Involvement of 2-deoxyglucose-6-phosphate phosphatases in facilitating resilience against ionic and osmotic stress in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae DOG genes, DOG1 and DOG2, encode for 2-deoxyglucose-6-phosphate phosphatases. These enzymes of the haloacid dehalogenase superfamily are known to utilize the non-natural 2-deoxyglucose-6-phosphate as their substrate. However, their physiological substrate and hence their biological role remain elusive. In this study, we investigated their potential role as enzymes in biosynthesizing glycerol through an alternative pathway, which involves the dephosphorylation of dihydroxyacetone phosphate into dihydroxyacetone, as opposed to the classical pathway which utilizes glycerol 3-phosphate. Overexpression of DOG1 or DOG2 rescued the osmotic and ionic stress-sensitive phenotype of gpp1∆ gpp2∆ or gpd1∆ gpd2∆ mutants, both affected in the production of glycerol. While small amounts of glycerol were observed in the DOG overexpression strains in the gpp1∆ gpp2∆ background, no glycerol was detected in the gpd1∆ gpd2∆ mutant background. This indicates that overexpression of the DOG enzymes can rescue the osmosensitive phenotype of the gpd1∆ gpd2∆ mutant independent of glycerol production. We also did not observe a drop in glycerol levels in the gpp1∆ gpp2∆ dog1∆ dog2∆ as compared to the gpp1∆ gpp2∆ mutant, indicating that the Dog enzymes are not involved in glycerol biosynthesis. This indicates that Dog enzymes have a distinct substrate and their function within the cell remains undiscovered. IMPORTANCE: Yeast stress tolerance is an important characteristic that is studied widely, not only regarding its fundamental insights but also for its applications within the biotechnological industry. Here, we investigated the function of two phosphatase encoding genes, DOG1 and DOG2, which are induced as part of the general stress response pathway, but their natural substrate in the cells remains unclear. They are known to dephosphorylate the non-natural substrate 2-deoxyglucose-6-phosphate. Here, we show that overexpression of these genes overcomes the osmosensitive phenotype of mutants that are unable to produce glycerol. However, in these overexpression strains, very little glycerol is produced indicating that the Dog enzymes do not seem to be involved in a previously predicted alternative pathway for glycerol production. Our work shows that overexpression of the DOG genes may improve osmotic and ionic stress tolerance in yeast.

Altered S-AdenosylMethionine availability impacts dNTP pools in Saccharomyces cerevisiae.

Saccharomyces cerevisiae has long been used as a model organism to study genome instability. The SAM1 and SAM2 genes encode AdoMet synthetases, which generate S-AdenosylMethionine (AdoMet) from Methionine (Met) and ATP. Previous work from our group has shown that deletions of the SAM1 and SAM2 genes cause changes to AdoMet levels and impact genome instability in opposite manners. AdoMet is a key product of methionine metabolism and the major methyl donor for methylation events of proteins, RNAs, small molecules, and lipids. The methyl cycle is interrelated to the folate cycle which is involved in de novo synthesis of purine and pyrimidine deoxyribonucleotides (dATP, dTTP, dCTP, and dGTP). AdoMet also plays a role in polyamine production, essential for cell growth and used in detoxification of reactive oxygen species (ROS) and maintenance of the redox status in cells. This is also impacted by the methyl cycle's role in production of glutathione, another ROS scavenger and cellular protectant. We show here that sam2∆/sam2∆ cells, previously characterized with lower levels of AdoMet and higher genome instability, have a higher level of each dNTP (except dTTP), contributing to a higher overall dNTP pool level when compared to wildtype. Unchecked, these increased levels can lead to multiple types of DNA damage which could account for the genome instability increases in these cells.

Genome sequence data of Saccharomyces cerevisiae CBS 493.94.

Whole genome sequencing (WGS) and data concerning identity and safety for Saccharomyces cerevisiae CBS 493.94 are reported. This strain was isolated from a British brewery in 1958 and deposited at the CBS culture collection Westerdijk Fungal Biodiversity Institute under the accession number CBS 493.94. The long-reads sequencing data, obtained via PacBio Sequel, and short-reads data, via Illumina NovaSeq 6000, were deposited at NCBI under accession number PRJNA1044661. The hybrid assembly was made publicly available via Zenodo and NCBI. For strain identification, data from 18S rRNA, ANI dendrogram and Core Genome single nucleotide polymorphism (SNP) Tree showed that the present isolate belongs to the genus Saccharomyces, species cerevisiae. The potential genes of concern, e.g. antimycotic resestance genes, were not detected. This strain is commonly used as a feed additive for animal health improvement and the present data summarise the unambiguous identity and strain's FKS1 gene does not code for any amino acid variants of concern.

Distinct regions of its first intracellular loop contribute to the proper localization, transport activity and substrate-affinity adjustment of the main yeast K+ importer Trk1.

Trk1 is the main K+ importer of Saccharomyces cerevisiae. Its proper functioning enables yeast cells to grow in environments with micromolar amounts of K+. Although the structure of Trk1 has not been experimentally determined, the transporter is predicted to be composed of four MPM (transmembrane segment - pore loop - transmembrane segment) motifs which are connected by intracellular loops. Of those, in particular the first loop (IL1) is unique in its length; it forms more than half of the entire protein. The deletion of the majority of IL1 does not abolish the transport activity of Trk1. However IL1 is thought to be involved in the modulation of the transporter's functioning. In this work, we prepared a series of internally shortened versions of Trk1 that lacked various parts of IL1, and we studied their properties in S. cerevisiae cells without chromosomal copies of TRK genes. Using this approach, we were able to determine that both N- and C-border regions of IL1 are necessary for the proper localization of Trk1. Moreover, the N-border part of IL1 is also important for the functioning of Trk1, as its absence resulted in a decrease in the transporter's substrate affinity. In addition, in the internal part of IL1, we newly identified a stretch of amino-acid residues that are indispensable for retaining the transporter's maximum velocity, and another region whose deletion affected the ability of Trk1 to adjust its affinity in response to external levels of K+.

Diet supplementation with Saccharomyces cerevisiae influences the electrophoretic parameters in blood in young Charolaise bulls.

Introduction: The objective of the research was to investigate the effect of Saccharomyces cerevisiae supplementation on some acute-phase proteins, haptoglobin and all electrophoretic parameters in young Charolaise bulls. Material and Methods: Sixty bulls were divided into two equal groups: the control group (CG) receiving the base diet without yeast supplementation and the diet supplementation group (YG) receiving the base diet with 5g of Saccharomyces cerevisiae supplementation. The base diet was total mixed ration allocated at 11.85 kg per animal per day. Blood samples were collected from all bulls on day 0 before the start of the diet supplementation, and on days 20 and 40 after the start. Total proteins, albumin, globulin fraction (α1-, α2-, ß1-, ß2- and γ-globulins), albumin: globulin ratio (A: G) and haptoglobin were determined. Results: Two-way analysis of variance showed a significant effect of the yeast feeding time on all studied parameters except α2-globulins in both groups. The YG showed a higher average concentration of total proteins, albumin and A: G and a lower average concentration of γ-globulins and haptoglobin than the CG. Conclusion: These results indicated the beneficial effect of the Saccharomyces cerevisiae on the inflammatory status of the young bulls, which showed an adequate response in serum levels of the acute-phase proteins tested.

Extracellular Self-DNA Effects on Yeast Cell Cycle and Transcriptome during Batch Growth.

The cell cycle and the transcriptome dynamics of yeast exposed to extracellular self-DNA during an aerobic batch culture on glucose have been investigated using cytofluorimetric and RNA-seq analyses. In parallel, the same study was conducted on yeast cells growing in the presence of (heterologous) nonself-DNA. The self-DNA treatment determined a reduction in the growth rate and a major elongation of the diauxic lag phase, as well as a significant delay in the achievement of the stationary phase. This was associated with significant changes in the cell cycle dynamics, with slower exit from the G0 phase, followed by an increased level of cell percentage in the S phase, during the cultivation. Comparatively, the exposure to heterologous DNA did not affect the growth curve and the cell cycle dynamics. The transcriptomic analysis showed that self-DNA exposure produced a generalized downregulation of transmembrane transport and an upregulation of genes associated with sulfur compounds and the pentose phosphate pathway. Instead, in the case of the nonself treatment, a clear response to nutrient deprivation was detected. Overall, the presented findings represent further insights into the complex functional mechanisms of self-DNA inhibition.

Short-term adaptation as a tool to improve bioethanol production using grass press-juice as fermentation medium.

Grass raw materials collected from grasslands cover more than 30% of Europe's agricultural area. They are considered very attractive for the production of different biochemicals and biofuels due to their high availability and renewability. In this study, a perennial ryegrass (Lolium perenne) was exploited for second-generation bioethanol production. Grass press-cake and grass press-juice were separated using mechanical pretreatment, and the obtained juice was used as a fermentation medium. In this work, Saccharomyces cerevisiae was utilized for bioethanol production using the grass press-juice as the sole fermentation medium. The yeast was able to release about 11 g/L of ethanol in 72 h, with a total production yield of 0.38 ± 0.2 gEthanol/gsugars. It was assessed to improve the fermentation ability of Saccharomyces cerevisiae by using the short-term adaptation. For this purpose, the yeast was initially propagated in increasing the concentration of press-juice. Then, the yeast cells were re-cultivated in 100%(v/v) fresh juice to verify if it had improved the fermentation efficiency. The fructose conversion increased from 79 to 90%, and the ethanol titers reached 18 g/L resulting in a final yield of 0.50 ± 0.06 gEthanol/gsugars with a volumetric productivity of 0.44 ± 0.00 g/Lh. The overall results proved that short-term adaptation was successfully used to improve bioethanol production with S. cerevisiae using grass press-juice as fermentation medium. KEY POINTS: • Mechanical pretreatment of grass raw materials • Production of bioethanol using grass press-juice as fermentation medium • Short-term adaptation as a tool to improve the bioethanol production.

Metabolic Engineering of Saccharomyces cerevisiae for Production of Canthaxanthin, Zeaxanthin, and Astaxanthin.

The sustainable production of natural compounds is increasingly important in today's industrial landscape. This study investigates the metabolic engineering of Saccharomyces cerevisiae for the efficient biosynthesis of valuable carotenoids: canthaxanthin, zeaxanthin, and astaxanthin. Utilizing a tailored parental yeast strain, Sp_Bc, we optimized the carotenoid pathway by screening and identifying CrtW and CrtZ enzymatic variants. The CrtW variant from Bradyrhizobium sp. achieved a canthaxanthin titer of 425.1 ± 69.1 µg/L, while the CrtZ variant from Pantoea ananatis achieved a zeaxanthin titer of 70.5 ± 10.8 µg/L. Additionally, we optimized carotenoid production by exploring enzyme fusion strategies for all three studied carotenoids and organelle compartmentalization specifically for enhancing astaxanthin synthesis. We further improved carotenoid production by integrating the optimal gene constructs into the yeast genome and deleting the GAL80 gene, enabling the use of sucrose as a carbon source. The engineered strain Sp_Bc-Can001 ∆gal80 was evaluated in a 5 L bioreactor fermentation, achieving a notable canthaxanthin titer of 60.36 ± 1.51 mg/L using sucrose. This research conclusively establishes S. cerevisiae as a viable platform for efficient carotenoid biosynthesis and, for the first time in this yeast system, illustrates sucrose's viability as a carbon source for canthaxanthin production. These findings pave the way for sustainable, cost-effective carotenoid production at an industrial scale.

The distribution of beneficial mutational effects between two sister yeast species poorly explains natural outcomes of vineyard adaptation.

Domesticated strains of Saccharomyces cerevisiae have adapted to resist copper and sulfite, two chemical stressors commonly used in winemaking. S. paradoxus, has not adapted to these chemicals despite being consistently present in sympatry with S. cerevisiae in vineyards. This contrast represents a case of apparent evolutionary constraints favoring greater adaptive capacity in S. cerevisiae. In this study, we used a comparative mutagenesis approach to test whether S. paradoxus is mutationally constrained with respect to acquiring greater copper and sulfite resistance. For both species, we assayed the rate, effect size, and pleiotropic costs of resistance mutations and sequenced a subset of 150 mutants isolated from our screen. We found that the distributions of mutational effects displayed by the two species were very similar and poorly explained the natural pattern. We also found that chromosome VIII aneuploidy and loss of function mutations in PMA1 confer copper resistance in both species, whereas loss of function mutations in REG1 were only a viable route to copper resistance in S. cerevisiae. We also observed a single de novo duplication of the CUP1 gene in S. paradoxus but none in S. cerevisiae. For sulfite, loss of function mutations in RTS1 and KSP1 confer resistance in both species, but mutations in RTS1 have larger average effects in S. paradoxus. Our results show that even when the distributions of mutational effects are largely similar, species can differ in the adaptive paths available to them. They also demonstrate that assays of the distribution of mutational effects may lack predictive insight concerning adaptive outcomes.

Dual auxotrophy coupled red labeling strategy for efficient genome editing in Saccharomyces cerevisiae.

The homologous recombination strategy has a long history of editing Saccharomyces cerevisiae target genes. The application of CRISPR/Cas9 strategy to editing target genes in S. cerevisiae has also received a lot of attention in recent years. All findings seem to indicate that editing relevant target genes in S. cerevisiae is an extremely easy event. In this study, we systematically analyzed the advantages and disadvantages of homologous recombination (HR) strategy, CRISPR/Cas9 strategy, and CRISPR/Cas9 combined homology-mediated repair (CRISPR/Case9-HDR) strategy in knocking out BY4742 ade2. Our data showed that when the ade2 was knocked out by HR strategy, a large number of clones appeared to be off-target, and 10 %-80 % of the so-called knockout clones obtained were heteroclones. When the CRISPR/Cas9 strategy was applied, 60% of clones were off-target and the rest were all heteroclones. Interestingly, most of the cells were edited successfully, but at least 60 % of the clones were heteroclones, when the CRISPR/Cas9-HDR strategy was employed. Our results clearly showed that the emergence of heteroclone seems inevitable regardless of the strategies used for editing BY4742 ade2. Given the characteristics of BY4742 defective in ade2 showing red on the YPD plate, we attempted to build an efficient yeast gene editing strategy, in which the CRISPR/Cas9 combines homology-mediated repair template carrying an ade2 expression cassette, BY4742(ade2Δ0) as the start strain. We used this strategy to successfully achieve 100 % knockout efficiency of trp1, indicating that technical challenges of how to easily screen out pure knockout clones without color phenotype have been solved. Our data showed in this study not only establishes an efficient yeast gene knockout strategy with dual auxotrophy coupled red labeling but also provides new ideas and references for the knockout of target genes in the monokaryotic mycelium of macrofungi.

Transcriptomic-metabolomic analysis reveals the effect of copper toxicity on fermentation properties in Saccharomyces cerevisiae.

Copper is one of the unavoidable heavy metals in wine production. In this study, the effects on fermentation performance and physiological metabolism of Saccharomyces cerevisiae under copper stress were investigated. EC1118 was the most copper-resistant among the six strains. The ethanol accumulation of EC1118 was 26.16-20 mg/L Cu2+, which was 1.90-3.15 times higher than that of other strains. The fermentation rate was significantly reduced by copper, and the inhibition was relieved after 4-10 days of adjustment. Metabolomic-transcriptomic analysis revealed that amino acid and nucleotide had the highest number of downregulated and upregulated differentially expressed metabolites, respectively. The metabolism of fructose and mannose was quickly affected, which then triggered the metabolism of galactose in copper stress. Pathways such as oxidative and organic acid metabolic processes were significantly affected in the early time, resulting in a significant decrease in the amount of carboxylic acids. The pathways related to protein synthesis and metabolism under copper stress, such as translation and peptide biosynthetic process, was also significantly affected. In conclusion, this study analyzed the metabolite-gene interaction network and molecular response during the alcohol fermentation of S. cerevisiae under copper stress, providing theoretical basis for addressing the influence of copper stress in wine production.

New advances in protein engineering for industrial applications: Key takeaways.

Recent advancements in protein/enzyme engineering have enabled the production of a diverse array of high-value compounds in microbial systems with the potential for industrial applications. The goal of this review is to articulate some of the most recent protein engineering advances in bacteria, yeast, and other microbial systems to produce valuable substances. These high-value substances include α-farnesene, vitamin B12, fumaric acid, linalool, glucaric acid, carminic acid, mycosporine-like amino acids, patchoulol, orcinol glucoside, d-lactic acid, keratinase, α-glucanotransferases, ß-glucosidase, seleno-methylselenocysteine, fatty acids, high-efficiency ß-glucosidase enzymes, cellulase, ß-carotene, physcion, and glucoamylase. Additionally, recent advances in enzyme engineering for enhancing thermostability will be discussed. These findings have the potential to revolutionize various industries, including biotechnology, food, pharmaceuticals, and biofuels.

[Advances in the biosynthesis and metabolic regulation of terpenoids in Saccharomyces cerevisiae].

Terpenoids are the one of most abundant natural products. With diverse varieties and biological activities, they are widely used in the food, medicine, chemical industry, and novel fuels. However, the conventional methods such as plant extraction and chemical synthesis cannot meet the current market demand for terpenoids. Efficient microbial cell factories, especially engineered Saccharomyces cerevisiae strains, have been constructed for the industrial production of terpenoids. In recent years, researchers have constructed multiple S. cerevisiae strains with increased yield and productivity via approaches of synthetic biology and metabolic engineering. This paper reviews the recent progress in the biosynthesis of terpenoids in S. cerevisiae cells and summarizes a variety of metabolic engineering strategies for the production of terpenoids in S. cerevisiae. These strategies include the construction and optimization of metabolic pathways, the mining and modification of key enzymes, the regeneration of cofactors, the engineering of cell localization and cell efflux, and the improvement of cell tolerance. Our review will provide information and strategies for the effective biosynthesis of terpenoids in S. cerevisiae.

[Stable integration sites in Saccharomyces cerevisiae: identification and application in the biosynthesis of valencene].

Valencene, a high-value sesquiterpene with a citrus aroma, is widely employed in the food and cosmetic fields and the industrial synthesis of nootkatone. In this study, 16 genomic loci in the intergenic regions (IGRs) of Saccharomyces cerevisiae were identified. A Ypet expression cassette was successfully integrated into various genomic loci by CRISPR-Cas9, with an impressive integration success rate of 87.50% and exhibiting expression variations of up to 1.91-fold depending on the insertion site. The study demonstrates that the positional effect exhibits relative stability in gene expression, and is essentially unaffected by changes in promoters and reporter genes. Furthermore, a high-expression element combination, PTDH3-TPRC1, was selected. The iterative integration of the valencene synthase gene VSm from Callitropsis nootkatensis at the selected loci increased the valencene yield to 254.67 mg/L. Overexpression of key genes tHMG1-ERG20 with multiple copies increased the valencene yield by 93.49%. The engineered strain L-13 achieved the valencene yield of 9 530.18 mg/L by two-stage fed-batch fermentation in a 3 L fermenter. This yield represents a nearly 100-fold increase compared with that of the starting strain, highlighting the significant potential of the screened genomic loci in optimizing valencene production.

[Construction and culture condition optimization of a Saccharomyces cerevisiae strain for production of galactitol].

Galactitol, a rare sugar alcohol, has promising potential in the food industry and pharmaceutical field. The available industrial production methods rely on harsh hydrogenation processes, which incur high costs and environmental concerns. It is urgent to develop environmentally friendly and efficient biosynthesis technologies. In this study, a xylose reductase named AnXR derived from Aspergillus niger CBS 513.88 was identified and characterized for the enzymatic properties. AnXR exhibited the highest activity at 25 ℃ and pH 8.0, and it belonged to the NADPH-dependent aldose reductase family. To engineer a strain for galactitol production, we deleted the galactokinase (GAL1) gene in Saccharomyes cerevisiae by using the recombinant gene technology, which significantly reduced the metabolic utilization of D-galactose by host cells. Subsequently, we introduced the gene encoding AnXR into this modified strain, creating an engineered strain capable of catalyzing the conversion of D-galactose into galactitol. Furthermore, we optimized the whole-cell catalysis conditions for the engineered strain, which achieved a maximum galactitol yield of 12.10 g/L. Finally, we tested the reduction ability of the strain for other monosaccharides and discovered that it could produce functional sugar alcohols such as xylitol and arabinitol. The engineered strain demonstrates efficient biotransformation capabilities for galactitol and other functional sugar alcohols, representing a significant advancement in environmentally sustainable production practices.

Oxidative effects of the human antifungal drug clotrimazole on the eucaryotic model organism Saccharomyces cerevisiae.

Clotrimazole is a type of antifungal medication developed from azole compounds. It exhibits several biological actions linked to oxidative stress. This study focuses on the oxidative effects of clotrimazole on the eukaryotic model yeast, Saccharomyces cerevisiae. Our results showed that although initial nitric oxide levels were above control in clotrimazole exposed cells, they showed decreasing tendencies from the beginning of incubation and dropped below control at 125 µM from the 60th min. The highest superoxide anion and hydrogen peroxide levels were 1.95- and 2.85-folds of controls at 125 µM after 15 and 60 min, respectively. Hydroxyl radical levels slightly increased throughout the incubation period in all concentrations and reached 1.3-fold of control, similarly at 110 and 125 µM in the 90th min. The highest level of reactive oxygen species was observed at 110 µM, 2.31-fold of control. Although NADH/NADPH oxidase activities showed similar tendencies for all conditions, the highest activities were found as 3.07- and 2.27-folds of control at 125 and 110 µM in the 15th and 30th min, respectively. The highest superoxide dismutase and catalase activities were 1.59- and 1.21-folds of controls at 110 µM clotrimazole in 30 and 90 min, respectively. While the drug generally induced glutathione-related enzyme activities, the ratios of glutathione to oxidized glutathione were above the control only at low concentrations of the drug. The levels of lipid peroxidation in all treated cells were significantly higher than the controls. The findings crucially demonstrate that this medicine can generate serious oxidative stress in organisms.

Overexpression of arginase gene CAR1 renders yeast Saccharomyces cerevisiae acetic acid tolerance.

Acetic acid is a common inhibitor present in lignocellulose hydrolysate, which inhibits the ethanol production by yeast strains. Therefore, the cellulosic ethanol industry requires yeast strains that can tolerate acetic acid stress. Here we demonstrate that overexpressing a yeast native arginase-encoding gene, CAR1, renders Saccharomyces cerevisiae acetic acid tolerance. Specifically, ethanol yield increased by 27.3% in the CAR1-overexpressing strain compared to the control strain under 5.0 g/L acetic acid stress. The global intracellular amino acid level and compositions were further analyzed, and we found that CAR1 overexpression reduced the total amino acid content in response to acetic acid stress. Moreover, the CAR1 overexpressing strain showed increased ATP level and improved cell membrane integrity. Notably, we demonstrated that the effect of CAR1 overexpression was independent of the spermidine and proline metabolism, which indicates novel mechanisms for enhancing yeast stress tolerance. Our studies also suggest that CAR1 is a novel genetic element to be used in synthetic biology of yeast for efficient production of fuel ethanol.

RNAi-based Boolean gates in the yeast Saccharomyces cerevisiae.

Boolean gates, the fundamental components of digital circuits, have been widely investigated in synthetic biology because they permit the fabrication of biosensors and facilitate biocomputing. This study was conducted to design and construct Boolean gates in the yeast Saccharomyces cerevisiae, the main component of which was the RNA interference pathway (RNAi) that is naturally absent from the budding yeast cells. We tested different expression cassettes for the siRNA precursor (a giant hairpin sequence, a DNA fragment-flanked by one or two introns-between convergent promoters or transcribed separately in the sense and antisense directions) and placed different components under the control of the circuit inputs (i.e., the siRNA precursor or proteins such as the Dicer and the Argonaute). We found that RNAi-based logic gates are highly sensitive to promoter leakage and, for this reason, challenging to implement in vivo. Convergent-promoter architecture turned out to be the most reliable solution, even though the overall best performance was achieved with the most difficult design based on the siRNA precursor as a giant hairpin.

Induction of point and structural mutations in engineered yeast Saccharomyces cerevisiae improve carotenoid production.

ß-Carotene is an attractive compound and that its biotechnological production can be achieved by using engineered Saccharomyces cerevisiae. In a previous study, we developed a technique for the efficient establishment of diverse mutants through the introduction of point and structural mutations into the yeast genome. In this study, we aimed to improve ß-carotene production by applying this mutagenesis technique to S. cerevisiae strain that had been genetically engineered for ß-carotene production. Point and structural mutations were introduced into ß-carotene-producing engineered yeast. The resulting mutants showed higher ß-carotene production capacity than the parental strain. The top-performing mutant, HP100_74, produced 37.6 mg/L of ß-carotene, a value 1.9 times higher than that of the parental strain (20.1 mg/L). Gene expression analysis confirmed an increased expression of multiple genes in the glycolysis, mevalonate, and ß-carotene synthesis pathways. In contrast, expression of ERG9, which functions in the ergosterol pathway competing with ß-carotene production, was decreased in the mutant strain. The introduction of point and structural mutations represents a simple yet effective method for achieving mutagenesis in yeasts. This technique is expected to be widely applied in the future to produce chemicals via metabolic engineering of S. cerevisiae.

Enzyme and Pathway Engineering for Improved Betanin Production in Saccharomyces cerevisiae.

Betanin is a water-soluble red-violet pigment belonging to the betacyanins family. It has become more and more attractive for its natural food colorant properties and health benefits. However, the commercial production of betanin, typically extracted from red beetroot, faces economic and sustainability challenges. Microbial heterologous production therefore offers a promising alternative. Here, we performed combinatorial engineering of plant P450 enzymes and precursor metabolisms to improve the de novo production of betanin in Saccharomyces cerevisiae. Semirational design by computer simulation and molecular docking was used to improve the catalytic activity of CYP76AD. Alanine substitution and site-directed saturation mutants were screened, with a combination mutant showing an approximately 7-fold increase in betanin titer compared to the wild type. Subsequently, betanin production was improved by enhancing the l-tyrosine pathway flux and UDP-glucose supply. Finally, after optimization of the fermentation process, the engineered strain BEW10 produced 134.1 mg/L of betanin from sucrose, achieving the highest reported titer of betanin in a shake flask by microbes. This work shows the P450 enzyme and metabolic engineering strategies for the efficient microbial production of natural complex products.