Maxence Holtz

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. Tête-à-Tête 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 b₅ 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 μM 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.

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 (EC50220 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.

The yeast Saccharomyces cerevisiae is widely employed in industrial biotechnology for chemical and pharmaceutical production. However, engineering yeast for high product titre remains challenging due to metabolic imbalances and competition for cellular resources. To address this, we developed an orthogonal quorum-sensing (QS) system based on N -acyl-homoserine lactones (AHLs) for cell density-dependent regulation in yeast. Using metabolic engineering, we established AHL production in yeast. Next, we improved AHL-biosensors via directed evolution and a novel growth-based screening strategy with amdS as a counter-selectable marker. We identified three LuxR variants with enhanced sensitivity and confirmed N86 to play an important role in their sensitivity to ligands, corroborating literature on the native system in bacteria. These sensitive LuxR variants were engineered for QS-controlled expression of a reporter gene, demonstrated by delayed autonomous expression of yeGFP. Additionally, we engineered LuxR to function as a repressor, achieving QS-dependent repression. The QS system was applied to enhance aloesone production, a plant-derived metabolite with cosmetic and pharmaceutical applications. The established system showed 51% increased production through QS-controlled repression of FAS1 . This work establishes a versatile QS-based regulatory platform to support dynamic pathway regulation for metabolic engineering in yeast.

Exploring metabolic and protein engineering solutions, this article discusses breakthroughs in enhancing cytochrome P450 performance, paving the way for more sustainable and efficient bioprocesses in natural product manufacturing.

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.