Saccharomyces Cerevisiae BY4741 Research Articles

Different transcriptomic and metabolomic analysis of Saccharomyces cerevisiae BY4742 and CEN.PK2-1C strains.

To establish efficient yeast cell factories, it is necessary to understand the transcriptional and metabolic changes among different yeasts. Saccharomyces cerevisiae BY4742 and CEN.PK2-1C strains are originated from different yeast strains and are commonly used as model organisms and chassis cells in molecular biology study and synthetic biology-based natural production. Metabolomic analysis showed that the BY4742 strain produced higher levels of phenylalanine, tyrosine than CEN.PK2-1C, while CEN.PK2-1C produced high levels of indoleacetaldehyde, indolepyruvate. Transcriptomic analysis showed that the two strains showed large differences in the glycolysis pathway and pyruvate metabolism pathway. CEN.PK2-1C had greater glycolysis flux than BY4742, whereas BY4742 has greater flux in the pathway of pyruvate metabolism to produce fumarate. These findings provide a basis knowledge of the metabolomic and transcriptomic differences between BY4742 and CEN.PK2-1C strains, and also provide preliminary information for strain selection for molecular biology study and synthetic biology-based natural product production.

Unveiling superior phenol detoxification and degradation ability in Candida tropicalis SHC-03: a comparative study with Saccharomyces cerevisiae BY4742.

This study examined the phenol degradation capabilities and oxidative stress responses of Candida tropicalis SHC-03, demonstrating its metabolic superiority and resilience compared to Saccharomyces cerevisiae BY4742 in a culture medium with phenol as the sole carbon source. Through comparative growth, transcriptomic, and metabolomic analyses under different phenol concentrations, this study revealed C. tropicalis SHC-03's specialized adaptations for thriving in phenol as the sole carbon source environments. These include a strategic shift from carbohydrate metabolism to enhanced phenol degradation pathways, highlighted by the significant upregulation of genes for Phenol 2-monoxygenase and Catechol 1,2-dioxygenase. Despite phenol levels reaching 1.8 g/L, C. tropicalis exhibits a robust oxidative stress response, efficiently managing ROS through antioxidative pathways and the upregulation of genes for peroxisomal proteins like PEX2, PEX13, and PMP34. Concurrently, there was significant upregulation of genes associated with membrane components and transmembrane transporters, enhancing the cell's capacity for substance exchange and signal transduction. Especially, when the phenol concentration was 1.6 g/L and 1.8 g/L, the degradation rates of C. tropicalis towards it were 99.47 and 95.91%, respectively. Conversely, S. cerevisiae BY4742 shows limited metabolic response, with pronounced growth inhibition and lack of phenol degradation. Therefore, our study not only sheds light on the molecular mechanisms underpinning phenol tolerance and degradation in C. tropicalis but also positions this yeast as a promising candidate for environmental and industrial processes aimed at mitigating phenol pollution.

Inhibitor Tolerance Capacity of Pichia kudriavzevii NBRC1279 and NBRC1664

The thermotolerant yeast Pichia kudriavzevii (previously known as Issatchenkia orientalis), can produce ethanol from a variety of carbon sources and grows at around 45 °C. Thus, this yeast is considered a useful biocatalyst for producing ethanol from lignocellulose through simultaneous saccharification and fermentation (SSF). SSF has several advantages, such as a simplified manufacturing process, ease of operation and reduced energy input. Using P. kudriavzevii NBRC1279 and NBRC1664, we previously succeeded in producing ethanol through SSF; however, the extent to which inhibitors by-produced from lignocellulose hydrolysis affect the growth and ethanol productivity of the two strains remains to be investigated. In this study, to better understand the inhibitor tolerance capacity of the two strains, spot assay, growth experiment, real-time quantitative PCR (RT-qPCR) analysis and multiple sequence alignment analysis were carried out. When P. kudriavzevii NBRC1279 and NBRC1664, as well as Saccharomyces cerevisiae BY4742 as a control, were cultured on SCD plates containing 17% ethanol, 42 mM furfural, 56 mM 5-hydroxymethylfurfural (HMF) or 10 mM vanillin, only P. kudriavzevii NBRC1664 was able to grow under all conditions. Moreover, the inhibitor tolerance capacity of P. kudriavzevii NBRC1664 was greater than those of other strains using SCD medium containing the same concentrations of various inhibitors. When an RT-qPCR analysis of seven gene sequences from aldehyde dehydrogenase and the aldehyde dehydrogenase family protein (ADHF) was performed using P. kudriavzevii NBRC1664 cultivated in the presence of 56 mM HMF, ADHF1 and ADHF2 were up-regulated in the early logarithmic growth phase. Moreover, a multiple sequence alignment of the amino acid sequences of ADHF1, ADHF2 and the known ADH suggested that ADHF1 and ADHF2 may catalyze the reversible NAD+-dependent oxidation of HMF. Our data may be useful for future studies on the metabolic engineering of more useful strains for ethanol production from lignocellulose.

Photopolymerization of L-DOPA-Alginate Immobilized Cell Wall Laccase for Textile Dye Decolorization

Alginate is a naturally occurring polymer derived from brown algae biomass, which has numerous applications in various fields. Chemical modification of alginate is widely used to improve alginate’s physicochemical properties and provide new potential for multiple applications. In this article, we modified alginate with L-DOPA, using periodate oxidation and reductive amination, to obtain more suitable biopolymer for biocatalyst immobilization and hydrogel formation. Obtained modified alginate was used for the immobilization of laccase on cell walls. For this purpose, laccase from Streptomyces cyaneus was expressed on the surface of Saccharomyces cerevisiae EBY100 cells. The obtained cell wall laccase was immobilized within L-DOPA-alginate beads by crosslinking the L-DOPA-alginate with calcium ions and laccase. The effect of additional crosslinking of beads by green light-induced photopolymerization with eosin Y was investigated. The immobilized laccase systems were used for dye decolorization and investigated in multiple treatment processes. Beads with L-DOPA-alginate with a higher degree of modification (5.0 mol%) showed higher enzymatic activity and better decolorization efficiency than those with a lower degree of modification (2.5 mol%). Obtained immobilized biocatalysts are suitable for decolorizing dye Evans Blue due to their high efficiency and reusability.

Lighting up Pyruvate Metabolism in Saccharomyces cerevisiae by a Genetically Encoded Fluorescent Biosensor.

Monitoring intracellular pyruvate is useful for the exploration of fundamental metabolism and for guiding the construction of yeast cell factories for chemical production. Here, we employed a genetically encoded fluorescent Pyronic biosensor to light up the pyruvate metabolic state in the cytoplasm, nucleus, and mitochondria of Saccharomyces cerevisiae BY4741. A strong correlation was observed between the pyruvate fluctuation in mitochondria and cytoplasm when exposed to different metabolites. Further metabolic analysis of pyruvate uptake and glycolytic dynamics showed that glucose and fructose dose-dependently activated cytoplasmic pyruvate levels more effectively than direct exposure to pyruvate. Meanwhile, the Pyronic biosensor could visually distinguish phenotypes of the wild-type S. cerevisiae BY4741 and the pyruvate-hyperproducing S. cerevisiae TAM at a single-cell resolution, having the potential for high-throughput screening. Overall, Pyronic biosensors targeting different suborganelles contribute to mapping and studying the central carbon metabolism in-depth and guide the design and construction of yeast cell factories.

Engineering of redox partners and cofactor NADPH supply of CYP68JX for efficient steroid two-step ordered selective hydroxylation activity

CYP68JX, a P450 hydroxylase, derived from Colletotrichum lini ST-1 is capable of biotransforming dehydroepiandrosterone (DHEA) to 3β,7α,15α-trihydroxy-5-androstene-17-one (7α,15α-diOH-DHEA). Redox partners and cofactor supply are important factors affecting the catalytic activity of CYP68JX. In this study, the heterologous expression of CYP68JX in Saccharomyces cerevisiae BY4741 was realized resulting in a 17.1% target product yield. In order to increase the catalytic efficiency of CYP68JX in S. cerevisiae BY4741, a complete cytochrome P450 redox system was constructed. Through the combination of CYP68JX and heterologous CPRs, the yield of the target product 7α,15α-diOH-DHEA in CYP68JX recombinant system was increased to 37.8%. Furthermore, by adding NADPH coenzyme precursor tryptophan of 40 mmol/L and co-substrate fructose of 20 g/L during the conversion process, the catalytic efficiency of CYP68JX was further improved, the target product yield reached 57.9% which was 3.39-fold higher than initial yield. Overall, this study provides a reference for improving the catalytic activity of P450s.

Optimization, Scale-Up, and Economic Analysis of the Ethanol Production Process Using Sargassum horneri

Recently, the extensive spread of some algae along coastlines has surged into unmanageable thick decomposition layers. This study aimed to demonstrate the use of Sargassum horneri as a biomass resource for ethanol production through the continuous hydrolysis, enzymatic saccharification, and fermentation process. Sugars from S. horneri were obtained using a combination of thermal acid hydrolysis and enzymatic saccharification. The optimal conditions for thermal acid hydrolysis involved a 10% (w/v) S. horneri slurry treated with 100 mM H2SO4 at 121 °C for 60 min; enzymatic saccharification using 16 U/mL Cellic CTec2 further boosted the monosaccharide concentration to 23.53 g/L. Fermentation experiments were conducted with mannitol-adapted Saccharomyces cerevisiae BY4741 using S. horneri hydrolysate. Enhanced ethanol production was observed in the hydrolysate, particularly with mannitol-adapted S. cerevisiae BY4741, which yielded 10.06 g/L ethanol. Non-adapted S. cerevisiae produced 8.12 g/L ethanol, as it primarily utilized glucose and not mannitol. Regarding ethanol fermentation using 5 L- and 500 L-scale fermenters, the ethanol concentrations reached 10.56 g/L and 7.88 g/L with yields of 0.51 and 0.45, respectively, at 48 h. This study confirmed the economic viability of ethanol production using waste seaweed with optimized pretreatment conditions and the adaptive evolution of S. cerevisiae to mannitol.

Evaluation of Cell Responses of Saccharomyces cerevisiae under Cultivation Using Wheat Bran as a Nutrient Resource by Analyses of Growth Activities and Comprehensive Gene Transcription Levels.

Wheat bran has high nutritional values and is also cheaper than yeast nitrogen base as an important component of a medium. Although its use in microbial cultivations is expected, research and development has hardly progressed so far. In this study, with experimental Saccharomyces cerevisiae BY4741, the cell responses to wheat bran as a nutrient were evaluated by analyses of cell growth, ethanol production, and comprehensive gene transcription levels. Comparing wheat bran and yeast nitrogen base, BY4741 showed specific growth rates of 0.277 ± 0.002 and 0.407 ± 0.035 as a significant difference. Additionally, wheat bran could be used as a restricted media component like yeast nitrogen base. However, in 24 h of cultivation with wheat bran and yeast nitrogen base, although conversion ratios of ethanol productions showed no significant difference at 63.0 ± 7.2% and 62.5 ± 8.2%, the ratio of cell production displayed a significant difference at 7.31 ± 0.04% and 4.90 ± 0.16%, indicating a different cell response. In fact, the comprehensive evaluation of transcription levels strongly suggested major changes in glucose metabolism. This study indicated that BY4741 could switch transcription levels efficiently to use wheat bran.

Reconstitution and Optimization of the Marmesin Biosynthetic Pathway in Yeast.

Marmesin is essential in plant defense systems and exhibits various biological activities. In this study, we reconstituted the marmesin biosynthetic pathway in the Saccharomyces cerevisiae BY4741 chassis. We engineered the aromatic amino acid (AAA) biosynthetic pathways by introducing Escherichia coli-derived ppsA to improve the availability of the AAA precursor phosphoenolpyruvate, overexpressing the feedback inhibition resistance genes ARO4K229L and ARO7G141S to direct the metabolic flux toward the tyrosine branch, and deleting ARO10, PDC5, and PDC6 to reduce the byproducts from the Ehrlich pathway. The umbelliferone 6-dimethylallyltransferase (U6DT) and marmesin synthase (MS) involved in marmesin synthesis were optimized to increase marmesin production. Marmesin production was improved by truncating the transmembrane domains of PcU6DT, FcMS, and AtCPR1 and increasing the copy numbers of the genes encoding the truncated enzymes. Finally, a marmesin titer of 27.7 mg/L was obtained in shake flasks using the engineered yeast strain MU5. The constructed marmesin-producing strain provides the foundation for the green and large-scale production of pharmaceutically important furanocoumarins.

Characterization and designing of an SAM riboswitch to establish a high-throughput screening platform for SAM overproduction in Saccharomyces cerevisiae.

S-adenosyl- l-methionine (SAM)is a high-value compound widely used in the treatment of various diseases. SAM can be produced through fermentation, but further enhancing the microbial production of SAM requires novel high-throughput screening methods for rapid detection and screening of mutant libraries. In this work, anSAM-OFF riboswitch capable of responding to the SAM concentration was obtained and a high-throughput platform for screening SAM overproducers was established. SAM synthase was engineered by semirational design and directed evolution, which resulted in the SAM2S203F,W164R,T251S,Y285F,S365R mutant with almost twice higher catalytic activity than the parental enzyme. The best mutant was then introduced into Saccharomyces cerevisiae BY4741, and the resulting strain BSM8 produced a sevenfold higher SAM titer in shake-flask fermentation, reaching 1.25 g L-1 . This work provides a reference for designing biosensors to dynamically detect metabolite concentrations for high-throughput screening and the construction of effective microbial cell factories.

Red Quinoa hydrolysates with antioxidant bioactive properties on oxidative stress-induced Saccharomyces cerevisiae

Quinoa (Chenopodium quinoa Willd) is a pseudo-cereal of great interest for its nutritional value. Specifically, enzymatic hydrolysis of quinoa proteins has shown several biological activities. The purpose of this study was to investigate the influence of enzymatic hydrolysis on antioxidant by the oxygen radical absorbance capacity (ORAC) method and sensory properties of white, red and black quinoa varieties, and to evaluate the in vivo antioxidant capacity of the most promising quinoa hydrolysate using Saccharomyces cerevisiae BY4741 as an experimental model. The results showed a hydrolysate from red quinoa seeds with a promising sensory profile and antioxidant activity. Although more studies in experimental models and human trials will be necessary to corroborate the antioxidant effect and the mechanisms of action involved, the results obtained may allow the development of new plant-based foods with antioxidant properties scientifically supported and useful in the prevention and/or the treatment of pathologies related to oxidative stress.

Screening of Ultraviolet-Induced Thermotolerant Yeast Mutants and Their Performance

The simultaneous saccharification and fermentation (SSF) technique holds promise for the conversion of lignocellulose to ethanol. However, the optimal fermentation temperature of yeast is lower than the enzymatic hydrolysis temperature of the saccharification process, which leads to the temperature of the actual production process of SSF usually being lower than 38 °C. In this work, two ultraviolet (UV)-induced mutations were performed step by step using Saccharomyces cerevisiae BY4742 as the original strain to enable the yeast to perform well at higher temperatures. Thermotolerant strains obtained through mutagenesis and screening, YUV1-1 and YUV2-2, were utilized for fermentation and SSF at a targeted temperature of 40 °C. They obtained ethanol yields comparable to those at 38 °C in SSF, whereas the ethanol yields of the original strain at 40 °C decreased by about 10% compared to those at 38 °C. This study proves that thermotolerant strains adapted to elevated fermentation and SSF temperatures can be obtained through UV mutagenesis and screening, thereby increasing the stability of the fermentation and SSF processes and lowering the subsequent distillation costs.

Transcription Analysis of the Acid Tolerance Mechanism of Pichia kudriavzevii NBRC1279 and NBRC1664

Simultaneous saccharification and fermentation (SSF) has been investigated for the efficient production of ethanol because it has several advantages such as simplifying the manufacturing process, operating easily, and reducing energy input. Previously, using lignocellulosic biomass as source materials, we succeeded in producing ethanol by SSF with Pichia kudriavzevii NBRC1279 and NBRC1664. However, various acids that fermentation inhibitors are also produced by the hydrolysis of lignocellulosic biomass, and the extent to which these acids affect the growth and ethanol productivity of the two strains has not yet been investigated. In this study, to better understand the acid tolerance mechanism of the two strains, a spot assay, growth experiment, and transcriptome analysis were carried out using Saccharomyces cerevisiae BY4742 as a control. When the three strains were cultured in SCD medium containing 15 mM formic acid, 35 mM sulfuric acid, 60 mM hydrochloric acid, 100 mM acetic acid, or 550 mM lactic acid, only P. kudriavzevii NBRC1664 could grow well under all conditions, and it showed the fastest growth rates. The transcriptome analysis showed that “MAPK signaling pathway-yeast” was significantly enriched in P. kudriavzevii NBRC1664 cultured with 60 mM hydrochloric acid, and most genes involved in the high osmolarity glycerol (HOG) pathway were up-regulated. Therefore, the up-regulation of the HOG pathway may be important for adapting to acid stress in P. kudriavzevii. Moreover, the log2-transformed fold change value in the expression level of Gpd1 was 1.3-fold higher in P. kudriavzevii NBRC1664 than in P. kudriavzevii NBRC1279, indicating that high Gpd1 expression may be accountable for the higher acid tolerance of P. kudriavzevii NBRC1664. The transcriptome analysis performed in this study provides preliminary knowledge of the molecular mechanism of acid stress tolerance in P. kudriavzevii. Our data may be useful for future studies on methods to improve the tolerance of P. kudriavzevii to acids produced from lignocellulose hydrolysis.

Engineered Yeast Displaying Specific Norovirus-Binding Nanobodies for the Concentration and Detection of Human Norovirus in Food Matrix.

Human noroviruses pose grave threats to public health and economy. In this study, we genetically engineered yeast (Saccharomyces cerevisiae EBY100) to display specific norovirus-binding nanobodies (Nano-26 and Nano-85) on cell surface to facilitate the concentration of noroviruses for improved detection. Binding of norovirus virus-like particles (VLPs) to these nanobody-displaying yeasts was confirmed and characterized using confocal microscopy and flow cytometry. The ability of our engineered yeasts to capture norovirus VLPs can reach up to 91.3%. Furthermore, this approach was applied to concentrate and detect norovirus VLPs in a real food matrix. A wide linear detection range (1-104 pg/g) was observed, and the detection limit on spiked spinach was calculated as low as 0.071 pg/g. Overall, our engineered yeasts could be a promising approach to concentrate and purify noroviruses in food samples for easy detection, which allows us to prevent the spread of food-borne virus in the food supply chain.

Design and optimization of artificial light-driven microbial consortia for the sustainable growth and biosynthesis of 2-phenylethanol

Synthetic light-driven microbial consortia can serve as a versatile and efficient platform for microbial growth and bio-chemicals production through the efficient labor division. Within the light-driven consortium system, photoautotrophic microbe can convert carbon dioxide into oxygen and organic carbons to support the sustainable growth and metabolites synthesis of the heterotrophic aerobic partners. In this study, a synthetic light-driven consortium composed of Saccharomyces cerevisiae BY4741 and Chlorella sp. GY-H4 was first established to achieve the 2-phenylethanol (2-PE) production. To further increase 2-PE production and stabilize this co-culture system, an aerobic phenylethylamine pathway was constructed in S. cerevisiae BY4741. Subsequently, a 3D bioprinting of living materials containing Chlorella sp. GY-H4 was printed, which provided defined spatial niches for Chlorella sp. GY-H4. The final 2-PE titer was improved to 2.13 g/L by the space-efficient light-driven microbial consortium, which was 1.9-fold than wild S. cerevisiae BY4741. This study provides a solid reference for the effective production of fine chemicals by using the synthetic light-driven microbial consortia.

High-yield production of protopanaxadiol from sugarcane molasses by metabolically engineered Saccharomyces cerevisiae

BackgroundGinsenosides are Panax plant-derived triterpenoid with wide applications in cardiovascular protection and immunity-boosting. However, the saponins content of Panax plants is fairly low, making it time-consuming and unsustainable by direct extraction. Protopanaxadiol (PPD) is a common precursor of dammarane-type saponins, and its sufficient supply is necessary for the efficient synthesis of ginsenoside.ResultsIn this study, a combinational strategy was used for the construction of an efficient yeast cell factory for PPD production. Firstly, a PPD-producing strain was successfully constructed by modular engineering in Saccharomyces cerevisiae BY4742 at the multi-copy sites. Then, the INO2 gene, encoding a transcriptional activator of the phospholipid biosynthesis, was fine-tuned to promote the endoplasmic reticulum (ER) proliferation and improve the catalytic efficiency of ER-localized enzymes. To increase the metabolic flux of PPD, dynamic control, based on a carbon-source regulated promoter PHXT1, was introduced to repress the competition of sterols. Furthermore, the global transcription factor UPC2-1 was introduced to sterol homeostasis and up-regulate the MVA pathway, and the resulting strain BY-V achieved a PPD production of 78.13 ± 0.38 mg/g DCW (563.60 ± 1.65 mg/L). Finally, sugarcane molasses was used as an inexpensive substrate for the first time in PPD synthesis. The PPD titers reached 1.55 ± 0.02 and 15.88 ± 0.65 g/L in shake flasks and a 5-L bioreactor, respectively. To the best of our knowledge, these results were new records on PPD production.ConclusionThe high-level of PPD production in this study and the successful comprehensive utilization of low-cost carbon source -sugarcane molassesindicate that the constructed yeast cell factory is an excellent candidate strain for the production of high-value-added PPD and its derivativeswith great industrial potential.Graphical

ACtivE: Assembly and CRISPR-Targeted in Vivo Editing for Yeast Genome Engineering Using Minimum Reagents and Time.

Thanks to its sophistication, the CRISPR/Cas system has been a widely used yeast genome editing method. However, CRISPR methods generally rely on preassembled DNAs and extra cloning steps to deliver gRNA, Cas protein, and donor DNA. These laborious steps might hinder its usefulness. Here, we propose an alternative method, Assembly and CRISPR-targeted in vivo Editing (ACtivE), that only relies on in vivo assembly of linear DNA fragments for plasmid and donor DNA construction. Thus, depending on the user's need, these parts can be easily selected and combined from a repository, serving as a toolkit for rapid genome editing without any expensive reagent. The toolkit contains verified linear DNA fragments, which are easy to store, share, and transport at room temperature, drastically reducing expensive shipping costs and assembly time. After optimizing this technique, eight loci proximal to autonomously replicating sequences (ARS) in the yeast genome were also characterized in terms of integration and gene expression efficiencies and the impacts of the disruptions of these regions on cell fitness. The flexibility and multiplexing capacity of the ACtivE were shown by constructing a β-carotene pathway. In only a few days, >80% integration efficiency for single gene integration and >50% integration efficiency for triplex integration were achieved on Saccharomyces cerevisiae BY4741 from scratch without using in vitro DNA assembly methods, restriction enzymes, or extra cloning steps. This study presents a standardizable method to be readily employed to accelerate yeast genome engineering and provides well-defined genomic location alternatives for yeast synthetic biology and metabolic engineering purposes.

Yeast Cell Surface Engineering of a Nicotinamide Riboside Kinase for the Production of β-Nicotinamide Mononucleotide via Whole-Cell Catalysis.

β-Nicotinamide mononucleotide (NMN) has been widely used as a nutraceutical for self-medication. The one-step conversion of nicotinamide riboside (NR) to β-NMN has been considered to be the most promising synthetic route for β-NMN. Here, human nicotinamide riboside kinase 2 (NRK-2) was functionally displayed on the cell surface of Saccharomyces cerevisiae EBY100, forming a whole-cell biocatalyst (Whole-cell NRK-2). Whole-cell NRK-2 could convert nicotinamide riboside (NR) to β-NMN efficiently in the presence of ATP and Mg2+, with a maximal activity of 64 IU/g (dry weight) and a Km of 3.5 μM, similar to that of free NRK-2 reported previously. To get the best reaction conditions for β-NMN synthesis, the amounts of NR, ATP, and Mg2+ used, pH, and temperature for the synthetic reaction were optimized. Using Whole-cell NRK-2 as the catalyst under the optimized conditions, β-NMN synthesized from NR reached a maximal conversion rate of 98.2%, corresponding to 12.6 g/L of β-NMN in the reaction mixture, which was much higher than those of synthetic processes reported. Additionally, Whole-cell NRK-2 had good pH stability and thermostability, required no complicated treatments before or after use, and could be reused in sequential production. Therefore, this study provided a safe, stable, highly effective, and low-cost biocatalyst for the preparation of β-NMN, which has great potential in industrial production.

CRISPR-Cas Assisted Shotgun Mutagenesis Method for Evolutionary Genome Engineering.

Genome mutagenesis drives the evolution of organisms. Here, we developed a CRISPR-Cas assisted random mutation (CARM) technique for whole-genome mutagenesis. The method leverages an entirely random gRNA library and SpCas9-NG to randomly damage genomes in a controllable shotgunlike manner that then triggers diverse and abundant mutations via low-fidelity repair. As a proof of principle, CARM was applied to evolve the capacity of Saccharomyces cerevisiae BY4741 to produce β-carotene. After seven rounds of iterative evolution over two months, a β-carotene hyperproducing strain, C7-143, was isolated with a 10.5-fold increase in β-carotene production and 857 diverse genomic mutations that comprised indels, duplications, inversions, and chromosomal rearrangements. Transcriptomic analysis revealed that the expression of 2541 genes of strain C7-143 was significantly altered, suggesting that the metabolic landscape of the strain was deeply reconstructed. In addition, CARM was applied to evolve industrially relevant S. cerevisiae CEN.PK2-1C for S-adenosyl-L-methionine production, which was increased 2.28 times after just one round. Thus, CARM can contribute to increasing genetic diversity to identify new phenotypes that could further be investigated by reverse engineering.

Expression of the GFP-mammalian pleckstrin homology (PH) domain of the phospholipase C δ1 in Saccharomyces cerevisiae BY4741.

Pleckstrin homology (PH) domains are common modules of ∼120 amino acids found in proteins involved in signalling, cytoskeletal organization, membrane transport, and modification of phospholipids. Previous live cell studies have involved the use of the green-fluorescent protein (GFP) labelling of PH-domain of phospholipase C δ1 (PLC δ1) to study the interactions of molecules at the membrane interface. For this study, the aim was to construct and express the GFP-PH domain of PLC δ1 in the Saccharomyces cerevisiae BY4741. The transformants expressing GFP-PH domain of PLC δ1 displayed localised fluorescence to the cell periphery (plasma membrane) while the negative control expressed GFP within the cytoplasm only. No GFP was observed in the non-transformed yeast cells. Thus, this technique could be useful in future molecular interactions studies targeted specifically at the yeast cell membrane interface in live yeast cells.