Cerevisiae Research Articles

Characterization of a High-Affinity Copper Transporter CTR1a in the White-Nose Syndrome Causing Fungal Pathogen Pseudogymnoascus destructans.

Copper is an essential micronutrient and the ability to scavenge tightly bound or trace levels of copper ions at the host-pathogen interface is vital for fungal proliferation in animal hosts. Recent studies suggest that trace metal ion acquisition is critical for the establishment and propagation of Pseudogymnoascus destructans, the fungal pathogen responsible for white-nose syndrome (WNS), on their bat host. However, little is known about these metal acquisition pathways in P. destructans. In this study, we report the characterization of the P. destructans high-affinity copper transporter VC83_00191 (PdCTR1a), which is implicated as a virulence factor associated with the WNS disease state. Using Saccharomyces cerevisiae as a recombinant expression host, we find that PdCTR1a can efficiently traffic Cu ions into the yeast cytoplasm. Complementary studies in the native P. destructans fungus provide evidence that PdCTR1a transcripts and protein levels are dictated by Cu-bioavailability in the growth media. Our study demonstrates that PdCTR1a is a functional high-affinity copper transporter and is relevant to Cu homeostasis pathways in P. destructans.

Intracellular dry mass density increases under growth-induced pressure

Cells that proliferate in confined environments develop mechanical compressive stress, referred to as growth-induced pressure, which inhibits growth and division across various organisms. Recent studies have shown that in these confined spaces, the diffusivity of intracellular nanoparticles decreases. However, the physical mechanisms behind this reduction remain unclear. In this study, we use quantitative phase imaging to measure the refractive index and dry mass density of Saccharomyces cerevisiae cells proliferating under confinement in a microfluidic bioreactor. Our results indicate that the observed decrease in diffusivity can be at least attributed to the intracellular accumulation of macromolecules. Furthermore, the linear scaling between cell content and growth-induced pressure suggests that the concentrations of macromolecules and osmolytes are maintained proportionally under such pressure in S. cerevisiae.

Engineering Saccharomyces cerevisiae for the Production of Punicic Acid-Rich Yeast Biomass.

Punicic acid (PuA), an unusual conjugated linolenic acid found in pomegranate, offers diverse health benefits and has potential applications in the food industry. Due to the limited availability of PuA from natural plant sources, there is growing interest in producing it through microbial fermentation. In this study, the yeast Saccharomyces cerevisiae, which is classified as "generally recognized as safe", was engineered to produce PuA using a results-driven approach. Genes potentially involved in PuA synthesis were integrated directly into the yeast genome, targeting Ty retrotransposon sites. Screening of the yeast transformants, followed by optimization of culture conditions, resulted in the production of 26.7% PuA within the yeast's total fatty acids. Further analysis revealed that the strain's triacylglycerol fraction contained over 22% PuA. By incorporating this health-promoting lipid into the nutritional profile of S. cerevisiae, the engineered strain could serve as a sustainable source of yeast biomass with enhanced nutritional value.

Optimization of Citrus Pulp Waste-Based Medium for Improved Bacterial Nanocellulose Production.

Bacterial nanocellulose (BC) has attracted significant attention across a wide array of applications due to its distinctive characteristics. Recently, there has been increasing interest in leveraging waste biomass to improve sustainability in BC biogenesis processes. This study focuses on optimizing the citrus pulp waste (CPW) medium to enhance BC production using Komagataeibacter sucrofermentans. The screening of initial medium pH, yeast extract, CPW sugar and inoculum concentrations was conducted using the Plackett-Burman design, with BC yield (mgDW/gCPW) as the model response. The significant parameters, i.e., CPW sugars and yeast extract concentrations, were optimized using response surface methodology, employing a five-level, two-factor central composite design. The optimized CPW-based growth medium resulted in a final yield of 66.7 ± 5.1 mgDW/gCPW, representing a 14-fold increase compared to non-optimized conditions (4.3 ± 0.4 mgBC/gCPW). Material characterization analysis indicated that the produced BC showed high thermal stability (30% mass retained at 600 °C) and a crystallinity index value of 71%. Additionally, to enhance process sustainability, spent baker's yeast hydrolysate (BYH) was assessed as a substitute for yeast extract, leading to a final BC titer of 9.3 ± 0.6 g/L.

Isolation, identification, and technological characteristics analysis of yeast strains from Pyracantha fortuneana fruits fermentation liquid.

Pyracantha fortuneana (P. fortuneana), as a medicinal and edible plant resource, is rich in nutrients. In order to screen the high quality yeast used in Firebone fruit wine, 12 strains of yeast were isolated and purified from P. fortuneana fermentation broth by traditional pure culture method. They were identified by molecular biology as Pichia kudriavzevii (P. kudriavzevii) and Saccharomyces cerevisiae (S. cerevisiae), respectively. Strain HJ-2 could grow normally at 30℃, alcohol content 15%, SO2 mass concentration 360mg/L, pH 3.2, sucrose mass concentration 400g/L and glucose mass concentration 250g/L. Strain HJ-6 could grow normally at 30℃, alcohol content 3%, SO2 concentration 360mg/L, pH 3.2, sucrose concentration 250g/L, glucose concentration 300g/L. Based on the technological characteristics of fruit wine, S. cerevisiae HJ-2 has the potential of brewing P. fortuneana fruit wine. P. kudriavzevii HJ-6 has a low tolerance to ethanol and is suitable for the production of fermented beverages such as low-alcohol wine or beer.

The Role of Keeving in Modulating Fermentation and the Flavour Profiles of Apple Brandy

Keeving is the removal of nutrients from apple musts due to their binding to pectin, resulting in a slower fermentation and spontaneous arrest. The aim of this study was to determine the effect of keeving on the chemical composition of fermented apple must and on the volatile profile and sensory analysis of apple brandies. We compared the application of keeving during spontaneous fermentation with fermentation carried out by Saccharomyces cerevisiae (SafSpirit HG-1). We evaluated the impact of adding different doses of calcium chloride on various parameters of fermented musts and distillates. Calcium chloride had a greater effect on the ethanol concentration, total extract, and fermentation efficiency than on the type of fermentation used. However, a different phenomenon was observed with respect to the volatiles. The concentration of most of the higher alcohols, acetaldehyde, dodecanal, and geranylaceton, decreased after spontaneous fermentation and increased during the fermentation carried out with Saccharomyces cerevisiae SafSpirit HG-1. In general, the application of keeving contributed to a decrease in the concentration of ethyl and methyl esters, but caused an increase in the concentration of all acetate esters and terpenoids. When the amount of nutrients in the environment is limited and starvation occurs, microorganisms use the available nutrients for basic metabolic processes that allow them to survive and limit the formation of side metabolites such as volatiles. However, most of the samples fermented after the faecal depletion achieved high scores for the floral, fruity, and “overall note” parameters in the sensory analysis. This means that this method, carried out with a properly selected yeast strain, could be feasible for the distilling industry.

New Plasmid Constructs for Production of Yeast Based Oral Vaccines

In order to avoid conventional vaccines, there is an urgent need to look for new ways of vaccine generation that can cut down the cost, time, and the need for special requirements during storage and distribution. The development of oral peptide/protein-based vaccines could be an ideal alternative. Five expression plasmids are designed in this work for the production of Saccharomyces cerevisiae-based oral vaccines. Therefore, in this work genetic cassette with different organizations of the required genetic components for efficient gene expression is constructed in the pYES2 plasmid. The construction of five new plasmids for surface display of heterologous recombinant proteins in S. cerevisiae would enable easy insertion of the gene of interest into an optimized genetic cassette consisting of a strong host promoter GAL1, HA-tag for detection of recombinant enzymes, GAS1 gene with/without binding sequence coded for GAS1 cell wall protein and signal sequence for directing the protein into the secretory pathway. After the construction of the genetic cassettes, E gene coded for envelope protein of Chikungunya and Dengue virus is inserted in the genetic cassettes. Genetic models pYES2HAGAS1EgeneChikungunya, pYES2HAGAS1 Egene Dengue, pYES2GAS1 Egene ChikungunyaHA, and pYES2GAS1EGeneDengueHA are constructed with different genetic organizations to display envelope protein of Chikungunya and Dengue virus in the cell surface of S. cerevisiae. In the pYES2SignalEGeneDengueHA construction, envelope protein will be secreted by S. cerevisiae into the media. The constructed vectors are specialized to regulate the expression and surface display of heterologous recombinant proteins in Saccharomyces cerevisiae and will be suitable for large-scale oral vaccine production.

The effect of indigenous non-Saccharomyces yeasts on the volatile profile of Maraština wine: Monoculture versus sequential fermentation

Non-Saccharomyces yeasts are presented as a new promising tool in winemaking to enhance aroma complexity in fermentation with Saccharomyces yeasts. Indigenous yeasts are recognized for their better adaptation to environmental conditions and for highlighting the unique terroir impact on wine aroma characteristics. To study the individual impact of ten indigenous non-Saccharomyces yeasts on the primary metabolites and volatile compounds of wine from autochthonous Croatian Maraština variety, Hyphopichia pseudoburtonii, Metschnikowia chrysoperlae, Metschnikowia sinensis/shanxiensis, Metschnikowia pulcherrima, Lachancea thermotolerans, Hanseniaspora uvarum, Hanseniaspora guilliermondii, Hanseniaspora pseudoguilliermondii, Pichia kluyveri, and Starmerella apicola were inoculated in sterile grape juice in monoculture fermentations. Additionally, seven of them were also studied in sequential fermentation of sterile grape juice with commercial Saccharomyces cerevisiae. A targeted approach was used for the identification of volatile compounds via headspace solid-phase microextraction coupled with gas chromatography and a mass spectrometer. P. kluyveri and H. uvarum produced higher total concentrations of terpenes in sequential versus monoculture fermentation. Sequential fermentation of M. chrysoperlae, L. thermotolerans, and P. kluyveri with S. cerevisiae resulted in higher production of C13-norisoprenoids. The esters concentration was higher in monocultures for L. thermotolerans, H. uvarum, and H. guilliermondii, whereas other isolates showed higher concentrations in sequential fermentations. The results highlighted different indigenous yeast metabolisms and provided promising insights into potential new non-Saccharomyces starter cultures as a first step in their selection, with several species characterized in terms of their potential effect on the aroma profile.

Automated yeast cultivation control using a biosensor and flow cytometry.

Effective microbial bioprocessing relies on maintaining ideal cultivation conditions, highlighting the necessity for tools that monitor and regulate cellular performance and robustness. This study evaluates a fed-batch cultivation control system based on at-line flow cytometry monitoring of intact yeast cells having a fluorescent transcription factor-based redox biosensor. Specifically, the biosensor assesses the response of an industrial xylose-fermenting Saccharomyces cerevisiae strain carrying the TRX2p-yEGFP biosensor for NADPH/NADP+ ratio imbalance when exposed to furfural. The developed control system successfully detected biosensor output and automatically adjusted furfural feed rate, ensuring physiological fitness at high furfural levels. Moreover, the single-cell measurements enabled the monitoring of subpopulation dynamics, enhancing control precision over traditional methods. The presented automated control system highlights the potential of combining biosensors and flow cytometry for robust microbial cultivations by leveraging intracellular properties as control inputs.

Improving CsOAC Activity in Saccharomyces cerevisiae for directed Production of Olivetolic Acid through Rational Design.

Olivetolic acid (OA) is an essential precursor in the cannabinoid biosynthesis. It is produced through a unique interaction between the two proteins, olivetol synthase (CsOLS) and olivetolic acid cyclase (CsOAC). When the OA biosynthesis is transferred to Saccharomyces cerevisiae, olivetol (OL) is produced as a side product, even with a high enhancement of copy number of CsOAC. In order to increase the OA titer while decreasing the OL titer in S. cerevisiae, rational design was applied to CsOAC using in silico approaches such as protein-ligand docking to find potential protein variants. In vivo screening and also testing different approaches for both proteins was applied to identify the best performing variants of CsOAC. Four variants were identified that gave the desired properties. The best CsOAC variant, G82A/L92Y, resulted in a 1.7-fold increase in OA production and a shift in the ratio between the two products towards OA.

Functional similarities and differences among subunits of the nascent polypeptide-associated complex (NAC) of Saccharomyces cerevisiae.

Protein factors bind ribosomes near the tunnel exit, facilitating protein trafficking and folding. In eukaryotes, the heterodimeric nascent polypeptide-associated complex (NAC) is the most abundant - equimolar to ribosomes. Saccharomyces cerevisiae has a minor β-type subunit (Nacβ2) in addition to abundant Nacβ1, and therefore two NAC heterodimers, α/β1 and α/β12. The additional beta NAC gene arose at the time of the whole genome duplication that occurred in the S. cerevisiae lineage. Nacβ2 has been implicated in regulating the fate of mRNA encoding ribosomal protein Rpl4 during translation via its interaction with the Caf130 subunit of the regulatory CCR4-Not complex. We found that Nacβ2 residues just C-terminal to the globular domain are required for its interaction with Caf130 and its negative effect on growth of cells lacking Acl4, the specialized chaperone for Rpl4. Substitution of these Nacβ2 residues at homologous positions in Nacβ1 results in a chimeric protein that interacts with Caf130 and slows the growth of ∆acl4 cells lacking Nacβ2. Furthermore, alteration of residues in the N-terminus of Nacβ2 or chimeric Nacβ1 previously shown to affect ribosome binding overcomes the growth defect of ∆acl4. Our results are consistent with a model in which Nacβ2's ribosome association per se, or its precise positioning, is necessary for productive recruitment of CCR4-Not via its interaction with the Caf130 subunit to drive Rpl4 mRNA degradation.

Evaluation of Ability of Inactivated Biomasses of Lacticaseibacillus rhamnosus and Saccharomyces cerevisiae to Adsorb Aflatoxin B1 In Vitro.

Biological decontamination strategies using microorganisms to adsorb aflatoxins have shown promising results for reducing the dietary exposure to these contaminants. In this study, the ability of inactivated biomasses of Lacticaseibacillus rhamnosus (LRB) and Saccharomyces cerevisiae (SCB) incorporated alone or in combination into functional yogurts (FY) at 0.5-4.0% (w/w) to adsorb aflatoxin B1 (AFB1) was evaluated in vitro. Higher adsorption percentages (86.9-91.2%) were observed in FY containing 1.0% LR + SC or 2.0% SC (w/w). The survival of mouse embryonic fibroblasts increased after exposure to yogurts containing LC + SC at 1.0-4.0% (w/w). No significant differences were noted in the physicochemical and sensory characteristics between aflatoxin-free FY and control yogurts (no biomass) after 30 days of storage. The incorporation of combined LRB and SCB into yogurts as vehicles for these inactivated biomasses is a promising alternative for reducing the exposure to dietary AFB1. The results of this trial support further studies to develop practical applications aiming at the scalability of using the biomasses evaluated in functional foods to mitigate aflatoxin exposure.

MiR-326 overexpression inhibits colorectal cancer cell growth and proteasome activity by targeting PNO1: unveiling a novel therapeutic intervention strategy

Proteasome inhibition emerges as a promising strategy for cancer prevention. PNO1, pivotal for colorectal cancer (CRC) progression, is involved in proteasome assembly in Saccharomyces cerevisiae. Hence, we aimed to explore the role of PNO1 in proteasome assembly and its up- and down-streams in CRC. Here, we demonstrated that PNO1 knockdown suppressed CRC cells growth, proteasome activities and assembly, as well as CDKN1B/p27Kip1 (p27) degradation. Moreover, p27 knockdown partially attenuated the inhibition of HCT116 cells growth by PNO1 knockdown. The up-stream studies of PNO1 identified miR-326 as a candidate miRNA directly targeting to CDS-region of PNO1 and its overexpression significantly down-regulated PNO1 protein expression, resulting in suppression of cell growth, decrease of proteasome activities and assembly, as well as increasing the stability of p27 in CRC cells. These findings indicated that miR-326 overexpression can suppress CRC cell growth, acting as an endogenous proteasome inhibitor by targeting PNO1.

Survival of Escherichia coli O157, Salmonella Enteritidis, Bacillus cereus and Clostridium botulinum in non-alcoholic beers

Why was the work done: To (i) determine whether microbial pathogens were present in packaged alcohol-free and low alcohol beers, (ii) to assess whether pathogens can survive or grow in non-alcoholic beers, and (iii) to determine the impact of pH and bitterness on their growth and survival of pathogens in alcohol-free beer. How was the work done: : 50 alcohol-free and low alcohol beers, available in the UK, were screened for pathogens and analysed for ABV, pH and bitterness (IBU). One of the alcohol-free beers (with the lowest IBU) was adjusted to 25 and 50 IBU and pH 3.8, 4.2, 4.6 and 4.9. Challenge testing of these beers was performed with Escherichia coli O157, Salmonella Enteritidis, Bacillus cereus and Clostridium botulinum. In addition, the heat resistance (D60 value) of the pathogens, spoilage bacteria and Saccharomyces cerevisiae ascospores in these beers was determined. What are the main findings: Salmonella, E. coli, Enterobacteriaceae, Bacillus cereus and sulphite reducing clostridia were not found in any of the 50 beers. However, two emerging opportunistic pathogens (Cupriavidus gilardii and Sphingomonas paucimobilis) were found in the low alcohol keg beers. None of the pathogens used in this study could grow in the alcohol-free beer at low pH (pH 3.8). E. coli O157 was unable to grow at pH 4.2 but could grow at pH 4.6 but only with reduced levels of carbon dioxide and increased oxygen. Salmonella Enteritidis was able to grow at pH 4.2 and 4.6 but also with reduced levels of CO2 and increased O2. Although Bacillus cereus and C. botulinum were unable to grow in any of the tested conditions, both pathogens were able to survive. Survival and/or growth of the microorganisms was impacted by pH; bitterness had no effect. Why is the work important: Salmonella Enteritidis and E. coli O157 only grew in alcohol free beer at a higher pH (4.2 and 4.6 for Salmonella and 4.6 for E. coli) together with with reduced levels of CO2 and increased O2. This suggests that packaged beer with appreciable levels of carbon dioxide and negligible levels of oxygen will not support the growth of pathogens. However, draught alcohol free beer may be vulnerable to pathogens.

Raman‐Enabled Predictions of Protein Content and Metabolites in Biopharmaceutical Saccharomyces cerevisiae Fermentations

ABSTRACTRaman spectroscopy, a robust and non‐invasive analytical method, has demonstrated significant potential for monitoring biopharmaceutical production processes. Its ability to provide detailed information about molecular vibrations makes it ideal for the detection and quantification of therapeutic proteins and critical control parameters in complex biopharmaceutical mixtures. However, its application in Saccharomyces cerevisiae fermentations has been hindered by the inherent strong fluorescence background from the cells. This fluorescence interferes with Raman signals, compromising spectral data accuracy. In this study, we present an approach that mitigates this issue by deploying Raman spectroscopy on cell‐free media samples, combined with advanced chemometric modeling. This method enables accurate prediction of protein concentration and key process parameters, fundamental for the control and optimization of biopharmaceutical fermentation processes. Utilizing variable importance in projection (VIP) further enhances model robustness, leading to lower relative root mean squared error of prediction (RMSEP) values across the six targets studied. Our findings highlight the potential of Raman spectroscopy for real‐time, on‐line monitoring and control of complex microbial fermentations, thereby significantly enhancing the efficiency and quality of S. cerevisiae‐based biopharmaceutical production.

Heat Shock Factor 1 forms nuclear condensates and restructures the yeast genome before activating target genes

In insects and mammals, 3D genome topology has been linked to transcriptional states yet whether this link holds for other eukaryotes is unclear. Using both ligation proximity and fluorescence microscopy assays, we show that in Saccharomyces cerevisiae, Heat Shock Response (HSR) genes dispersed across multiple chromosomes and under the control of Heat Shock Factor (Hsf1) rapidly reposition in cells exposed to acute ethanol stress and engage in concerted, Hsf1-dependent intergenic interactions. Accompanying 3D genome reconfiguration is equally rapid formation of Hsf1-containing condensates. However, in contrast to the transience of Hsf1-driven intergenic interactions that peak within 10–20 min and dissipate within 1 hr in the presence of 8.5% (v/v) ethanol, transcriptional condensates are stably maintained for hours. Moreover, under the same conditions, Pol II occupancy of HSR genes, chromatin remodeling, and RNA expression are detectable only later in the response and peak much later (>1 hr). This contrasts with the coordinate response of HSR genes to thermal stress (39°C) where Pol II occupancy, transcription, histone eviction, intergenic interactions, and formation of Hsf1 condensates are all rapid yet transient (peak within 2.5–10 min and dissipate within 1 hr). Therefore, Hsf1 forms condensates, restructures the genome and transcriptionally activates HSR genes in response to both forms of proteotoxic stress but does so with strikingly different kinetics. In cells subjected to ethanol stress, Hsf1 forms condensates and repositions target genes before transcriptionally activating them.

Heat Shock Factor 1 forms nuclear condensates and restructures the yeast genome before activating target genes.

In insects and mammals, 3D genome topology has been linked to transcriptional states yet whether this link holds for other eukaryotes is unclear. Using both ligation proximity and fluorescence microscopy assays, we show that in Saccharomyces cerevisiae, Heat Shock Response (HSR) genes dispersed across multiple chromosomes and under the control of Heat Shock Factor (Hsf1) rapidly reposition in cells exposed to acute ethanol stress and engage in concerted, Hsf1-dependent intergenic interactions. Accompanying 3D genome reconfiguration is equally rapid formation of Hsf1-containing condensates. However, in contrast to the transience of Hsf1-driven intergenic interactions that peak within 10-20 min and dissipate within 1 hr in the presence of 8.5% (v/v) ethanol, transcriptional condensates are stably maintained for hours. Moreover, under the same conditions, Pol II occupancy of HSR genes, chromatin remodeling, and RNA expression are detectable only later in the response and peak much later (>1 hr). This contrasts with the coordinate response of HSR genes to thermal stress (39°C) where Pol II occupancy, transcription, histone eviction, intergenic interactions, and formation of Hsf1 condensates are all rapid yet transient (peak within 2.5-10 min and dissipate within 1 hr). Therefore, Hsf1 forms condensates, restructures the genome and transcriptionally activates HSR genes in response to both forms of proteotoxic stress but does so with strikingly different kinetics. In cells subjected to ethanol stress, Hsf1 forms condensates and repositions target genes before transcriptionally activating them.

Manipulating the nucleolar serine-rich protein Srp40p in Saccharomyces cerevisiae may improve isobutanol production.

Isobutanol represents a promising second-generation biofuel. Saccharomyces cerevisiae can produce minor quantities of isobutanol as a byproduct. Increasing yeast tolerance to isobutanol is a crucial step toward achieving higher production levels. Previously, we discovered that expression of the srp40 gene could increase S. cerevisiae isobutanol tolerance. In this study, we explored the impact of overexpressing srp40 on isobutanol production. We used the CEN/ARS plasmid YCplac22-srp40 to overexpress srp40 in S. cerevisiae strain W303-1A. The resulting strain was named W303-1A-srp40. We subsequently performed metabolic engineering of isobutanol synthesis by overexpressing ILV2, ILV3 and ARO10 in W303-1A-srp40. The resulting strain was named 303V2V3A10-22-srp40. Our findings revealed that, compared with the control strain, the 303V2V3A10-22-srp40 strain amplified isobutanol production by 50%. A transcriptome analysis revealed that upregulated genes associated with aminoacyl-tRNA biosynthesis or downregulated genes associated with phenylalanine, tyrosine, and tryptophan biosynthesis might yield increased isobutanol production in 303V2V3A10-22-srp40. Moreover, the decreases in the biosynthesis of amino acids and oxidative phosphorylation might play pivotal roles in the increased isobutanol tolerance of strain W303-1A-srp40. In summary, the overexpression of srp40 could increase isobutanol production and tolerance in S. cerevisiae. This study offers novel insights regarding strategies for increasing isobutanol production.

Yeast Solutions and Hyperpolarization Enable Real-Time Observation of Metabolized Substrates Even at Natural Abundance.

Metabolic changes in an organism often occur much earlier than macroscopic manifestations of disease, such as invasive tumors. Therefore, noninvasive tools to monitor metabolism are fundamental as they provide insights into in vivo biochemistry. NMR represents one of the gold standards for such insights by observing metabolites. Using nuclear spin hyperpolarization greatly increases the NMR sensitivity, enabling μmol/L sensitivity with a time resolution of about one second. However, a metabolic phantom with reproducible, rapid, and human-like metabolism is needed to progress research in this area. Using baker's yeast as a convenient metabolic factory, we demonstrated in a single study that yeast cells provide a robust and rapidly metabolizing phantom for pyruvate and fumarate, including substrates with a natural abundance of 13C: we observed the production of ethanol, carbon dioxide, bicarbonate, lactate, alanine from pyruvate, malate, and oxaloacetate from fumarate. For observation, we hyperpolarized pyruvate and fumarate via the dissolution dynamic nuclear polarization (dDNP) technique to about 30% 13C polarization that is equivalent to 360,000 signal enhancement at 1 T and 310 K. Major metabolic pathways were observed using tracers at a natural abundance of 13C, demonstrating that isotope labeling is not always essential in vitro. Enriched [1-13C]pyruvate revealed minor lactate production, presumably via the D-lactate dehydrogenase (DLD) enzyme pathway, demonstrating the sensitivity gain using a dense yeast solution. We foresee that yeast as a metabolic factory can find application as an abundant MRI phantom standard to calibrate and optimize molecular MRI protocols. Our study highlights the potential of using hyperpolarization to probe the metabolism of yeast and other microorganisms even with naturally abundant substrates, offering valuable insights into their response to various stimuli such as drugs, treatment, nourishment, and genetic modification, thereby advancing drug development and our understanding of biochemical processes.

Innovative Oral Nano/Gene Delivery System Based on Engineered Modified Saccharomyces cerevisiae for Colorectal Cancer Therapy.

The efficient delivery of RNA-based drugs to solid tumors remains a formidable obstacle. We aim to develop a safe and efficient oral drug delivery system compatible with RNA-based drugs that is urgently needed to overcome challenges such as enzymatic degradation and gastrointestinal barriers to facilitate effective treatment for treating colorectal cancer (CRC). To address these challenges, we utilized engineered modified Saccharomyces cerevisiae to evaluate the delivery efficacy of miR21-antagomir for treating CRC in preclinical mouse models, including adenomatosis polyposis coli mutant transgenic mice ApcMin/+ and in situ tumor-bearing mice. An orally deliverable gene delivery system, YS@NPs21, was designed. This gene delivery system demonstrated effectively suppressed tumor growth in both ApcMin/+ and in situ tumor-bearing mice models. This system exhibited tumor-targeting capability, effective inhibition of tumor growth, and low toxicity toward nontumor cells. Successful implementation of this innovative oral drug delivery system could offer a straightforward, safe, and RNA drug-compatible approach to CRC treatment, ultimately improving patient outcomes and reducing medical costs.