Biology subjects

Crocoll, C.

Publications and source records attributed to Crocoll, C..

16 recordsLinked to original sources

bioRxiv2026

Biocatalytic Production of Galantamine in Yeast through Cytochrome P450 Optimization

Galantamine is a pharmaceutically relevant Amaryllidaceae alkaloid used for the treatment of Alzheimer's disease. Its structural complexity and low abundance in plants motivate the development of alternative manufacturing routes. Here, we established the first engineered yeast platform for the biocatalytic production of galantamine from 4OMe-norbelladine. Heterologous expression of the downstream pathway enzymes NtCYP96T6, NtNMT1, and NtAKR1 from Narcissus cv. Tete-a-Tete in Saccharomyces cerevisiae was complemented by optimization of cultivation temperature, carbon source, and medium pH enabling the first demonstration of galantamine production in yeast. Systematic screening of cytochrome P450 reductase and cytochrome b partners to boost NtCYP96T6 activity led to the identification of a novel reductase mined from the Narcissus pseudonarcissus transcriptome, NpCPR, which supported the highest pathway flux and yielded 7.0 {+/-} 0.6 mg/L galantamine, corresponding to a 7.6 % molar yield from 250 M 4OMe-norbelladine and an approximately 173-fold improvement over the parental strain. We further exploited this yeast cell factory for the precursor-directed biosynthesis of 7F-galantamine, highlighting the potential of pathway enzyme promiscuity to access new-to-nature GAL analogues that may be challenging to produce through conventional chemical synthesis. Together, this work establishes a foundation for microbial galantamine production and biosynthetic diversification of its pharmaceutically relevant scaffold.

synthetic biology↗

bioRxiv2026

Water stress adaptive responses in plants require movement of ABA and AB-aldehyde from vascular to target tissues

Vascular plants rapidly coordinate root and shoot responses to water stress. Abscisic acid (ABA) mediates these adaptations; however, it remains unclear which cells produce ABA, whether ABA synthesis shifts during stress, and whether ABA movement is required for its adaptive functions. Here, we map ABA biosynthesis at cellular resolution in Arabidopsis and report that water-stress adaptive responses in roots and shoots require movement of ABA and its precursor AB-aldehyde from vascular tissues to target cells. We suggest that ABA accumulation arises from two parallel routes: (i) ABA synthesized in the vasculature via ABA2 and AAO3, then moving to guard cells, and (ii) phloem-derived AB-aldehyde being converted to ABA in the epidermis or bundle sheath by AAO1 and AAO2. Finally, we predict that tightly packed cells beneath leaf veins facilitate efficient ABA delivery to guard cells, an anatomical arrangement that has enabled angiosperms to evolve the use of ABA to rapidly close stomata.

plant biology↗

bioRxiv2026

Characterisation of gene expression markers and glucosinolates during discrete infection stages of Pyrenopeziza brassicae in Brassica napus

Light leaf spot (LLS), caused by subcuticular hemibiotrophic ascomycete fungus Pyrenopeziza brassicae, is a major constraint on oilseed rape (Brassica napus) production, yet the genetic and biochemical mechanisms of quantitative disease resistance (QDR) remain poorly defined. Here, disease phenotyping, pathogen quantification, microscopy, gene expression profiling and glucosinolate (GSL) analysis were integrated to dissect resistance mechanisms in B. napus. Disease assays of 19 diverse lines revealed clear contrasts between susceptible and resistant genotypes, with the commercial cultivar Ambassador showing a phenotype inconsistent with the UK Recommended List rating. Microscopy demonstrated that resistance within doubled haploid line Cubs Root does not inhibit spore germination or penetration but restricts hyphal branching and subcuticular colonisation from 4 to 8 days post-inoculation. Expression profiling of seven candidate gene expression markers (GEMs) and pathogenesis-related PR1 showed that cinnamate-4-hydroxylase, phospholipase C4, {beta}-adaptin, universal stress protein and the 40S ribosomal subunit protein S24 were strongly pathogen-induced in resistant lines, whereas a BAHD acyltransferase, a putative susceptibility factor, was induced only in susceptible cultivars. GSL profiling identified negative correlations between disease severity and total GSLs, particularly aliphatic and aromatic GSLs, with 2{square}phenylethyl and 7-methylsulfinyl heptyl GSLs showing the strongest associations with resistance. Together, these results highlight coordinated transcriptional and metabolic responses that limit pathogen proliferation and provide targets for breeding durable LLS resistance in B. napus.

plant biology↗

bioRxiv2026

Engineering orthogonal quorum sensing circuits using LuxR-type systems in yeast consortia

Engineered microbial communities hold significant biotechnological potential because their collective metabolism can produce functions beyond those achievable by individual strains. However, multicellular synthetic gene circuits require orthogonal communication systems that enable precise, programmable signaling between cells. Quorum sensing (QS), where cells both produce and detect small diffusible signal molecules, offers a natural framework for such intercellular communication. However, the construction of complex multicellular circuits for applications such as biobased production is currently hampered by the limited number of orthogonal QS channels available in yeast. Here, we expand the QS toolkit in Saccharomyces cerevisiae by characterizing four LuxR-type biosensors based on EsaR, LasR, TraR and RpaR, alongside the previously established LuxR biosensor. We functionally expressed acyl-CoA-dependent HSL synthases in yeast, producing a diverse range of aliphatic and aromatic HSL signals. LuxR and RpaR, were compatible with in vivo ligand production and established as orthogonal QS signaling pair with synthases MesI and RpaI, respectively. Co-culture experiments demonstrated QS-dependent intercellular signaling, with 3.9-fold and 6.4-fold induction relative to monocultures. Together, these results establish a modular and extensible platform for orthogonal intercellular communication in yeast, enabling the construction of multicellular synthetic gene circuits.

synthetic biology↗

bioRxiv2026

Utilizing raw rapeseed press cake in foods: A case study on sensory quality and profile of selected bitter compounds in snack bars

Todays canola quality rapeseed press cake (RPC) is a protein-rich co-product with potential as human food, but its application is limited due to antinutritional compounds and bitter taste. It remains, however, unknown how introduction of raw RPC to a food matrix affects sensory perception and which metabolites drive the sensation. Here, raw RPC from whole or dehulled seeds was introduced into snack bars at 0%, 7%, 14%, and 21%, and sensory responses were correlated to selected known RPC-derived bitter compounds. A trained panel evaluated 13 RPC-characteristic sensory attributes, and the bitter compounds sinapic acid, kaempferol 3-O-(2'''-O-sinapoyl-{beta}-sophoroside) (KSS), KSS-hexose, selected bitter glucosinolates, and goitrin were quantified using targeted LC-MS/MS. Most dose-dependent sensory responses increased up to 14% RPC and then plateaued, whereas astringent mouthfeel increased almost linearly across the full dose range. Dehulling intensified several odor- and flavor-related attributes but did not increase bitterness or protein content in the final product. Principal component analysis linked bitterness and astringency positively with KSS, KSS-hexose, and goitrin. Dose-over-threshold analysis further showed that goitrin, but not progoitrin, reached concentrations relevant for bitterness perception. Together, the results demonstrate that raw RPC contributes distinct dose-dependent sensory attributes and that metabolite transformations in the food matrix shape final sensory profiles. These findings provide a basis for developing RPC-containing foods and for breeding rapeseed lines with improved sensory characteristics. HIGHLIGHTSO_LIThis study presents the first sensory panel assessment of rapeseed press cake (RPC)-containing in food products (snack bars) made from whole and dehulled seeds. C_LIO_LI13 RPC-characteristic sensory attributes are identified. C_LIO_LISensory profiles of the tasted snack bars differed significantly, influenced by the dosage of RPC and by the dehulling treatment. Bitterness and astringency are positively correlated with the RPC dosage. C_LIO_LIGoitrin, kaempferol 3-O-(2'''-O-sinapoyl-{beta}-sophoroside) (KSS) and sinapic acid are RPC-derived bitter compounds that correlate with bitter taste of RPC-containing snack bars. C_LIO_LIApproximately 90% of glucosinolates introduced with the RPC are not detected in the snack bars, and goitrin levels in snack bars accounts for only [~]10% of introduced progoitrin. C_LIO_LIGoitrin is - for the first time - reported to contribute to the perceived bitterness of an RPC-containing food product. C_LI

plant biology↗

bioRxiv2026

Optimization of the glucosinolate core pathway for production of simple glucosinolates in Escherichia coli

Microbial biosynthesis of plant secondary metabolites aims to provide access to compounds of medicinal or industrial value independently of the native plant. Glucosinolates are plant secondary metabolites characteristic of brassicaceous plants and recognized as promoters of human health. However, plants often contain a complex mixture of glucosinolates with insufficient amounts to elicit a clinical effect through diet. Here, we demonstrate the biosynthesis of defined glucosinolate products in Escherichia coli through combinatorial screening of pathway enzyme homologs, tailoring the optimal biosynthetic route for each individual product. To achieve high product titers, we establish efficient P450 expression by membrane anchor truncation and engineer sulfate assimilation to increase the supply of a sulfate donor 3-phosphoadenosine-5-phosphosulfate. We use benzyl glucosinolate pathway as a model to test the engineering strategies and improve the titer 37-fold over our previous study. Extrapolating the best approaches to other simple glucosinolates, we establish the first microbial synthesis of tyrosine-derived p-glucosinolate. Showing the highest titer overall, we report production of 1250 {+/-} 91 {micro}M indolyl-3-methyl glucosinolate, a 500-fold increase over biosynthesis in yeast.

synthetic biology↗

bioRxiv2025

A versatile method to expand and compare transporter substrate spectra

Membrane transport proteins (transporters) are critical players in the physiology of complex organisms. The range of metabolites a transporter translocates across a membrane (substrate spectra) determines the membrane selectivity and, consequently, the transporters physiologic function. Nevertheless, determining the full substrate spectra of a transporter, and hence its role(s) in the organism, remains a daunting challenge. Many transporters lack an experimentally determined substrate, and many show a puzzling substrate promiscuity. Substrate promiscuity raises questions about which experimentally defined substrates are physiologic, which are not, and which are missing. Targeted approaches to determine transporter substrate spectra present limitations: Compound availability, lack of unexpected cofactors, and the researchers biases. To tackle these issues, transporters can be screened with complex native metabolites to generate new hypotheses on their function. Here, we describe an untargeted method to expand and compare the substrate spectra of sets of plant transporters. This method compares the substrate spectra of different transporters when expressed in Xenopus laevis oocytes and exposed to plant metabolic extracts. Comparing the transport-mediated accumulation of metabolites enables us to contextualize the relative transporter activity towards the same set of metabolites from undefined mixtures. Additionally, metabolite accumulation patterns across transporter sets allow us to annotate novel compounds. We validate the method with three well-characterized transporters, where we find unreported substrate spectra specificities and detect the transport of tens of metabolites at a time. We screen a set of fourteen transporters and find unreported activities for eight of them, some of which are confirmed from pure compounds. Finally, we propose to reinterpret the role of two plant transporters previously linked to developmental regulation (phytohormone transport) in light of their substrate promiscuity.

molecular biology↗

bioRxiv2023

Mechanistic insight into substrate specificity of plant glucosinolate transporters

Plants depend on transport processes for correct allocation of specialized metabolites. This is important for optimal defense, avoidance of autotoxicity, connecting compartmented biosynthetic modules and more. Transport of a wide variety of specialized metabolites is mediated by transporters from the Nitrate and Peptide transporter Family (NPF), which belongs to the Major Facilitator Superfamily (MFS). However, the mechanism by which NPF members recognize and transport specialized metabolites remains unknown. Here we mutate eight residues to reciprocally swap the substrate-preference of two closely related glucosinolate transporters (GTRs). Seven of these residues assemble in a ring-like structure in all conformations of the transporters. We labeled the ring-like structure a selectivity filter and based on docking studies, we propose that the interaction between the selectivity filter and the glucosinolate side chain determines whether a given glucosinolate is recognized as a substrate. Besides partly explaining the distinct substrate preference of GTR1 (NPF2.10) and GTR3 (NPF2.9), this study proposes fundamental principles of substrate recognition in the NPF and establishes the GTR subclade as a novel model system for studying structure function relationships in the NPF.

molecular biology↗

bioRxiv2022

Systematic engineering of plant cytochrome P450 system identifies a comprehensive strategy for expression of highly functional P450 enzymes in Escherichia coli

Cytochrome P450s catalyse diverse and unique chemical reactions, which makes them invaluable enzymes in nature and industry. Metabolic engineers leverage these unique catalytic properties when refactoring plant biosynthetic pathways into microbial cell factories. However, due to their hydrophobic anchor, microbial expression of membrane-bound cytochrome P450s is challenging. An arsenal of protein engineering strategies was developed to improve their expression in Escherichia coli, but extensive screening is often necessary to tailor the engineering approach to an individual enzyme. Here, we propose a universal strategy that allows the expression of highly active cytochrome P450s in E. coli by systematically evaluating six common N-terminal modifications and their effect on in vivo activity of enzymes from the CYP79 and CYP83 families. We identified transmembrane domain truncation as the only strategy that had a significantly positive effect on all seven tested enzymes, increasing product titres between 2- to 170-fold. When comparing the changes in protein titre and product generation, we show that higher expression does not always translate to higher in vivo activity, thus making protein titre an unreliable screening target. Our results demonstrate that transmembrane domain truncation improves in vivo activity across a broad range of cytochrome P450s with diverse N-terminal sequences and could be applied as the modification-of-choice to avoid the time-consuming screening process and accelerate the future design of E. coli cell factories.

bioengineering↗

bioRxiv2021

Herbivore feeding behavior validates optimal defense theory for specialized metabolites within plants

Numerous plants protect themselves from attackers using specialized metabolites. The biosynthesis of these deterrent, often toxic metabolites is costly, as their synthesis diverts energy and resources on account of growth and development. How plants diversify investments into growth and defense is explained by the optimal defense theory. The central prediction of the optimal defense theory is that plants maximize growth and defense by concentrating specialized metabolites in tissues that are decisive for fitness. To date, supporting physiological evidence merely relies on the correlation between plant metabolite distribution and animal feeding preference. Here, we use glucosinolates as a model to examine the effect of changes in chemical defense distribution on actual feeding behavior. Taking advantage of the uniform glucosinolate distribution in transporter mutants, we show that high glucosinolate accumulation in tissues important to fitness protects them by guiding larvae of a generalist herbivore to feed on other tissues. Moreover, we show that mature leaves of Arabidopsis thaliana supply young leaves with glucosinolates to optimize defense against herbivores. Our study provides physiological evidence for the central hypothesis of the optimal defense theory and sheds light on the importance of integrating glucosinolate biosynthesis and transport for optimizing plant defense.

plant biology↗

bioRxiv2021

Sugar transporters enable a leaf beetle to accumulate plant defense compounds

Many herbivorous insects selectively accumulate plant toxins for defense against predators; however, little is known about the transport processes that enable insects to absorb and store defense compounds in the body. Here, we investigate how a specialist herbivore, the horseradish flea beetle, accumulates high amounts of glucosinolate defense compounds in the hemolymph. Using phylogenetic analyses of coleopteran membrane transporters of the major facilitator superfamily, we identified a clade of glucosinolate-specific transporters (PaGTRs) belonging to the sugar porter family. PaGTR expression was predominantly detected in the excretory system, the Malpighian tubules. Silencing of PaGTRs led to elevated glucosinolate excretion, significantly reducing the levels of sequestered glucosinolates in beetles. This suggests that PaGTRs reabsorb glucosinolates from the Malpighian tubule lumen to prevent their loss by excretion. Ramsay assays performed with dissected Malpighian tubules confirmed a selective retention of glucosinolates. Thus, the selective accumulation of plant defense compounds in herbivorous insects can depend on the ability to prevent excretion.

physiology↗

bioRxiv2020

Identification and characterization of phlorizin transporter from Arabidopsis thaliana and its application for phlorizin production in Saccharomyces cerevisiae

Bioengineering aimed at producing complex and valuable plant specialized metabolites in microbial hosts requires efficient uptake of precursor molecules and export of final products to alleviate toxicity and feedback inhibition. Plant genomes encode a vast repository of transporters of specialized metabolites that-- due to lack of molecular knowledge--remains largely unexplored in bioengineering. Using phlorizin as a case study--an anti-diabetic and anti-cancerous flavonoid from apple--we demonstrate that brute-force functional screening of plant transporter libraries in Xenopus oocytes is a viable approach to identify transporters for bioengineering. By screening 600 Arabidopsis transporters, we identified and characterized purine permease 8 (AtPUP8) as a bidirectional phlorizin transporter. Functional expression in the plasma membrane of a phlorizin-producing yeast strain increased phlorizin titer by more than 80 %. This study provides a generic approach for identifying plant exporters of specialized metabolites and demonstrates the potential of transport-engineering for improving yield in bioengineering approaches.

molecular biology↗

bioRxiv2020

Engineering and optimization of the 2-phenylethylglucosinolate production in Nicotiana benthamiana by combining biosynthetic genes from Barbarea vulgaris and Arabidopsis thaliana

Among the glucosinolate (GLS) defense compounds characteristic of the Brassicales order, several have been shown to promote human health. This includes 2-phenylethylglucosinolate (2PE) derived from homophenylalanine (HPhe). In this study, we used transient expression in Nicotiana benthamiana to validate and characterize previously predicted key genes in the 2PE biosynthetic pathway from Barbarea vulgaris and demonstrate the feasibility of engineering 2PE production. We used genes from B. vulgaris and Arabidopsis thaliana, in which the biosynthesis of GLSs is predominantly derived from HPhe and dihomomethionine, respectively. The resulting GLS profiles partially mirrored GLS profiles in the gene donor plant, but in both cases the profiles in N. benthamiana were wider than in the native plants. We found that BvBCAT4 is a more efficient entry enzyme for biosynthesis of both HPhe and dihomomethionine and that MAM1 enzymes determine the chain-elongated profile. Co-expression of the chain elongation pathway and CYP79F6 from B. vulgaris with the remaining aliphatic GLS core pathway genes from A. thaliana, demonstrated the feasibility of engineering production of 2PE in N. benthamiana. Noticeably, the HPhe-converting enzyme BvCYP79F6 in the core GLS pathway belongs to the CYP79F subfamily, a family believed to have substrate specificity towards chain-elongated methionine derivatives. Replacing the B. vulgaris chain elongation pathway with a chimeric pathway consisting of BvBCAT4, BvMAM1, AtIPMI and AtIPMDH1 resulted in an additional 2-fold increase in 2PE production, demonstrating that chimeric pathway with genes from different species can increase flux and boost production in an engineered pathway. Our study provides a novel approach to produce the important HPhe and 2PE in a heterologous host. Chimeric engineering of a complex biosynthetic pathway enabled detailed understanding of catalytic properties of individual enzymes - a prerequisite for understanding biochemical evolution - and with biotechnological and plant breeding potentials of new-to-nature gene combinations.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsGLSglucosinolate;2PE2-phenylethylglucosinolate;HPhehomophenylalanine;HMhomomethionine;DHMdihomomethionine;THMtrihomomethionine;HLhomoleucine;DHLdihomoleucine;PEITC2-phenylethyl isothiocyanate;3MTP3-(methylthio)propylglucosinolate;3MSP3-(methylsulfinyl)propylglucosinolate;4MTB4-(methylthio)butylglucosinolate;4MSB4-(methylsulfinyl)butylglucosinolate;BGLSbenzylglucosinolateView Full Text

synthetic biology↗

bioRxiv2020

VC1 catalyzes a key step in the biosynthesis of vicine from GTP in faba bean

Faba bean is a widely adapted and high-yielding legume cultivated for its protein-rich seeds1. However, the seeds accumulate the anti-nutritional pyrimidine glucosides vicine and convicine, which can cause haemolytic anaemia--favism--in the 400 million individuals genetically predisposed by a deficiency in glucose-6-phosphate dehydrogenase2. Here, we identify the first enzyme associated with vicine and convicine biosynthesis, which we name VC1. We show that VC1 co-locates with the major QTL for vicine and convicine content and that the expression of VC1 correlates highly with vicine content across tissues. We also show that low-vicine varieties express a version of VC1 carrying a small, frame-shift insertion, and that overexpression of wild-type VC1 leads to an increase in vicine levels. VC1 encodes a functional GTP cyclohydrolase II, an enzyme normally involved in riboflavin biosynthesis from the purine GTP. Through feeding studies, we demonstrate that GTP is a precursor of vicine both in faba bean and in the distantly related plant bitter gourd. Our results reveal an unexpected biosynthetic origin for vicine and convicine and pave the way for the development of faba bean cultivars that are free from these anti-nutrients, providing a safe and sustainable source of dietary protein.

biochemistry↗

bioRxiv2019

De novo indol-3-ylmethyl glucosinolate biosynthesis, and not long-distance transport, contributes to defence of Arabidopsis against powdery mildew

Powdery mildew is a fungal disease that affects a wide range of plants and reduces crop yield worldwide. As obligate biotrophs, powdery mildew fungi manipulate living host cells to suppress defence responses and to obtain nutrients. Members of the plant order Brassicales produce indole glucosinolates that effectively protect them from attack by non-adapted fungi. Indol-3-ylmethyl glucosinolates are constitutively produced in the phloem and transported to epidermal cells for storage. Upon attack, indol-3-ylmethyl glucosinolates are activated by CYP81F2 to provide broad-spectrum defence against fungi. How de novo biosynthesis and transport contribute to defence of powdery mildew-attacked epidermal cells is unknown. Bioassays and glucosinolate analysis indicate that GTR glucosinolate transporters are not involved in antifungal defence. Using quantitative live-cell imaging of fluorophore-tagged markers, we show that accumulation of the glucosinolate biosynthetic enzymes CYP83B1 and SUR1 is induced in epidermal cells attacked by the non-adapted barley powdery mildew Blumeria graminis f.sp. hordei. By contrast, glucosinolate biosynthesis is attenuated during interaction with the virulent powdery mildew Golovinomyces orontii. Interestingly, SUR1 induction is delayed during the Golovinomyces orontii interaction. We conclude that epidermal de novo synthesis of indol-3-ylmethyl glucosinolate contributes to CYP81F2-mediated broad-spectrum antifungal resistance and that adapted powdery mildews may target this process.

plant biology↗

bioRxiv2019

An Optimized Screen Reduces the Number of GA Transporters and provides Insights into NPF Substrate Determinants

Based on recent in vitro data, a relatively large number of the plant Nitrate transporter 1/Peptide transporter Family (NPF) proteins has been suggested to function as gibberellic acid (GA) transporters. Most GA transporting NPF proteins also appear to transport other structurally unrelated phytohormones or metabolites. Several of the GAs used in previous in vitro assays are membrane permeable weak organic acids whose movement across membranes are influenced by the pH-sensitive ion-trap mechanism. Moreover, a large proportion of in vitro GA transport activities have been demonstrated indirectly via long-term yeast-based GA-dependent growth assays that are limited to detecting transport of bioactive GAs. Thus, there is a need for an optimized transport assay for identifying and characterizing GA transport. Here, we develop an improved transport assay in Xenopus laevis oocytes wherein we directly measure movement of six different GAs across oocyte membranes over short time. We show that membrane permeability of GAs in oocytes can be predicted based on number of oxygen atoms and that several GAs do not diffuse over membranes regardless of changes in pH values. In addition, we show that small changes in internal cellular pH can result in strongly altered distribution of membrane permeable phytohormones. This prompts caution when interpreting heterologous transport activities. We use our transport assay to screen all Arabidopsis thaliana NPF proteins for transport activity towards six GAs (two membrane permeable and four non-permeable). The results presented here, significantly reduce the number of bona fide NPF GA transporters in Arabidopsis and narrow the activity to fewer subclades within the family. Furthermore, to gain first insight into the molecular determinants of substrate specificities towards organic molecules transported in the NPF, we charted all surface exposed amino acid residues in the substrate-binding cavity and correlated them to GA transport. This analysis identified distinct residues within the substrate-binding cavity that are shared between GA transporting NPF proteins; the potential roles of these residues in determining substrate specificity are discussed.

biochemistry↗