncreased white matter hyperintensities (WMHs) have been reported in genetic frontotemporal dementia (FTD) in small studies, but the sequence of WMH abnormalities relative to other biomarkers is unclear. METHODSUsing a large dataset (n=763 GENFI2 participants), we measured WMHs and examined them across genetic FTD variants and stages. Cortical and subcortical volumes were parcellated, and serum neurofilament light chain (NfL) levels were measured. Biomarker progression was assessed with discriminative event-based and regression modeling. RESULTSSymptomatic GRN carriers showed elevated WMHs, primarily in the frontal lobe, while no significant increase was observed in C9orf72 or MAPT carriers. WMH abnormalities preceded NfL elevation, ventricular enlargement, and cortical atrophy. Longitudinally, baseline WMHs predicted subcortical changes, while subcortical volumes did not predict WMH changes, suggesting WMHs may precede neurodegeneration. DISCUSSIONWMHs are elevated in a subset of GRN-related FTD. When present, they appear early and should be considered in disease progression models. HighlightsO_LIElevated WMH volumes in symptomatic GRN carriers, but not in other mutations. C_LIO_LIWMH accumulation is mostly observed in the frontal lobe. C_LIO_LIWMH abnormalities appear early in GRN-FTD, before NfL, atrophy, and ventriculomegaly. C_LIO_LILongitudinally, WMH volumes can predict subcortical changes, but not vice versa. C_LIO_LIWMHs are key early markers in GRN-FTD and should be included in progression models. C_LI RESEARCH IN CONTEXTO_ST_ABSSystematic reviewC_ST_ABSWe systematically reviewed the literature on white matter hyperintensities (WMHs) in frontotemporal dementia (FTD) using PubMed. While a few small studies reported increased WMHs in GRN mutation carriers, their sample sizes were limited, and they did not assess the timing of WMHs within disease progression or their temporal relationship to other biomarkers. InterpretationWe identified a sequence of key biomarkers in GRN-related FTD and demonstrated that WMHs are among the earliest biomarkers, preceding cortical and subcortical atrophy as well as blood biomarkers. This aligns with neuropathological evidence of early white matter involvement in FTLD-GRN. Additionally, using a larger dataset, we validated previous reports of elevated WMHs in GRN carriers, confirming their reliability. Future directionsFuture studies should integrate WMHs into FTD progression models to enhance early diagnosis. Understanding why only a subset of GRN carriers exhibit high WMH volumes remains a key research priority.

neuroscience↗

bioRxiv2025

A staged approach using ants as test organism in ecotoxicology

Although ants (Hymenoptera: Formicidae) are ecological key components, they are currently not included in ecotoxicity testing. Based on their social organization, ecology and exposure routes to pesticides, we propose a multilevel testing scheme for ants starting from isolated workers (level-1), workers and brood (level-2) and founding queens or entire colonies (level-3). To evaluate feasibility, we tested three ant species (Camponotus maculatus, Crematogaster sp., Lasius niger) using the neonicotinoid imidacloprid as a test substance. Ants were orally exposed through liquid food. At level-1, worker survival showed a clear concentration response and allowed to estimate LC50 with sufficiently narrow confidence intervals. In level-2, worker mortality and sublethal effects negatively affected larval survival and development, e.g. occurrence of naked pupae, with a lower NOEC for larvae (<0.05 mg/L feeding solution) than workers (1.7 mg/L). At level-3, the test substance significantly reduced the reproductive output of newly mated L. niger queens over 21 days after a single exposure to 0.5 mg/L. Our results confirm that ants can be easily handled and tested in the laboratory, making them a valuable extension for ecotoxicity testing. Given their ecological relevance, easy culture in laboratory settings and promising first results, we recommend further research using our new testing scheme, e.g. testing other stressors, species and endpoints. Establishing a robust test protocol for ants would provide coverage of a new and ecologically important group of non-target insects, which contributes to a broader protection of biodiversity and ecosystems. HighlightsO_LIAnts play key roles in many ecosystems but are not covered in ecotoxicity testing. C_LIO_LIWe propose a testing scheme for ants with 3 different levels of complexity. C_LIO_LIAnts can be cultured and tested cost- and space-efficient in the laboratory. C_LIO_LIThree ant species were tested using imidacloprid in parts of our testing scheme. C_LIO_LILethal and sublethal endpoints can be linked to individual and colony fitness. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/641392v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@11387feorg.highwire.dtl.DTLVardef@1456368org.highwire.dtl.DTLVardef@8171b5org.highwire.dtl.DTLVardef@504536_HPS_FORMAT_FIGEXP M_FIG C_FIG

ecology↗

bioRxiv2024

Most L1CAM is not associated with extracellular vesicles in human biofluids and iPSC-derived neurons

Transmembrane L1 cell adhesion molecule (L1CAM) is widely used as a marker to enrich for neuron-derived extracellular vesicles (EVs), especially in plasma. However, this approach lacks sufficient robust validation. This study aimed to assess whether human biofluids are indeed enriched for EVs, particularly neuron-derived EVs, by L1CAM immunoaffinity, utilizing multiple sources (plasma, CSF, conditioned media from iPSC-derived neurons [iNCM]) and different methods (mass spectrometry [MS], nanoparticle tracking analysis [NTA]). Following a systematic multi-step validation approach, we confirmed isolation of generic EV populations using size-exclusion chromatography (SEC) and polymer-aided precipitation (PPT) - two most commonly applied EV isolation methods - from all sources. Neurofilament light (NfL) was detected in both CSF and blood-derived EVs, indicating their neuronal origin. However, L1CAM immunoprecipitation did not yield enrichment of L1CAM in EV fractions. Instead, it was predominantly found in its free-floating form. Additionally, MS-based proteomic analysis of CSF-derived EVs also did not show L1CAM enrichment. Our study validates EV isolation from diverse biofluid sources by several isolation approaches and confirms that some EV subpopulations in human biofluids are of neuronal origin. Thorough testing across multiple sources by different orthogonal methods, however, does not support L1CAM as a marker to reliably enrich for a specific subpopulation of EVs, particularly of neuronal origin.

neuroscience↗

bioRxiv2024

Amygdala-predominant α-synuclein pathology exacerbates hippocampal neuron loss in Alzheimer's disease

Misfolded -synuclein (Syn) protein accumulates in 43-63% of individuals with symptomatic Alzheimers disease (AD). Two main patterns of co-morbid Syn pathology have been identified: caudo-rostral and amygdala-predominant, with the latter being more common in AD. Syn pathology has been shown to interact with DNA-binding protein 43 (TDP-43) and abnormally phosphorylated Tau protein (pTau). These proteins tend to accumulate in the amygdala, yet the specific role of amygdala-predominant Syn pathology in the progression of AD and hippocampal degeneration remains unclear. In this cross-sectional study, we analyzed 291 autopsy brains from both demented and non-demented elderly individuals neuropathologically. Neuronal density in the CA1 region of the hippocampus was assessed using hematoxylin-stained sections for all cases. We semi-quantitatively evaluated Syn pathology severity in six brain regions and stratified the cases into the two spreading patterns. In 99 AD cases, we assessed limbic-predominant age-related TDP-43 neuropathological changes (LATE-NC), CA1 pTau density, and cerebral amyloid angiopathy (CAA). Structural equation modeling analysis was conducted based on the assessed pathological parameters in AD patients. We identified an association between the amygdala-predominant Syn pathology pattern and decreased neuronal density in the CA1 region. AD patients with an amygdala-predominant Syn pattern exhibited the most severe pTDP-43 pathology among all groups, while those with the caudo-rostral pattern had the lowest severity of AD-related pathological changes including CAA type 1. Our model revealed that the relationship between Syn pathology and CA1 neuron loss is mediated through pTau and LATE-NC. Our results indicate that amygdala-predominant Syn pathology, in contrast to Syn pathology with a caudo-rostral pattern, significantly contributes to hippocampal neuron loss, potentially by accelerating TDP-43 and pTau pathologies. This finding, along with observed neuropathological differences between AD patients with these two Syn spreading patterns, underscores the need for precise stratification of patients. The stratification should consider not only the molecular and morphological identity of co-pathologies but also the distribution pattern of the respective co-pathologies.

neuroscience↗

bioRxiv2023

Distinguishing the roles of adaptation and demography in gene copy number changes in human populations

Genes with multiple copies are likely to be maintained by stabilizing selection that puts a bound to unlimited expansion of copy number. We designed a model where copy number variation is generated by unequal recombination, which fits well to a number of genes, surveyed in three human populations. Based on this theoretical model and on computer simulations, we were interested in answering the question whether gene copy number distribution in the derived European and Asian populations can be explained by a purely demographic scenario or whether shifts in the distribution are signatures of adaptation. Although copy number distribution in most of the analyzed gene clusters can be explained by a bottleneck as in the out of Africa expansion of homo sapiens 60-10kyrs ago, we identified several candidate genes, for instance AMY1A and PGA3, whose copy numbers are likely to be selected differently among African, Asian and European populations.

evolutionary biology↗

bioRxiv2023

Engineering Yeast to Improve Heterologous Abscisic Acid Production

Abscisic acid (ABA) is a high-value product with agricultural, medical and nutritional applications. We previously constructed an ABA cell factory by expressing the ABA metabolic pathway from Botrytis cinerea in the biotechnological workhorse Saccharomyces cerevisiae. In this study, we aimed to improve ABA production and explored various rational engineering targets mostly focusing on increasing the activity of two rate-limiting cytochrome P450 monooxygenases of the ABA pathway, BcABA1 and BcABA2. We evaluated the effects of overexpression and knock-down of cell membrane transporters, expression of heterologous cytochrome b5, overexpression of a rate-limiting heme biosynthesis gene and overexpression or knock-out of genes involved in ER membrane homeostasis. One of the genes involved in ER membrane homeostasis, PAH1, was identified as the most promising engineering target. Knock-out of PAH1 improved ABA titers, but also caused a sever growth defect. By replacing the PAH1 promoter with a weak minimal promoter, it was possible to mediate the growth defect while still improving ABA production. In this report we were able to improve the ABA cell factory and furthermore provide valuable insights for future studies aiming to engineer cytochrome P450 monooxygenases. One-sentence summaryIn this study we explored various strategies to improve heterologous abscisic acid production in Saccharomyces cerevisiae and identified fine-tuning of the PAH1 gene as a promising engineering strategy.

bioengineering↗

bioRxiv2023

Sense and Screen-ability: Development of tuneable, biosensor-based screening platforms for abscisic acid

The activities of heterologous enzymes often limit the production titers, rates and yields of cell factories. With the help of biosensors, large random mutagenesis libraries can be screened for improved enzyme variants in a high-throughput manner, even if the enzyme-of-interest is poorly characterised. We previously constructed a Saccharomyces cerevisiae cell factory for the heterologous production of abscisic acid (ABA), a high-value product with a broad range of applications in medicine, agriculture and nutrition. In the current study, we developed high-throughput screening platform strains for two rate-limiting cytochrome P450 monooxygenases, BcABA1 and BcABA2, in the ABA biosynthetic pathway. The screening platforms are designed to minimize the occurrence of false positives during screening experiments. We thoroughly characterised two plant protein-based ABA biosensor candidates. Furthermore, we designed a simple genetic switch, based on the thiamine-repressible promoter pTHI4, to regulate the expression level of enzyme variants. We demonstrated that ABA production can be fine-tuned by varying thiamine concentration in the media. In-depth analysis of the platform strains revealed that screening conditions can be optimized by varying thiamine concentration and cultivation time, making it possible to utilize the full dynamic and operational range of the biosensor. In the future, the constructed strains can be used to screen for improved BcABA1 and BcABA2 variants.

bioengineering↗