Biology subjects

Arnesen, J. A.

Publications and source records attributed to Arnesen, J. A..

4 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

Engineering Biosensors to Enhance Monoterpene Indole Alkaloid Production in Yeast

Monoterpene Indole Alkaloids (MIAs) are a diverse family of plant natural products with various medicinal applications. Although MIAs, such as vinblastine and reserpine, are clinically validated, sourcing of MIAs for clinical use or drug discovery from natural resources or via chemical synthesis is hampered due to their scarcity and chemical complexity. Refactoring MIA biosynthesis pathways in microbial cell factories could offer an alternative, more stable and potentially sustainable manufacturing route for alkaloid medicines and novel therapies. However, reaching commercially attractive titers, rates and yields remains challenging owing to the length and complexity of these metabolic pathways. One critical bottleneck is the low screening throughput and very high cost of the analytical methods used to quantify MIA for optimizing production. In this study, we evolved RamR, a promiscuous bacterial transcription factor to respond to five different MIAs, resulting in highly sensitive and selective sensor variants (EC50<10 M). X-ray crystallography and computational modeling provided insight into the MIA binding of the evolved biosensor variants. The RamR biosensing platform was functionalized in yeast and subsequently applied in a cost-effective semi-throughput screening campaign of a 188-gene overexpression library to identify high-performing cell factory designs for strictosidine, the common precursor for all MIAs. The fluorescent biosensor signal correlated with HPLC quantification (r2 = 0.932) allowing identification of single metabolic engineering hits which when combined yielded a maximum titer of >220 mg/L strictosidine, 3-fold higher than the parental reference strain. This study demonstrates the development of selective biosensors for MIAs and the cost-effective identification of novel metabolic engineering hits for optimizing MIA production in microbial cell factories.

synthetic biology↗

bioRxiv2024

Essential sterols from engineered yeast prevent honeybee colony population decline

Honeybees, the worlds most important crop pollinators, are increasingly facing pollen starvation arising from agricultural intensification and climate change. Frequent flowering dearth periods and high-density rearing conditions weaken colonies, often leading to their demise. Beekeepers provide colonies with pollen substitutes, but these feeds cannot sustain brood production because they lack essential sterols found in pollen. Here, we describe a technological breakthrough in honeybee nutrition with wide-reaching impacts on global food security. We first measured the quantity and proportion of sterols found in honeybee tissues. Using this information, we genetically engineered a strain of the oleaginous yeast, Yarrowia lipolytica, to produce a mixture of essential sterols for bees and incorporated it into an otherwise nutritionally complete diet. Colonies fed exclusively with this diet reared brood for significantly longer than those fed diets without suitable sterols. Incorporating sterol supplements into pollen substitutes using this method will enable beekeepers to rear healthier, longer-lived colonies to meet the growing demands for global crop pollination. It could also reduce competition between bee species for access to natural floral resources, stemming the decline of wild bee populations.

synthetic biology↗

bioRxiv2018

Early diverging insect pathogenic fungi of the order Entomophthorales possess diverse and unique subtilisin-like serine proteases

Insect-pathogenic fungi use subtilisin-like serine proteases (SLSPs) to degrade chitin-associated proteins in the insect procuticle. Most insect-pathogenic fungi in the order Hypocreales (Ascomycota) are generalist species with a broad host-range, and most species possess a high number of SLSPs. The other major clade of insect-pathogenic fungi is part of the subphylum Entomophthoromycotina (Zoopagomycota, formerly Zygomycota) which consists of high host-specificity insect-pathogenic fungi that naturally only infect a single or very few host species. The extent to which insect-pathogenic fungi in the order Entomophthorales rely on SLSPs is unknown. Here we take advantage of recently available transcriptomic and genomic datasets from four genera within Entomophthoromycotina: the saprobic or opportunistic pathogens Basidiobolus meristosporus, Conidiobolus coronatus, C. thromboides, C. incongruus, and the host-specific insect pathogens Entomphthora muscae and Pandora formicae, specific pathogens of house flies (Muscae domestica) and wood ants (Formica polyctena), respectively. We use phylogenetics and protein domain analysis to show that the obligate biotrophic fungi E. muscae, P. formicae and the saprobic human pathogen C. incongruus all contain \"classical\" fungal SLSPs and a unique group of SLSPs that loosely resembles bacillopeptidase F-like SLSPs. This novel group of SLSPs is found in the genomes of obligate insect pathogens and a generalist saprobic opportunistic pathogen why they are unlikely to be responsible for the host specificity of Entomophthorales. However, this class represent a unique group of SLSPs so far only observed among Bacteria, Oomycetes and early diverging fungi such as Cryptomycota, Microsporidia, and Entomophthoromycotina and missing in the sister fungal lineages of Kickxellomycotina or the fungal phyla Mucoromyocta, Ascomycota and Basidiomycota fungi suggesting interesting gene loss patterns.

evolutionary biology↗