Cerevisiae BY4741 Research Articles

Estimation of Carbon Metabolism in Saccharomyces cerevisiae Acclimatized to Glycerol Assimilation with Quantitative PCR.

Saccharomyces cerevisiae has the potential to produce value-added chemicals; however, this strain is restricted by using glycerol as a carbon source. Although acclimatization of S. cerevisiae as a glycerol-assimilating strain was confirmed so far, the reason why S. cerevisiae can be acclimatized was not clear in detail with limited information on the metabolic changes. In this report, glycerol-assimilating strains from S. cerevisiae BY4741 were isolated, and the biomass production, ethanol fermentation, and transcription levels related to glycolysis and the tricarboxylic acid cycle under aerobic and slightly anaerobic conditions were analyzed. As the results show, although µmax was equal to 0.15 h−1 between wildtype and glycerol-assimilating strains in an aerobic culture including glucose, the differences in max biomass production and percentage yields of ethanol and transcription levels between the two strains were shown. In slightly anaerobic culture, the differences in transcription levels downstream of glycolysis were also displayed. In the case of the glycerol-assimilating strain with glycerol under aerobic conditions, although the transcription levels related to ethanol production were sufficient, the ethanol production was not detected. Additionally, the biomass production reached a plateau even in the culture containing sufficient glycerol, indicating that the redox imbalance even in the cells of the glycerol-acclimatized strain could disturb the utilization of glycerol. The obtained knowledge will promote the use of glycerol resources with the glycerol-acclimatized S. cerevisiae in view of carbon recycling.

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.

Saccharomyces cerevisiae yeast cells as a test system for assessing Zeocin toxicity

Having unique genetic machinery and a high degree of conservation with higher eukaryotes, the yeast Saccharomyces cerevisiae is recognised as a smart experimental system for studying the modes of chemical toxicity. The present study was undertaken to elucidate the changes in the intracellular redox homeostasis and key macromolecule structure following exposure to Zeocin. Cell populations of logarithmic, quiescent (Q) and non-quiescent (NQ) cells of Saccharomyces cerevisiae BY4741 were used as a model to examine the cytotoxic effect of this radiomimetic. The levels of endogenous ROS, oxidized lipids, carbonylated proteins, and glutathione were analysed after treatment with Zeocin (IC50). An increase in ROS production and respectively increased oxidative stress was detected in all three types of cell populations, with the highest degree being observed in proliferating S. cerevisiae BY4741 cells. The stress response of both proliferating and stationary phase (Q and NQ) cells to Zeocin included an overexpression of glutathione. The quiescent cells also showed very low DNA susceptibility to high Zeocin concentration (100–300 µg/ml), presented as no induced double-strand breaks (DSBs) in the macromolecule. Based on our research it could be concluded that the cellular physiological state is a critical factor determining the resistance to environmental stress with Q cells being the most robust.

Integrative Gene Expression and Metabolic Analysis Tool IgemRNA.

Genome-scale metabolic modeling is widely used to study the impact of metabolism on the phenotype of different organisms. While substrate modeling reflects the potential distribution of carbon and other chemical elements within the model, the additional use of omics data, e.g., transcriptome, has implications when researching the genotype–phenotype responses to environmental changes. Several algorithms for transcriptome analysis using genome-scale metabolic modeling have been proposed. Still, they are restricted to specific objectives and conditions and lack flexibility, have software compatibility issues, and require advanced user skills. We classified previously published algorithms, summarized transcriptome pre-processing, integration, and analysis methods, and implemented them in the newly developed transcriptome analysis tool IgemRNA, which (1) has a user-friendly graphical interface, (2) tackles compatibility issues by combining previous data input and pre-processing algorithms in MATLAB, and (3) introduces novel algorithms for the automatic comparison of different transcriptome datasets with or without Cobra Toolbox 3.0 optimization algorithms. We used publicly available transcriptome datasets from Saccharomyces cerevisiae BY4741 and H4-S47D strains for validation. We found that IgemRNA provides a means for transcriptome and environmental data validation on biochemical network topology since the biomass function varies for different phenotypes. Our tool can detect problematic reaction constraints.

Antioxidant, antimicrobial, cytotoxic and protective effects of truffles

Fungi can be used as a potent chemotherapeutic agent to treat various cancers. In current study acetone and methanol extracts of Terfezia claveryi, Terfezia boudieri, Terfezia olbiensis, Picoa lefebvrei, Picoa juniperi were used to assess total phenolic contents, antioxidant activity, ion-chelating impact, antimicrobial activity, the cytotoxic and protective effects. Both methanol and acetone extracts of T. boudieri had the highest FRAP and DPPH scavenging abilities. Dose-dependent increased ion-chelating impact of all tested truffles species was found. Extracts of T. boudieri, T. claveryi, and T. albiensis exhibited higher antimicrobial activities. T. claveryi and T. boudieri showed the highest protective effects against H2O2-induced genotoxicity (P < 0.05), in S. cerevisiae BY4741. The least protective effect was showed by the acetone extracts of T. olbiensis (144 ± 8); methanol extracts of P. lefebvrei (140 ± 8) and P. juniperi (140 ± 10). MCF 7 cells showed more sensitivity against to methanol extracts of T. boudieri at 10–100 μg/mL concentrations. HepG2 cells showed more sensitivity against the methanolic extracts of T. boudieri at both doses. Overall, P. lefebvrei and P. juniperi extracts had the least cytotoxic effects. The species of Terfezia exhibit significant protective effects against DNA damage and also have the potential of cytotoxicity effects.

Overexpression of a leucine transfer RNA gene tL(CAA)K improves the acetic acid tolerance of Saccharomyces cerevisiae

Acetic acid is a common inhibitor present in lignocellulosic hydrolysate. Development of acetic acid tolerant strains may improve the production of biofuels and bio-based chemicals using lignocellulosic biomass as raw materials. Current studies on stress tolerance of yeast Saccharomyces cerevisiae have mainly focused on transcription control, but the role of transfer RNA (tRNA) was rarely investigated. We found that some tRNA genes showed elevated transcription levels in a stress tolerant yeast strain. In this study, we further investigated the effects of overexpressing an arginine transfer RNA gene tR(ACG)D and a leucine transfer RNA gene tL(CAA)K on cell growth and ethanol production of S. cerevisiae BY4741 under acetic acid stress. The tL(CAA)K overexpression strain showed a better growth and a 29.41% higher ethanol productivity than that of the control strain. However, overexpression of tR(ACG)D showed negative influence on cell growth and ethanol production. Further studies revealed that the transcriptional levels of HAA1, MSN2, and MSN4, which encode transcription regulators related to stress tolerance, were up-regulated in tL(CAA)K overexpressed strain. This study provides an alternative strategy to develop robust yeast strains for cellulosic biorefinery, and also provides a basis for investigating how yeast stress tolerance is regulated by tRNA genes.

Construction of synthetic microbial consortia for 2-keto-L-gulonic acid biosynthesis

Currently, the establishment of synthetic microbial consortia with rational strategies has gained extensive attention, becoming one of the important frontiers of synthetic biology. Systems biology can offer insights into the design and construction of synthetic microbial consortia. Taking the high-efficiency production of 2-keto-l-gulonic acid (2-KLG) as an example, we constructed a synthetic microbial consortium “Saccharomyces cerevisiae-Ketogulonigenium vulgare” based on systems biology analysis. In the consortium, K. vulgare was the 2-KLG producing strain, and S. cerevisiae acted as the helper strain. Comparative transcriptomic analysis was performed on an engineered S. cerevisiae (VTC2) and a wild-type S. cerevisiae BY4741. The results showed that the up-regulated genes in VTC2, compared with BY4741, were mainly involved in glycolysis, TCA cycle, purine metabolism, and biosynthesis of amino acids, B vitamins, and antioxidant proteases, all of which play important roles in promoting the growth of K. vulgare. Furthermore, Vitamin C produced by VTC2 could further relieve the oxidative stress in the environment to increase the production of 2-KLG. Therefore, VTC2 would be of great advantage in working with K. vulgare. Thus, the synthetic microbial consortium "VTC2-K. vulgare" was constructed based on transcriptomics analyses, and the accumulation of 2-KLG was increased by 1.49-fold compared with that of mono-cultured K. vulgare, reaching 13.2 ± 0.52 g/L. In addition, the increased production of 2-KLG was accompanied by the up-regulated activities of superoxide dismutase and catalase in the medium and the up-regulated oxidative stress-related genes (sod, cat and gpd) in K. vulgare. The results indicated that the oxidative stress in the synthetic microbial consortium was efficiently reduced. Thus, systems analysis confirmed a favorable symbiotic relationship between microorganisms, providing guidance for further engineering synthetic consortia.

Characterization and Heterologous Expression of UDP-Glucose 4-Epimerase From a Hericium erinaceus Mutant with High Polysaccharide Production.

Hericium erinaceus is an important medicinal fungus in traditional Chinese medicine because of its polysaccharides and other natural products. Compared terpenoids and polyketides, the analysis of synthetic pathway of polysaccharides is more difficult because of the many genes involved in central metabolism. In previous studies, A6180, encoding a putative UDP-glucose 4-epimerase (UGE) in an H. erinaceus mutant with high production of active polysaccharides, was significantly upregulated. Since there is no reliable genetic manipulation technology for H. erinaceus, we employed Escherichia coli and Saccharomyces cerevisiae to study the function and activity of A6180. The recombinant overexpression vector pET22b-A6180 was constructed for heterologous expression in E. coli. The enzymatic properties of the recombinant protein were investigated. It showed that the recombinant A6180 could strongly convert UDP-α-D-glucose into UDP-α-D-galactose under optimal conditions (pH 6.0, 30°C). In addition, when A6180 was introduced into S. cerevisiae BY4742, xylose was detected in the polysaccharide composition of the yeast transformant. This suggested that the protein coded by A6180 might be a multifunctional enzyme. The generated polysaccharides with a new composition of sugars showed enhanced macrophage activity in vitro. These results indicate that A6180 plays an important role in the structure and activity of polysaccharides. It is a promising strategy for producing polysaccharides with higher activity by introducing A6180 into polysaccharide-producing mushrooms.

Optimized biosynthesis of santalenes and santalols in Saccharomyces cerevisiae.

Santalenes and santalols from Santalum album are the main components of the valuable spice sandalwood essential oil, which also has excellent pharmacological activities such as antibacterial, anti-inflammatory, and antitumor. Firstly, we constructed biosynthesis pathways of santalenes by synthetic biology strategy. The assembled biosynthetic cassettes were integrated into the multiple copy loci of δ gene in S. cerevisiae BY4742 with assistance of pDi-CRISPR, and 94.6 mg/L santalenes was obtained by shake flask fermentation of engineered yeast. Secondly, a selected optimized P450-CPR redox system was integrated into the chromosome of the santalenes-producing strain with a single copy, and 24.6 mg/L santalols were obtained. Finally, the yields of santalenes and santalols were increased to 164.7 and 68.8 mg/L, respectively, by downregulating ERG9 gene. This is the first report on the de novo synthesis of santalols by P450-CPR chimera in S. cerevisiae. Meanwhile, the optimized chimeric CYP736A167opt-46tATR1opt exhibits higher activity to oxidize santalenes into santalols. It would provide a feasible solution for the optimal biosynthesis of santalols. KEY POINTS: • First-time de novo synthesis of santalols by P450-CPR chimera in S. cerevisiae. • Truncated 46tATR1 has higher activity than that of CPR2. • Yields of santalenes and santalols were increased by downregulating ERG9 gene.

Comparative metabolite profiling of wild type and thermo-tolerant mutant of Saccharomyces cerevisiae

Simultaneous saccharification and fermentation (SSF) is a process of enzymatic saccharification and yeast fermentation used for producing lignocellulosic ethanol, and is performed at high temperature for effective enzymatic saccharification. Thermo-tolerant yeast is needed for high ethanol yield from yeast fermentation. A thermo-tolerant mutant yeast, SSK2, derived from Saccharomyces cerevisiae BY4741, has been generated previously. However, the thermo-tolerance mechanisms in SSK2 remain unclear. In this study, to investigate the change of metabolisms in thermo-tolerant mutant, metabolomics was performed based on gas chromatography/time-of-flight mass spectrometry (GC/TOF MS). In total, 91 intracellular metabolites were identified by GC/TOF MS; the metabolite profiles of BY4741 and SSK2, cultivated at 30 °C and 42 °C, were clearly discriminated by principal component analysis (PCA) and hierarchical clustering analysis (HCA). The metabolomics results indicated that the metabolism of amino acids, cell wall and membrane, and fatty acids were changed by heat stress and were key factors in yeast thermo-tolerance. In this study, metabolomics was successfully exploited to reveal the changes of metabolisms in the thermo-tolerant yeast, and these metabolic changes could be used as the clues for future development of thermo-tolerant yeasts.

Minority potassium-uptake system Trk2 has a crucial role in yeast survival of glucose-induced cell death.

The existence of programmed cell death in Saccharomyces cerevisiae has been reported for many years. Glucose induces the death of S. cerevisiae in the absence of additional nutrients within a few hours, and the absence of active potassium uptake makes cells highly sensitive to this process. S. cerevisiae cells possess two transporters, Trk1 and Trk2, which ensure a high intracellular concentration of potassium, necessary for many physiological processes. Trk1 is the major system responsible for potassium acquisition in growing and dividing cells. The contribution of Trk2 to potassium uptake in growing cells is almost negligible, but Trk2 becomes crucial for stationary cells for their survival of some stresses, e.g. anhydrobiosis. As a new finding, we show that both Trk systems contribute to the relative thermotolerance of S. cerevisiae BY4741. Our results also demonstrate that Trk2 is much more important for the cell survival of glucose-induced cell death than Trk1, and that stationary cells deficient in active potassium uptake lose their ATP stocks more rapidly than cells with functional Trk systems. This is probably due to the upregulated activity of plasma-membrane Pma1 H+-ATPase, and consequently, it is the reason why these cells die earlier than cells with functional active potassium uptake.

Functional characterization and expression profile of microsomal FAD2 and FAD3 genes involved in linoleic and α-linolenic acid production in Leucas cephalotes.

The online version contains supplementary material available at 10.1007/s12298-021-01016-z.

Expression cassette and plasmid construction for Yeast Surface Display in Saccharomyces cerevisiae

Develop a Cell Surface Display system in Saccharomyces cerevisiae, based on the construction of an expression cassette for pYES2 plasmid. The construction of an expression cassette containing the α-factor signal peptide and the C-terminal portion of the α-agglutinin protein was made and its sequence inserted into a plasmid named pYES2/gDαAgglutinin. The construction allows surface display of bovine herpesvirus type 5 (BoHV-5) glycoprotein D (gD) on S. cerevisiae BY4741 strain. Recombinant protein expression was confirmed by dot blot, and indirect immunofluorescence using monoclonal anti-histidine antibodies and polyclonal antibodies from mice experimentally vaccinated with a recombinant gD. These results demonstrate that the approach and plasmid used represent not only an effective system for immobilizing proteins on the yeast cell surface, as well as a platform for immunobiologicals development.

Anhydrobiosis in yeast: role of cortical endoplasmic reticulum protein Ist2 in Saccharomyces cerevisiae cells during dehydration and subsequent rehydration.

Two Saccharomyces cerevisiae strains, BY4741 and BY4741-derived strain lacking the IST2 gene (ist2Δ), were used to characterise the possible role of cortical endoplasmic reticulum (ER) protein Ist2 upon cell dehydration and subsequent rehydration. For the first time, we show that not only protein components of the plasma membrane (PM), but also at least one ER membrane protein (Ist2) play an important role in the maintenance of the viability of yeast cells during dehydration and subsequent rehydration. The low viability of the mutant strain ist2∆ upon dehydration-rehydration stress was related to the lack of Ist2 protein in the ER. We revealed that the PM of ist2∆ strain is not able to completely restore its molecular organisation during reactivation from the state of anhydrobiosis. As the result, the permeability of the PM remains high regardless of the type of reactivation (rapid or gradual rehydration). We conclude that ER protein Ist2 plays an important role in ensuring the stability of molecular organisation and functionality of the PM during dehydration-rehydration stress. These results indicate an important role of ER-PM interactions during cells transition into the state of anhydrobiosis and the subsequent restoration of their physiological activities.

Saccharomyces cerevisiae as a model to study synthetic cannabinoids: the impact of using different carbon sources

Introduction Synthetic cannabinoids (SC) are potent agonists of cannabinoid receptors, that mime the psychoactive effect of Δ9-tetrahydrocannabinol (Δ9-THC), the principal psychoactive component of cannabis [1]. JWH-018 was the first novel psychoactive SC found in the recreational drug marketplace in 2008. A few years later, JWH-018 started to be controlled by authorities, so alternative molecules started to emerge. THJ-018 was designed to replace JWH-018, having a similar structural skeleton and also a naphthalene and pentyl chain connected via a middle core substructure. Eventually, THJ-018 was scheduled and alternatives emerged, such as EG-018 [2]. This practice makes almost impossible to characterise SC toxicological profiles on an acceptable time scale, mostly due to the time-consuming experiments that must be held in animal models or human cells by standard methods. The yeast Saccharomyces cerevisiae shares highly conserved molecular and cellular mechanisms with human cells and has been used before for synthetic cathinones [3]. The present work has studied the best carbon source (glucose or galactose) to measure the impact of synthetic cannabinoids on S. cerevisiae growth, aiming to develop a method able to profile synthetic cannabinoids toxicity in a short time scale. The difference between carbon sources is that in Crabtree-positive yeast strains, glucose induce a strong inhibition of mitochondrial oxidative phosphorylation [4]. Materials and methods The effect of EG-018 was evaluated by its impact on S. cerevisiae BY4741 (WT) growth on minimal medium with 2% Glucose or 1% Galactose as sole carbon sources, in the presence and absence of the SC, followed at OD600nm. Growth rates at each condition was fitted to a logistic equation. Results Figure 1 shows OD600max obtained from the non-linear regression to the logistic equation in the presence of different concentrations of EG-018. While in galactose there is no effect, in 2% Glucose the results points to a growth decrease with EG-018. Discussion and conclusions: Comparing the two carbon sources, yeast growth on glucose is more susceptible to EG-018. Recent studies points that JWH-018, a similar SC, exerts an effect on glycolytic and pentose phosphate pathway at high concentrations of glucose (personal communication). As yeast growth in galactose proceeds simultaneously via respiration and fermentation due to the “Crabtree effect” [4] an impact of EG-018 on glycolysis might be less effective. These results, suggest that glucose is the best carbon source to develop a yeast based toxicity sensor for SC. Figure 1. OD600max results in S. cerevisiae BY4741 in the presence of 0, 25 and 50 µM of EG-018 in glucose and galactose.

Reciprocal hemizygosity analysis reveals that the Saccharomyces cerevisiae CGI121 gene affects lag time duration in synthetic grape must.

It is standard practice to ferment white wines at low temperatures (10–18°C). However, low temperatures increase fermentation duration and risk of problem ferments, leading to significant costs. The lag duration at fermentation initiation is heavily impacted by temperature; therefore, identification of Saccharomyces cerevisiae genes influencing fermentation kinetics is of interest for winemaking. We selected 28 S. cerevisiae BY4743 single deletants, from a prior list of open reading frames (ORFs) mapped to quantitative trait loci (QTLs) on Chr. VII and XIII, influencing the duration of fermentative lag time. Five BY4743 deletants, Δapt1, Δcgi121, Δclb6, Δrps17a, and Δvma21, differed significantly in their fermentative lag duration compared to BY4743 in synthetic grape must (SGM) at 15 °C, over 72 h. Fermentation at 12.5°C for 528 h confirmed the longer lag times of BY4743 Δcgi121, Δrps17a, and Δvma21. These three candidates ORFs were deleted in S. cerevisiae RM11-1a and S288C to perform single reciprocal hemizygosity analysis (RHA). RHA hybrids and single deletants of RM11-1a and S288C were fermented at 12.5°C in SGM and lag time measurements confirmed that the S288C allele of CGI121 on Chr. XIII, encoding a component of the EKC/KEOPS complex, increased fermentative lag phase duration. Nucleotide sequences of RM11-1a and S288C CGI121 alleles differed by only one synonymous nucleotide, suggesting that intron splicing, codon bias, or positional effects might be responsible for the impact on lag phase duration. This research demonstrates a new role of CGI121 and highlights the applicability of QTL analysis for investigating complex phenotypic traits in yeast.

Simultaneous saccharification and bioethanol production from underutilized biomass, cowpea haulm using co-cultures of Saccharomyces cerevisiae (BY4743) and Scheffersomyces stipitis (PsY633)

Cowpea cultivation generates large quantities of biomass after pod harvest which are underutilized and could be exploited for energy generation. In this study, evaluation of sugar production by dilute sulfuric acid pretreatment of cowpea haulm for potential ethanolic fermentation was carried out. A central composite design (CCD) was used to investigate the effects of temperature (100–120 °C), time (30–90 min), and acid concentration (1.0–4.0%) on sugar yield and inhibitor formation. The model F-values of 25.42, 78.81, and 6.96 for xylose yield, glucose yield, and total inhibitor concentration, respectively, with low probability value (p < 0.05) suggest a high significance of the models. While the R2-values of 0.9581, 0.9861, and 0.8424 indicated a satisfactory agreement of the quadratic models in predicting the responses. The quadratic model predicted optimum conditions for maximum xylose (76.5%) and glucose yield (28.6%), at minimum inhibitor concentration (2.34 g/L) and temperature (110 °C), with an acid concentration of 3.1% and reaction time of 55 min. These conditions were validated experimentally suggesting the effectiveness of experimental design towards process optimization. The resulting sugar-rich prehydrolysate was detoxified and fermented to ethanol using co-cultures of Saccharomyces cerevisiae BY4743 and Scheffersomyces stipitis (PsY633), while the recovered pretreated solid was subjected to simultaneous saccharification and fermentation with prehydrolysis (PSSF). A total ethanol titer of 15.67 g/L was obtained which corresponds to an overall conversion efficiency of 75%. This suggests that cowpea haulm could also be potentially exploited for bioethanol production either singly or in combination with other lignocellulosic biomass.

Transcriptomic analysis of nonylphenol effect on Saccharomyces cerevisiae.

Nonylphenol (NP) is a bioaccumulative environmental estrogen that is widely used as a nonionic surfactant. We have previously examined short-term effects of NP on yeast cells using microarray technology. In the present study, we investigated the adaptive response of Saccharomyces cerevisiae BY4742 cells to NP exposure by analyzing genome-wide transcriptional profiles using RNA-sequencing. We used 2 mg/L NP concentration for 40 days of exposure. Gene expression analysis showed that a total of 948 genes were differentially expressed. Of these, 834 genes were downregulated, while 114 genes were significantly upregulated. GO enrichment analysis revealed that 369 GO terms were significantly affected by NP exposure. Further analysis showed that many of the differentially expressed genes were associated with oxidative phosphorylation, iron and copper acquisition, autophagy, pleiotropic drug resistance and cell cycle progression related processes such as DNA and mismatch repair, chromosome segregation, spindle checkpoint activity, and kinetochore organization. Overall, these results provide considerable information and a comprehensive understanding of the adaptive response to NP exposure at the gene expression level.

Pathway engineering of Saccharomyces cerevisiae for efficient lycopene production.

To construct a Saccharomyces cerevisiae strain for efficient lycopene production, we used a pathway engineering strategy based on expression modules comprising fusion proteins and a strong constitutive promoter. The two recombinant plasmids pEBI encoding the fusion genes with an inducible promoter, as well as pIETB with a constitutive promoter and terminator were introduced into S. cerevisiae YPH499 and BY4741 to obtain the four recombinant strains ypEBI, ypIETB, byEBI and byIETB. The lycopene production and the transcription levels of key genes were higher in the BY4741 chassis than in YPH499. Accordingly, the content of total and unsaturated fatty acids was also higher in BY4741, which also exhibited a decrease of glucose, increase of trehalose, increase of metabolite in citrate cycle, and low levels of amino acids. These changes rerouted metabolic fluxes toward lycopene synthesis, indicating that the BY4741 chassis was more suitable for lycopene synthesis. The lycopene content of bpIETB in SG-Leu medium supplemented with 100mg/L of linolenic acid reached 10.12mg/g dry cell weight (DCW), which was 85.7% higher than without the addition of unsaturated fatty acids. The constitutive promoter expression strategy employed in this study achieved efficient lycopene synthesis in S. cerevisiae, and the strain bpIETB was obtained a suitable chassis host for lycopene production, which provides a basis for further optimization of lycopene production in artificial synthetic cells and a reference for the multi-enzyme synthesis of other similar complex terpenoids.