Yeast Saccharomyces Cerevisiae Research Articles

Optimizing hexanoic acid biosynthesis in Saccharomyces cerevisiae for the de novo production of olivetolic acid

Medium chain fatty acids (MCFAs) are valuable platform compounds for the production of biotechnologically relevant chemicals such as biofuels and biochemicals. Two distinct pathways have been implemented in the yeast Saccharomyces cerevisiae for the biosynthetic production of MCFAs: (i) the mutant fatty acid biosynthesis (FAB) pathway in which the fatty acid synthase (FAS) complex is mutated and (ii) a heterologous multispecies-derived reverse β-oxidation (rBOX) pathway. Hexanoic acid has become of great interest as its acyl-CoA ester, hexanoyl-CoA, is required for the biosynthesis of olivetolic acid (OA), a cannabinoid precursor. Due to insufficient endogenous synthesis of hexanoyl-CoA, recombinant microbial systems to date require exogenous supplementation of cultures with hexanoate along with the overexpression of an acyl-CoA ligase to allow cannabinoid biosynthesis. Here, we engineer a recombinant S. cerevisiae strain which was metabolically optimized for the production of hexanoic acid via the FAB and rBOX pathways and we combine both pathways in a single strain to achieve titers of up to 120 mg L−1. Moreover, we demonstrate the biosynthesis of up to 15 mg L−1 OA from glucose using hexanoyl-CoA derived from the rBOX pathway.

A fully automated morphological analysis of yeast mitochondria from wide-field fluorescence images

Mitochondrial morphology is an important parameter of cellular fitness. Although many approaches are available for assessing mitochondrial morphology in mammalian cells, only a few technically demanding and laborious methods are available for yeast cells. A robust, fully automated and user-friendly approach that would allow (1) segmentation of tubular and spherical mitochondria in the yeast Saccharomyces cerevisiae from conventional wide-field fluorescence images and (2) quantitative assessment of mitochondrial morphology is lacking. To address this, we compared Global thresholding segmentation with deep learning MitoSegNet segmentation, which we retrained on yeast cells. The deep learning model outperformed the Global thresholding segmentation. We applied it to segment mitochondria in strain lacking the MMI1/TMA19 gene encoding an ortholog of the human TCTP protein. Next, we performed a quantitative evaluation of segmented mitochondria by analyses available in ImageJ/Fiji and by MitoA analysis available in the MitoSegNet toolbox. By monitoring a wide range of morphological parameters, we described a novel mitochondrial phenotype of the mmi1Δ strain after its exposure to oxidative stress compared to that of the wild-type strain. The retrained deep learning model, all macros applied to run the analyses, as well as the detailed procedure are now available at https://github.com/LMCF-IMG/Morphology_Yeast_Mitochondria.

Antioxidant content following fermentation of lemongrass for herbal beverage development.

Consumers have increasingly favoured fermented drinks due to their high content of probiotic secondary metabolites. These beverages are believed to possess the capacity to safeguard against non-communicable ailments such as coronary heart disease, cancer, diabetes, antimicrobial infections, and other dietary-related disorders. Lemongrass (Cymbopogon citratus) is a commonly used botanical ingredient in therapeutic tea production. It is renowned for its highly valuable essential oil, which has significant commercial demand. This study examines the functional content and antioxidant effects of fermented beverages derived from lemongrass. We employed the yeast Saccharomyces cerevisiae to carry out the fermentation process on the lemongrass compositions, extending the duration from t = 24 to t = 96h. We used non-fermented samples as control. This investigation identified numerous active biomolecules and polyphenols in the fermented samples, including flavonoids, tannins, cardiac glycosides, coumarins, terpenoids, steroids, saponins, and reducing sugars. After t = 24h fermentation, the radical-scavenging activity reached its maximum level of 89.1%, and the antioxidant content reached 13.06µg/ml, which is equivalent to the amount of ascorbic acid. After t = 36h fermentation, the total phenolic content reached a concentration of 237.19µg/ml, while the flavonoid content reached its peak at 55.21µg/ml after t = 72h fermentation. Lemongrass fermentation exhibits a wide range of phytochemicals and bioactive components that effectively eliminate free radicals, despite the antioxidant content fluctuation throughout the fermentation period of t = 24 to t = 96h.

Engineering Komagataella phaffii for ethylene glycol production from xylose

Ethylene glycol (EG) is a versatile molecule produced in the petrochemical industry and is widely used to manufacture plastic polymers, anti-freeze, and automotive fluids. Biotechnological production of EG from xylose, a pentose present in lignocellulose biomass hydrolysates, has been achieved by the engineering of bacteria, such as Escherichia coli and Enterobacter cloacae, and the yeast Saccharomyces cerevisiae with synthetic pathways. In the present work, the Dahms pathway was employed to construct Komagataella phaffii strains capable of producing EG from xylose. Different combinations of the four enzymes that compose the synthetic pathway, namely, xylose dehydrogenase, xylonate dehydratase, dehydro-deoxy-xylonate aldolase, and glycolaldehyde reductase, were successfully expressed in K. phaffii. Increased production of EG (1.31 g/L) was achieved by employing a newly identified xylonate dehydratase (xylD-HL). This xylonate dehydratase allowed 30% higher EG production than a previously known xylonate dehydratase (xylD-CC). Further strain engineering demonstrated that K. phaffii possesses native glycolaldehyde reduction and oxidation activities, which lead to pathway deviation from EG to glycolic acid (GA) production. Finally, cultivation conditions that favor the production of EG over GA were determined.

Improved growth and immunity in Nile tilapia Oreochromis niloticus fed a fermented rice bran supplement

Improvement of growth performance and disease resistance of cultured species is an important objective of the aquaculture industry. In this study, solid-state fermentation (SSF) was applied for increasing the nutritional value of rice bran with baker's yeast (Saccharomyces cerevisiae). Four diets containing different levels of fermented rice bran (FRB) at 0, 100, 200 and 300 g/kg (FRB0, FRB10, FRB20 and FRB30, respectively) were tested using juvenile Nile tilapia Oreochromis niloticus (average body weight = 5.22±0.02 g) for 56 days. Compared to FRB0, all diets improved growth performance of the experimental fish (P<0.05). Intestinal amylase and protease amounts were significantly increased (P<0.05). The experimental fish were intraperitoneally injected with Streptococcus agalactiae and the cumulative mortality rate was monitored for 10 days. All FRB-supplemented diets resulted in greater survival rates in challenge fish. The FRB20 and FRB30 diets promoted expression of insulin-like growth factor I (IGF-I) transcripts and enhanced non-specific immunity; lysozyme and antioxidant enzyme activities; myeloperoxidase (MPO), catalase (CAT), and superoxide dismutase (SOD) (P<0.05). The expression level of interleukin 8 (IL-8) was down-regulated in fish fed FRB20 and FRB30 (P<0.05) but IL-10 was up-regulated in fish fed FRB10 and FRB30 (P<0.05) while IL-1β was up-regulated in fish fed FRB20 (P<0.05). The expression of complementary 3 (C3) transcripts was significantly increased while nuclear factor-kappa B (NF-κB) was decreased in fish fed all FRB-supplemented diets (P<0.05). Conventional histology revealed increased villus height following FRB30 treatment (P<0.05). These results suggest the beneficial use of FRB supplementation on growth, immune defense and stress tolerance for juvenile O. niloticus.

Expression of Brassica napus cell number regulator 6 (BnCNR6) in Arabidopsis thaliana confers tolerance to copper

Copper is an essential but potential toxic micro-nutrient in rapeseed. So far, little is known about the mechanism of rapeseed Cu transport and detoxification. Here, we determined the function of Cu transporter, Brassica napus cell number regulator 6 (BnCNR6), in regulating Cu homeostasis. BnCNR6 exhibited higher expression level in euphylla and root tips. It was found that in protoplasts and transgenic plants expressing Pro35S:BnCNR6-GFP, BnCNR6 was localized to the plasma membrane (PM). Expression of BnCNR6 in the yeast (Saccharomyces cerevisiae), compensated the Cu hypersensitivity of Δcup2 by promoting Cu2+ efflux. The overexpression of BnCNR6 in Arabidopsis athma5 mutant restored its growth, increased its photosynthesis, and reduced Cu2+ concentration in the roots. Furthermore, the roots of BnCNR6 overexpression lines had lower net Cu influx than in those of the athma5 mutant. These results revealed that BnCNR6 is a PM protein which is useful for detoxification to increase tolerance to Cu toxicity. Collectively, our study provides a theoretical basis for reducing Cu stress in rapeseed.

Biological Importance of Complex Sphingolipids and Their Structural Diversity in Budding Yeast Saccharomyces cerevisiae.

Complex sphingolipids are components of eukaryotic biomembranes and are involved in various physiological functions. In addition, their synthetic intermediates and metabolites, such as ceramide, sphingoid long-chain base, and sphingoid long-chain base 1-phosphate, play important roles as signaling molecules that regulate intracellular signal transduction systems. Complex sphingolipids have a large number of structural variations, and this structural diversity is considered an important molecular basis for their various physiological functions. The budding yeast Saccharomyces cerevisiae has simpler structural variations in complex sphingolipids compared to mammals and is, therefore, a useful model organism for elucidating the physiological significance of this structural diversity. In this review, we focus on the structure and function of complex sphingolipids in S. cerevisiae and summarize the response mechanisms of S. cerevisiae to metabolic abnormalities in complex sphingolipids.

Chemical structure characterization of polysaccharides using diffusion ordered NMR spectroscopy (DOSY)

The potential of DOSY NMR spectroscopy to distinguish the linkage pattern of chemically related polysaccharides was evaluated using β-glucans isolated from yeast (Saccharomyces cerevisiae) and mushroom (Pleurotus eryngii). Laminarin from Laminaria digitata was included for chemical shift comparison. Characterization through methylation and 1D/2D NMR analysis showed that all the samples were constituted by →3)-Glcp-(1→; →3,6)-Glcp-(1→; Glcp-(1→ and →6)-Glcp-(1→ linkages. The results obtained allowed the identification of the well-known chemical structure of laminarin. Moreover, DOSY demonstrated that the units →3)-Glcp-(1→ and →6)-Glcp-(1→ from yeast β-glucans, presented the same diffusion time. For the mushrooms β-glucans, the diffusion time of these units were different, confirming that they belong to distinct polysaccharides. The yeast and the mushroom polysaccharide samples presented the same NMR correlations, but after DOSY analysis, different structures could be proposed. Therefore, DOSY NMR spectroscopy could be a tool for the identification of different linkage patterns of polysaccharides belonging to the same group and may be a useful contribution to the chemical structure and biological activity correlation studies of such structures.

Saccharomyces Genome Database: Advances in Genome Annotation, Expanded Biochemical Pathways, and Other Key Enhancements.

Budding yeast (Saccharomyces cerevisiae) is the most extensively characterized eukaryotic model organism and has long been used to gain insight into the fundamentals of genetics, cellular biology, and the functions of specific genes and proteins. The Saccharomyces Genome Database (SGD) is a scientific resource that provides information about the genome and biology of S. cerevisiae. For more than 30 years, SGD has maintained the genetic nomenclature, chromosome maps, and functional annotation for budding yeast along with search and analysis tools to explore these data. Here we describe recent updates at SGD, including the two most recent reference genome annotation updates, expanded biochemical pathways representation, changes to SGD search and data files, and other enhancements to the SGD website and user interface. These activities are part of our continuing effort to promote insights gained from yeast to enable the discovery of functional relationships between sequence and gene products in fungi and higher eukaryotes.

Porous PEG Scaffold Fabricated via Emulsion-Templating Technique Towards Immobilization of Saccharomyces cerevisiae Cells

The cell immobilization technique, which restricts living cells to a certain space, has received widespread attention as an emerging biotechnology. In this study, a yeast (Saccharomyces cerevisiae)-loaded highly open-cell emulsion-templated polyethylene glycol (PEG-polyHIPE) was synthesized to be a reusable enzymatic catalyst. An emulsion was prepared with polyethylene glycol diacrylate (PEGDA) aqueous solution, cyclohexane, and polyethylene-polypropylene glycol (F127) as the continuous phase, dispersed phase, and surfactant, respectively. Then PEG-polyHIPE was obtained by polymerization of the PEGDA in emulsion. The highly porous materials obtained by the emulsion-templating method are suitable for use as carrier materials for yeast immobilization, due to their favorable structural designability. During the activation process, the yeast S. cerevisiae can readily gain access to the interior of the material via the interconnected pores and immobilize itself inside the voids. The yeast-loaded polyHIPE was then used to ferment glucose for ethanol production. The yeast immobilized inside the polyHIPE has high fermentation efficiency, good recoverability, and storage stability. After seven cycles, the yeast maintained 70% initial fermentation efficiency. The S. cerevisiae kept more than 90% of the initial cellular activity after one week of storage both in the dry state and in yeast extract peptone dextrose medium (YPD) at 4 °C. This study strongly demonstrates the feasibility of using high-throughput porous materials as cell immobilization carriers to efficiently osmotically immobilize cells in polyHIPEs for high-performance fermentation.

Graph-based machine learning model for weight prediction in protein–protein networks

Proteins interact with each other in complex ways to perform significant biological functions. These interactions, known as protein–protein interactions (PPIs), can be depicted as a graph where proteins are nodes and their interactions are edges. The development of high-throughput experimental technologies allows for the generation of numerous data which permits increasing the sophistication of PPI models. However, despite significant progress, current PPI networks remain incomplete. Discovering missing interactions through experimental techniques can be costly, time-consuming, and challenging. Therefore, computational approaches have emerged as valuable tools for predicting missing interactions. In PPI networks, a graph is usually used to model the interactions between proteins. An edge between two proteins indicates a known interaction, while the absence of an edge means the interaction is not known or missed. However, this binary representation overlooks the reliability of known interactions when predicting new ones. To address this challenge, we propose a novel approach for link prediction in weighted protein–protein networks, where interaction weights denote confidence scores. By leveraging data from the yeast Saccharomyces cerevisiae obtained from the STRING database, we introduce a new model that combines similarity-based algorithms and aggregated confidence score weights for accurate link prediction purposes. Our model significantly improves prediction accuracy, surpassing traditional approaches in terms of Mean Absolute Error, Mean Relative Absolute Error, and Root Mean Square Error. Our proposed approach holds the potential for improved accuracy in predicting PPIs, which is crucial for better understanding the underlying biological processes.

Phenotyping of a new yeast mapping population reveals differences in the activation of the TORC1 signalling pathway between wild and domesticated yeast strains

Domestication can be understood as a symbiotic relationship that benefits both domesticator and domesticated species, involving multiple genetic changes that configure the phenotype of the domesticated species. One of the most important domesticated species is the yeast Saccharomyces cerevisiae, with both domesticated strains used for different fermentations processes for thousands of years and wild strains existing only in environments without human intervention; however, little is known about the phenotypic effects associated with its domestication. In the present work, we studied the effect of domestication on yeast TORC1 activation, a pleiotropic signalling pathway conserved across the eukaryotic domain. To achieve this goal, we improved a previously generated methodology to assess TORC1 activation, which turned out to be as effective as the original one but also presents several practical advantages for its application (such as facilitating confirmation of transformants and putting the Luc reporter gene under the control of the same PRPL26A promoter for each transformed strain). We then generated a mapping population, the so-called TOMAN-G population, derived from the “1002 Yeast Genomes Project” population, the most comprehensive catalogue of the genetic variation in yeasts. Finally, strains belonging to the TOMAN-G population were phenotyped for TORC1 activation, and then we compared the results obtained between yeast strains with different ecological origins, finding differences in TORC1 activation between wild and domesticated strains, particularly wine strains. These results are indicative of the effect of domestication on TORC1 activation, specifically that the different evolutionary trajectories of wild and domesticated strains have in fact caused differences in the activation of this pathway; furthermore, the phenotypic data obtained in this work could be used to continue underlying the genetic bases of TORC1 activation, a process that is still not fully understood, using techniques such as GWAS to search for specific genetic variants underlying the observed phenotypic variability and phylogenetic tree inferences to gain insight into the evolutionary relationships between these genetic variants.

Functional analysis of a S-adenosylmethionine-insensitive methylenetetrahydrofolate reductase identified in methionine-accumulating yeast mutants.

Essential amino acids (EAAs) are important for the maintenance of brain functions. Therefore, the yeast Saccharomyces cerevisiae that accumulate EAAs would help elderly people ingest appropriate levels of EAAs, which in turn could slow neurodegeneration, extend the healthy lifespan and improve quality of life. Here, we isolated two mutant strains, ETH-80 and ETH-129, that accumulate the EAA methionine. Both strains were derived from a diploid laboratory yeast by conventional mutagenesis and carry a novel mutation in the MET13 gene, which encodes the Ser443Phe variant of methylenetetrahydrofolate reductase. Enzymatic analysis revealed that the Ser443Phe substitution abolished the sensitivity to S-adenosyl methionine (SAM)-mediated inhibition even in the presence of 2mM SAM, while increasing the activity for NADPH-dependent reduction. Furthermore, yeast cells expressing the Ser443Phe variant showed a fourfold increase in intracellular methionine content compared to the wild-type Met13. These findings will be useful for the future development of methionine-accumulating yeast strains.

Essential Oils of The Leaves of Syzygium hemilamprum (F. Muell.) Craven & Biffin.: Chemical Analysis, Antimicrobial, Mosquito Larvicidal, Molecular Docking, and ADMET Studies.

The chemical composition of the essential oil and n-hexane extract from Syzygium hemilamprum leaves was first performed. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that the essential oil was predominantly composed of monoterpene hydrocarbons (71.5 %) and oxygenated derivatives (20.2 %), with β-pinene (31.5 %), limonene (19.4 %), α-pinene (12.3 %), and α-terpineol (7.4 %) being the principal constituents. The n-hexane extract contained monoterpene hydrocarbons (42.2 %) and non-terpenic compounds (34.0 %), with β-pinene (32.8 %) and n-hexadecane (10.2 %) as the major components. Antimicrobial and mosquito larvicidal assays demonstrated that both samples exhibited antimicrobial activity against Staphylococcus aureus (Gram-positive), Pseudomonas aeruginosa (Gram-negative), and Saccharomyces cerevisiae (yeast), with a minimum inhibitory concentration (MIC) of 128 μg/mL for all tested organisms. Both samples also showed significant mosquito larvicidal activity against Aedes aegypti and Culex quinquefasciatus, with LC50 and LC90 values below 20 μg/mL at 24 and 48 hours post-treatment. Molecular docking studies suggested that limonene and α-terpineol could serve as potent inhibitors of mosquito odorant binding proteins. Additionally, an in silico analysis was performed to evaluate the physicochemical and ADMET (absorption, distribution, metabolism, and toxicity) properties of the major constituents of the essential oil.

Impact of live yeast and selenium supplementation on blood metabolites and rumen pH of young bulls after long-transport to the fattening unit

Long-distance transport and the receiving phase at the fattening unit are sources of stress for young beef cattle. This randomised controlled study involved 80 Charolais young bulls that underwent 12 h of transport from France to Italy and aimed at testing whether the animals have some benefits from the supplementation of live yeast and selenium through slow-release boluses and diet. The bulls were randomly allocated into two supplementation groups of 40 animals each, named Yeast and Control groups. Bulls of the Yeast group received a supplementation of Saccharomyces cerevisiae and selenium-enriched yeast (1.5 × 1010 CFU/bull per day of live yeast and 1.5 mg/bull per day of selenium) by two slow-release ruminal boluses 1 day before leaving France, and a live yeast supplemented diet once in Italy (8 × 109 CFU/bull per day of live yeast). Yeast and control bulls underwent the same manipulations. Individual BW and complete blood metabolic profile were assessed at the arrival to the Italian fattening unit (day 0), after 7 days (day 7), and at the end of the receiving phase (day 30). The rumen environment was continuously monitored through reticulum-rumen sensors that measured several parameters in a subsample of 60 bulls, equally distributed between Yeast and Control groups. Saccharomyces cerevisiae and selenium supplementation did not affect growth performance and metabolic profile. However, the supplementation stabilised the rumen environment by limiting the daily pH amplitude and SD and the inter-animal variability. The Yeast group increased the time spent ruminating (+39 min/day) at day 30 compared to Control group. More stable ruminal conditions are important to support beef cattle health during the receiving period at the fattening unit, when animals face the delicate transition to high-energy diets.

Exploring the Potential of Nyonya Kuih Residues as the Substrate for Yeast Cell Protein Production

Nyonya kuih, a popular Malaysian traditional cuisine that have short shelf life due to their high moisture and fat contents. The unsold kuihs usually ended up as food residue. Thus, kuih talam(KT) and kuih lapis (KL), two varieties of Nyonya kuih were chosen as the investigative substrates to explore the recycling potential of this food residue. Initially, the effect of fat content on the enzymatic hydrolysability of the residues to release fermentable sugar was investigated. The efficiency of lipase-pretreatment (LP), polyvinylpyrrolidone-post-treatment (PVP) and their combination in lipid removal was determined. Then, the potential of the resulting glucose-rich hydrolysates for Saccharomyces cerevisiae yeast cell biomass production was assessed, with molasses as the positive control. Results indicated that combined LP and PVP (LP-PVP) significantly (p&lt;0.05) reduced crude fat content, while increasing the total sugar content of KT and KL hydrolysates by approximately 3.4 and 26.3 times, respectively. Yeast cell biomass produced from KT and KL hydrolysates were increased by 23% and 39%, respectively after LP-PVP treatment. Despite the yeast cells’ yield (by dry cell weight) from molasses fermentation being 19% and 11% higher than those from KT and KL hydrolysates, respectively, KT and KL hydrolysates were proven superior due to the significantly higher (p&lt;0.05) crude protein content in yeast cell produced from both hydrolysates. Further study is needed to enhance yeast cell yield from the fermentation of KT and KL hydrolysates, thereby improving the overall protein yield from the fermentation process. This study provides new insight into a novel food residue recycle strategy, promoting sustainable food production aligned with SDG 12.

Using Native and Synthetic Genes to Disrupt Inositol Pyrophosphates and Phosphate Accumulation in Plants.

Inositol pyrophosphates are eukaryotic signaling molecules that have been recently identified as key regulators of plant phosphate sensing and homeostasis. Given the importance of phosphate to current and future agronomic practices, we sought to design plants which could be used to sequester phosphate, as a step in a phytoremediation strategy. To achieve this, we expressed Diadenosine and Diphosphoinositol Polyphosphate Phosphohydrolase (DDP1), a yeast (Saccharomyces cerevisiae) enzyme demonstrated to hydrolyze inositol pyrophosphates, in Arabidopsis thaliana and pennycress (Thlaspi arvense), a spring annual cover crop with emerging importance as a biofuel crop. DDP1 expression in Arabidopsis decreased inositol pyrophosphates, activated Phosphate Starvation Response marker genes, and increased phosphate accumulation. These changes corresponded with alterations in plant growth and sensitivity to exogenously applied phosphate. Pennycress plants expressing DDP1 displayed increases in phosphate accumulation, suggesting that these plants could potentially serve to reclaim phosphate from phosphate-polluted soils. We also identified a native Arabidopsis gene, Nucleoside diphosphate-linked moiety X 13 (NUDIX13), which we show encodes an enzyme homologous to DDP1 with similar substrate specificity. Arabidopsis transgenics overexpressing NUDIX13 had lower inositol pyrophosphate levels and displayed phenotypes similar to DDP1-overexpressing transgenics, while nudix13-1 mutants had increased levels of inositol pyrophosphates. Taken together, our data demonstrates that DDP1 and NUDIX13 can be used in strategies to regulate plant inositol pyrophosphates and could serve as potential targets for engineering plants to reclaim phosphate from polluted environments.

Not all cytochrome b5s are created equal: How a specific CytB5 boosts forskolin biosynthesis in Saccharomyces cerevisiae

Cytochrome B5s, or CytB5s, are small heme-binding proteins, ubiquitous across all kingdoms of life that serve mainly as electron donors to enzymes engaged in oxidative reactions. They often function as redox partners of the cytochrome P450s (CYPs), a superfamily of enzymes participating in multiple biochemical processes. In plants, CYPs catalyze key reactions in the biosynthesis of plant specialized metabolites with their activity dependent on electron donation often from cytochrome P450 oxidoreductases (CPRs or PORs). In eukaryotic microsomal CYPs, CytB5s frequently participate in the electron transfer process although their exact role remains understudied, especially in plant systems. In this study, we assess the role of CytB5s in the heterologous biotechnological production of plant specialized metabolites in yeast. For this, we used as a case-study the biosynthesis of forskolin - a bioactive diterpenoid produced exclusively from the plant Coleus forskohlii. The complete biosynthetic pathway for forskolin is known and includes three CYP enzymes. We reconstructed the entire forskolin pathway in the yeast Saccharomyces cerevisiae, and upon co-expression of the three CytB5s - identified in C. forskohlii transcriptomes - alleviation of a CYP-related bottleneck step was noticed only when a specific CytB5, CfCytB5A, was used. Co-expression of CfCytB5A in yeast, in combination with forskolin pathway engineering, resulted in forskolin production at titers of 1.81 g/L in a bioreactor. Our findings demonstrate that CytB5s not only play an important role in plant specialized metabolism but also, they can interact with precision with specific CYPs, indicating that the properties of CytB5s are far from understood. Moreover, our work highlights how CytB5s may act as indispensable components in the sustainable microbial production of plant metabolites, when their biosynthetic pathways involve CYP enzymes.

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.

Influence of yeasts on wine acidity: new insights into &lt;i&gt;Saccharomyces cerevisiae&lt;/i&gt;

Climate change is strongly affecting the winemaking sector, notably by decreasing wine acidity due to lower malic acid levels in the grapes. Wine-related microorganisms can greatly affect the organic acid contained in wines as they are able to metabolise or synthesise different acids. Major advances in biochemistry, ecophysiology and molecular biology have led to numerous yeast strains being selected for that have specific oenological properties, including acidity modulation. The yeast Saccharomyces cerevisiae is the most extensively studied species, harbouring both malic acid-consuming and producing strains which are of interest in various vinification itineraries. Yeast-derived acidification of wines can indeed be achieved via malic acid production by S. cerevisiae, as well as via lactic acid production by Lachancea thermotolerans. Co-fermentations of these two species become promising tools to manage wine acidity while ensuring fermentation completion and wine quality. Deacidification of wines via malic acid consumption is relevant in cooler winemaking regions, and/or for shortening malolactic fermentation and thereby increasing wine stability. This review delivers an in-depth overview of the effect of various oenologically relevant yeasts on wine acidity, with a focus on the latest findings on novel (de)acidifying S. cerevisiae strains.