Biology subjects

van Aalst, A. C. A.

Publications and source records attributed to van Aalst, A. C. A..

7 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↗

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↗

bioRxiv2023

Co-cultivation of Saccharomyces cerevisiae strains combines advantages of different metabolic engineering strategies for improved ethanol yield

Glycerol is the major organic byproduct of industrial ethanol production with the yeast Saccharomyces cerevisiae. Improved ethanol yields have been achieved with engineered S. cerevisiae strains in which heterologous pathways replace glycerol formation as the predominant mechanism for anaerobic re-oxidation of surplus NADH generated in biosynthetic reactions. Functional expression of heterologous phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase (RuBisCO) genes enables yeast cells to couple a net oxidation of NADH to the conversion of glucose to ethanol. In another strategy, NADH-dependent reduction of exogenous acetate to ethanol is enabled by introduction of a heterologous acetylating acetaldehyde dehydrogenase (A-ALD). This study explores potential advantages of co-cultivating engineered PRK-RuBisCO-based and A-ALD-based strains in anaerobic bioreactor batch cultures. Co-cultivation of these strains, which in monocultures showed reduced glycerol yields and improved ethanol yields, strongly reduced the formation of acetaldehyde and acetate, two byproducts that were formed in anaerobic monocultures of a PRK-RuBisCO-based strain. In addition, co-cultures on medium with low acetate-to-glucose ratios that mimicked those in industrial feedstocks completely removed acetate from the medium. Kinetics of co-cultivation processes and glycerol production could be optimized by tuning the relative inoculum sizes of the two strains. Co-cultivation of a PRK-RuBisCO strain with a{Delta} gpd1 {Delta}gpd2 A-ALD strain, which was unable to grow in the absence of acetate and evolved for faster anaerobic growth in acetate-supplemented batch cultures, further reduced glycerol formation but led to extended fermentation times. These results demonstrate the potential of using defined consortia of engineered S. cerevisiae strains for high-yield, minimal-waste ethanol production.

microbiology↗

bioRxiv2023

Optimizing the balance between heterologous acetate- and CO2-reduction pathways in anaerobic cultures of Saccharomyces cerevisiae strains engineered for low glycerol production

In anaerobic Saccharomyces cerevisiae cultures, NADH-cofactor balancing by glycerol formation constrains ethanol yields. Introduction of an acetate-to-ethanol reduction pathway based on heterologous acetylating acetaldehyde dehydrogenase (A-ALD) can replace glycerol formation as redox-sink and improve ethanol yields in acetate-containing media. Acetate concentrations in feedstock for first-generation bioethanol production are, however, insufficient to completely replace glycerol formation. An alternative glycerol-reduction strategy bypasses the oxidative reaction in glycolysis by introducing phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase (RuBisCO). For optimal performance in industrial settings, yeast strains should ideally first fully convert acetate and, subsequently, continue low-glycerol fermentation via the PRK-RuBisCO pathway. However, anaerobic batch cultures of a strain carrying both pathways showed inferior acetate reduction relative to a strain expressing only the A-ALD pathway. Complete A-ALD-mediated acetate reduction by a dual-pathway strain, grown anaerobically on 50 g L-1 glucose and 5 mmol L-1 acetate, was achieved upon reducing PRK abundance by a C-terminal extension of its amino-acid sequence. Yields of glycerol and ethanol on glucose were 55% lower and 6% higher, respectively, than those of a non-engineered reference strain. The negative impact of the PRK-RuBisCO pathway on acetate reduction was attributed to sensitivity of the reversible A-ALD reaction to intracellular acetaldehyde concentrations.

bioengineering↗

bioRxiv2023

Loss of kinase Atg1 increases yeast maintenance energy requirement

Maintenance of cellular homeostasis underlies healthy aging. The processes involved in homeostasis rely on the so-called maintenance energy requirement and changes in this maintenance energy requirement impact aging and survival. Among maintenance processes, autophagy plays a crucial role as it is involved in the turn-over and recycling of damaged cellular material, such as organelles or proteins. The contribution of autophagy to the maintenance energy requirement is however unknown. Taking advantage of the high degree of conservation of autophagy between humans and Saccharomyces cerevisiae, we have used this yeast as a model organism to study the impact of macroautophagy on the maintenance energy requirement. The combination of the GFP-Atg8 cleavage assay with yeast retentostat cultures showed that autophagy is highly active in chronologically aging yeast cells, in non-dividing, but non-starving conditions. Deletion of the autophagy-activating kinase ATG1, homolog of human ULK1, resulted in a 60% increase in the maintenance energy requirement and doubled the specific death rate. Both these increases cannot be solely attributed to an observed increase in loss of respiratory capacity. Intriguingly, loss of Atg1 only reduced GFP-Atg8 cleavage by 20% under these conditions, indicating that Atg1-indendent modes of autophagy might be active. Overall, we illustrate the importance of autophagy on the energetics of aging cells and propose an alternative system for the widely applied yeast stationary phase cultures in chronological aging studies.

cell biology↗

bioRxiv2018

Laboratory evolution of a Saccharomyces cerevisiae x S. eubayanus hybrid under simulated lager-brewing conditions: genetic diversity and phenotypic convergence

Saccharomyces pastorianus lager-brewing yeasts are domesticated hybrids of S. cerevisiae x S. eubayanus that display extensive inter-strain chromosome copy number variation and chromosomal recombinations. It is unclear to what extent such genome rearrangements are intrinsic to the domestication of hybrid brewing yeasts and whether they contribute to their industrial performance. Here, an allodiploid laboratory hybrid of S. cerevisiae and S. eubayanus was evolved for up to 418 generations on wort under simulated lager-brewing conditions in six independent sequential batch bioreactors. Characterization of 55 single-cell isolates from the evolved cultures showed large phenotypic diversity and whole-genome sequencing revealed a large array of mutations. Frequent loss of heterozygosity involved diverse, strain-specific chromosomal translocations, which differed from those observed in domesticated, aneuploid S. pastorianus brewing strains. In contrast to the extensive aneuploidy of domesticated S. pastorianus strains, the evolved isolates only showed limited (segmental) aneuploidy. Specific mutations could be linked to calcium-dependent flocculation, loss of maltotriose utilisation and loss of mitochondrial activity, three industrially relevant traits that also occur in domesticated S. pastorianus strains. This study indicates that fast acquisition of extensive aneuploidy is not required for genetic adaptation of S. cerevisiae x S. eubayanus hybrids to brewing environments. In addition, this work demonstrates that, consistent with the diversity of brewing strains for maltotriose utilization, domestication under brewing conditions can result in loss of this industrially relevant trait. These observations have important implications for the design of strategies to improve industrial performance of novel laboratory-made hybrids.

evolutionary biology↗