A synthetic promoter system for well-controlled protein expression with different carbon sources in Saccharomyces cerevisiae

Abstract Background Saccharomyces cerevisiae is an important synthetic biology chassis for microbial production of valuable molecules. Promoter engineering has been frequently applied to generate more synthetic promoters with a variety of defined characteristics in order to achieve a well-regulated genetic network for high production efficiency. Galactose-inducible (GAL) expression systems, composed of GAL promoters and multiple GAL regulators, have been widely used for protein overexpression and pathway construction in S. cerevisiae. However, the function of each element in synthetic promoters and how they interact with GAL regulators are not well known. Results Here, a library of synthetic GAL promoters demonstrate that upstream activating sequences (UASs) and core promoters have a synergistic relationship that determines the performance of each promoter under different carbon sources. We found that the strengths of synthetic GAL promoters could be fine-tuned by manipulating the sequence, number, and substitution of UASs. Core promoter replacement generated synthetic promoters with a twofold strength improvement compared with the GAL1 promoter under multiple different carbon sources in a strain with GAL1 and GAL80 engineering. These results represent an expansion of the classic GAL expression system with an increased dynamic range and a good tolerance of different carbon sources. Conclusions In this study, the effect of each element on synthetic GAL promoters has been evaluated and a series of well-controlled synthetic promoters are constructed. By studying the interaction of synthetic promoters and GAL regulators, synthetic promoters with an increased dynamic range under different carbon sources are created.

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Pervasive and Dynamic Transcription Initiation in Saccharomyces cerevisiae

10.1101/450429 ◽

2018 ◽

Cited By ~ 3

Author(s):

Zhaolian Lu ◽

Zhenguo Lin

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Transcription Initiation ◽

Core Promoter ◽

Model Organism ◽

Start Codon ◽

Yeast Genome ◽

Core Promoters ◽

Nucleotide Resolution ◽

Yeast Genes

AbstractTranscription initiation is finely regulated to ensure the proper expression and function of these genes. The regulated transcription initiation in response to various environmental cues in the model organism Saccharomyces cerevisiae has not been systematically investigated. In this study, we generated quantitative maps of transcription start site (TSS) at a single-nucleotide resolution for S. cerevisiae grown in nine different conditions using no-amplification non-tagging Cap analysis of gene expression (nAnT-iCAGE) sequencing. Based on 337 million uniquely mapped CAGE tags, we mapped ~1 million well-supported TSSs, suggesting highly pervasive transcription initiation in the compact genome of yeast. The comprehensive TSS maps allowed us to identify core promoters for ~96% verified protein-coding genes and to revise the predicted translation start codon for 183 genes. We found that 56% of yeast genes have at least two core promoters and alternative usage of different core promoters in a gene is widespread in response to changing environments. More importantly, most core promoter shifts are coupled with differential gene expression, indicating that core promoter shift might play an important role in controlling transcriptional activity of yeast genes. Based on their dynamic activities, we divided yeast core promoters as constitutive core promoters (55%) and inducible core promoters (45%). The two classes of core promoters exhibit distinctive patterns in transcriptional abundance, chromatin structure, promoter shape, and sequence context. In summary, the quantitative TSS maps generated by this study improved the annotation of yeast genome, and revealed a highly pervasive and dynamic nature of transcription initiation in yeast.

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Directed Evolution of Saccharomyces cerevisiae for Increased Selenium Accumulation

Microorganisms ◽

10.3390/microorganisms6030081 ◽

2018 ◽

Vol 6 (3) ◽

pp. 81 ◽

Cited By ~ 3

Author(s):

Masafumi Yoshinaga ◽

Stephanie How ◽

Damien Blanco ◽

Ian Murdoch ◽

Matteo Grudny ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Cost Efficiency ◽

Production Efficiency ◽

Carbon Sources ◽

Nitrogen Sources ◽

Selenium Yeast ◽

Transgenic Yeast ◽

Yeast Strains ◽

Selenium Accumulation ◽

Nutrient Supplements

Selenium-enriched yeast (selenium yeast) are one of the most popular sources of selenium supplementation used in the agriculture and human nutritional supplements industries. To enhance the production efficiency of selenium yeast, we sought to develop a method to identify, and ultimately select for, strains of yeast with enhanced selenium accumulation capabilities. Selenite resistance of four genetically diverse strains of Saccharomyces cerevisiae was assayed in various conditions, including varying carbon sources, nitrogen sources, and phosphate amounts, and they were correlated with selenium accumulation in a commercially relevant selenium-containing growth medium. Glycerol- and selenite-containing media was used to select for six yeast isolates with enhanced selenite resistance. One isolate was found to accumulate 10-fold greater selenium (0.13 to 1.4 mg Se g−1 yeast) than its parental strain. Glycerol- and selenium-containing medium can be used to select for strains of yeast with enhanced selenium accumulation capability. The methods identified can lead to isolation of industrial yeast strains with enhanced selenium accumulation capabilities that can result in greater cost efficiency of selenium yeast production. Additionally, the selection method does not involve the construction of transgenic yeast, and thus produces yeasts suitable for use in human food and nutrient supplements.

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Isolation and characterization of temperature-sensitive mutations in the RAS2 and CYR1 genes of Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/123.4.739 ◽

1989 ◽

Vol 123 (4) ◽

pp. 739-748 ◽

Cited By ~ 3

Author(s):

H Mitsuzawa ◽

I Uno ◽

T Oshima ◽

T Ishikawa

Keyword(s):

Saccharomyces Cerevisiae ◽

Carbon Sources ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Extra Copy ◽

Yeast Saccharomyces Cerevisiae ◽

Isolation And Characterization ◽

Low Concentrations ◽

Gal1 Promoter ◽

Dependent Growth

Abstract The yeast Saccharomyces cerevisiae contains two ras homologues, RAS1 and RAS2, whose products have been shown to modulate the activity of adenylate cyclase encoded by the CYR1 gene. To isolate temperature-sensitive mutations in the RAS2 gene, we constructed a plasmid carrying a RAS2 gene whose expression is under the control of the galactose-inducible GAL1 promoter. A ras1 strain transformed with this plasmid was subjected to ethyl methanesulfonate mutagenesis and nystatin enrichment. Screening of approximately 13,000 mutagenized colonies for galactose-dependent growth at a high temperature (37 degrees) yielded six temperature-sensitive ras2 (ras2ts) mutations and one temperature-sensitive cyr1 (cyr1ts) mutation that can be suppressed by overexpression or increased dosage of RAS2. Some ras2ts mutations were shown to be suppressed by an extra copy of CYR1. Therefore increased dosage of either RAS2 or CYR1 can suppress the temperature sensitivity caused by a mutation in the other. ras1 ras2ts and ras1 cyr1ts mutants arrested in the G1 phase of the cell cycle at the restrictive temperature, and showed pleiotropic phenotypes to varying degrees even at a temperature permissive for growth (25 degrees), including slow growth, sporulation on rich media, increased accumulation of glycogen, impaired growth on nonfermentable carbon sources, heat-shock resistance, impaired growth on low concentrations of glucose, and lithium sensitivity. Of these, impaired growth on low concentrations of glucose and sensitivity to lithium are new phenotypes, which have not been reported for mutants defective in the cAMP pathway.

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Novel S. cerevisiae Hybrid Synthetic Promoters Based on Foreign Core Promoter Sequences

International Journal of Molecular Sciences ◽

10.3390/ijms22115704 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5704

Author(s):

Xiaofan Feng ◽

Mario Andrea Marchisio

Keyword(s):

Transcription Initiation ◽

Core Promoter ◽

Tata Box ◽

Yeast Saccharomyces Cerevisiae ◽

Transcription Start Sites ◽

Core Promoters ◽

Promoter Sequences ◽

Synthetic Promoters ◽

Synthetic Gene Circuits ◽

Enhance Protein Synthesis

Promoters are fundamental components of synthetic gene circuits. They are DNA segments where transcription initiation takes place. New constitutive and regulated promoters are constantly engineered in order to meet the requirements for protein and RNA expression into different genetic networks. In this work, we constructed and optimized new synthetic constitutive promoters for the yeast Saccharomyces cerevisiae. We started from foreign (e.g., viral) core promoters as templates. They are, usually, unfunctional in yeast but can be activated by extending them with a short sequence, from the CYC1 promoter, containing various transcription start sites (TSSs). Transcription was modulated by mutating the TATA box composition and varying its distance from the TSS. We found that gene expression is maximized when the TATA box has the form TATAAAA or TATATAA and lies between 30 and 70 nucleotides upstream of the TSS. Core promoters were turned into stronger promoters via the addition of a short UAS. In particular, the 40 nt bipartite UAS from the GPD promoter can enhance protein synthesis considerably when placed 150 nt upstream of the TATA box. Overall, we extended the pool of S. cerevisiae promoters with 59 new samples, the strongest overcoming the native TEF2 promoter.

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Promoter Architecture and Promoter Engineering in Saccharomyces cerevisiae

Metabolites ◽

10.3390/metabo10080320 ◽

2020 ◽

Vol 10 (8) ◽

pp. 320 ◽

Cited By ~ 3

Author(s):

Hongting Tang ◽

Yanling Wu ◽

Jiliang Deng ◽

Nanzhu Chen ◽

Zhaohui Zheng ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Dynamic Range ◽

Regulation Of Gene Expression ◽

Fine Tuning ◽

Genetic Circuits ◽

Future Directions ◽

Promoter Engineering ◽

Synthetic Promoters ◽

Promoter Architecture ◽

Recent Advances

Promoters play an essential role in the regulation of gene expression for fine-tuning genetic circuits and metabolic pathways in Saccharomyces cerevisiae (S. cerevisiae). However, native promoters in S. cerevisiae have several limitations which hinder their applications in metabolic engineering. These limitations include an inadequate number of well-characterized promoters, poor dynamic range, and insufficient orthogonality to endogenous regulations. Therefore, it is necessary to perform promoter engineering to create synthetic promoters with better properties. Here, we review recent advances related to promoter architecture, promoter engineering and synthetic promoter applications in S. cerevisiae. We also provide a perspective of future directions in this field with an emphasis on the recent advances of machine learning based promoter designs.

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Identification of a Functional Domain Within the Essential Core of Histone H3 That Is Required for Telomeric and HM Silencing in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/163.1.447 ◽

2003 ◽

Vol 163 (1) ◽

pp. 447-452 ◽

Cited By ~ 1

Author(s):

Jeffrey S Thompson ◽

Marilyn L Snow ◽

Summer Giles ◽

Leslie E McPherson ◽

Michael Grunstein

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Amino Acid Substitution ◽

Histone H3 ◽

Single Amino Acid ◽

Functional Domain ◽

Partial Loss ◽

Histone Fold ◽

Gal1 Promoter ◽

Substitution Mutations

Abstract Fourteen novel single-amino-acid substitution mutations in histone H3 that disrupt telomeric silencing in Saccharomyces cerevisiae were identified, 10 of which are clustered within the α1 helix and L1 loop of the essential histone fold. Several of these mutations cause derepression of silent mating locus HML, and an additional subset cause partial loss of basal repression at the GAL1 promoter. Our results identify a new domain within the essential core of histone H3 that is required for heterochromatin-mediated silencing.

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Multicopy FZF1 (SUL1) Suppresses the Sulfite Sensitivity but Not the Glucose Derepression or Aberrant Cell Morphology of a grr1 Mutant of Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/144.2.511 ◽

1996 ◽

Vol 144 (2) ◽

pp. 511-521 ◽

Cited By ~ 3

Author(s):

Dorina Avram ◽

Alan T Bakalinsky

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Factor ◽

Glucose Metabolism ◽

Cell Morphology ◽

Glucose Repression ◽

Carbon Sources ◽

Single Copy ◽

Aberrant Cell ◽

Growth Defects ◽

Putative Transcription Factor

Abstract An ssu2 mutation in Sacccharomyces cermisiae, previously shown to cause sulfite sensitivity, was found to be allelic to GRR1, a gene previously implicated in glucose repression. The suppressor rgt1, which suppresses the growth defects of grr1 strains on glucose, did not fully suppress the sensitivity on glucose or nonglucose carbon sources, indicating that it is not strictly linked to a defect in glucose metabolism. Because the Cln1 protein was previously shown to be elevated in grr1 mutants, the effect of CLN1 overexpression on sulfite sensitivity was investigated. Overexpression in GRR1 cells resulted in sulfite sensitivity, suggesting a connection between CLN1 and sulfite metabolism. Multicopy FZF1, a putative transcription factor, was found to suppress the sulfite sensitive phenotype of grr1 strains, but not the glucose derepression or aberrant cell morphology. Multicopy FZF1 was also found to suppress the sensitivity of a number of other unrelated sulfite-sensitive mutants, but not that of ssu1 or met20, implying that FZF1 may act through Ssulp and Met20p. Disruption of FZF1 resulted in sulfite sensitivity when the construct was introduced in single copy at the FZF1 locus in a GRR1 strain, providing evidence that FZF1 is involved in sulfite metabolism.

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Screening and Genetic Network Analysis of Genes Involved in Freezing and Thawing Resistance in DaMDHAR—Expressing Saccharomyces cerevisiae Using Gene Expression Profiling

Genes ◽

10.3390/genes12020219 ◽

2021 ◽

Vol 12 (2) ◽

pp. 219

Author(s):

Il-Sup Kim ◽

Woong Choi ◽

Jonghyeon Son ◽

Jun Hyuck Lee ◽

Hyoungseok Lee ◽

...

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Transcription Factors ◽

Stress Tolerance ◽

Genetic Network ◽

Yeast Cells ◽

Enzymatic Antioxidants ◽

Freezing And Thawing ◽

Metal Ion Homeostasis ◽

Transgenic Yeast

The cryoprotection of cell activity is a key determinant in frozen-dough technology. Although several factors that contribute to freezing tolerance have been reported, the mechanism underlying the manner in which yeast cells respond to freezing and thawing (FT) stress is not well established. Therefore, the present study demonstrated the relationship between DaMDHAR encoding monodehydroascorbate reductase from Antarctic hairgrass Deschampsia antarctica and stress tolerance to repeated FT cycles (FT2) in transgenic yeast Saccharomyces cerevisiae. DaMDHAR-expressing yeast (DM) cells identified by immunoblotting analysis showed high tolerance to FT stress conditions, thereby causing lower damage for yeast cells than wild-type (WT) cells with empty vector alone. To detect FT2 tolerance-associated genes, 3′-quant RNA sequencing was employed using mRNA isolated from DM and WT cells exposed to FT (FT2) conditions. Approximately 332 genes showed ≥2-fold changes in DM cells and were classified into various groups according to their gene expression. The expressions of the changed genes were further confirmed using western blot analysis and biochemical assay. The upregulated expression of 197 genes was associated with pentose phosphate pathway, NADP metabolic process, metal ion homeostasis, sulfate assimilation, β-alanine metabolism, glycerol synthesis, and integral component of mitochondrial and plasma membrane (PM) in DM cells under FT2 stress, whereas the expression of the remaining 135 genes was partially related to protein processing, selenocompound metabolism, cell cycle arrest, oxidative phosphorylation, and α-glucoside transport under the same condition. With regard to transcription factors in DM cells, MSN4 and CIN5 were activated, but MSN2 and MGA1 were not. Regarding antioxidant systems and protein kinases in DM cells under FT stress, CTT1, GTO, GEX1, and YOL024W were upregulated, whereas AIF1, COX2, and TRX3 were not. Gene activation represented by transcription factors and enzymatic antioxidants appears to be associated with FT2-stress tolerance in transgenic yeast cells. RCK1, MET14, and SIP18, but not YPK2, have been known to be involved in the protein kinase-mediated signalling pathway and glycogen synthesis. Moreover, SPI18 and HSP12 encoding hydrophilin in the PM were detected. Therefore, it was concluded that the genetic network via the change of gene expression levels of multiple genes contributing to the stabilization and functionality of the mitochondria and PM, not of a single gene, might be the crucial determinant for FT tolerance in DaMDAHR-expressing transgenic yeast. These findings provide a foundation for elucidating the DaMDHAR-dependent molecular mechanism of the complex functional resistance in the cellular response to FT stress.

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Maintenance of Mitochondrial Morphology Is Linked to Maintenance of the Mitochondrial Genome in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/162.3.1147 ◽

2002 ◽

Vol 162 (3) ◽

pp. 1147-1156 ◽

Cited By ~ 1

Author(s):

Theodor Hanekamp ◽

Mary K Thorsness ◽

Indrani Rebbapragada ◽

Elizabeth M Fisher ◽

Corrine Seebart ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Growth Rate ◽

Mitochondrial Dna ◽

Carbon Sources ◽

Mitochondrial Morphology ◽

Yeast Saccharomyces Cerevisiae ◽

Slow Growth Rate ◽

Dna Migration ◽

Mitochondrial Dna Stability ◽

Extragenic Suppressors

Abstract In the yeast Saccharomyces cerevisiae, certain mutant alleles of YME4, YME6, and MDM10 cause an increased rate of mitochondrial DNA migration to the nucleus, carbon-source-dependent alterations in mitochondrial morphology, and increased rates of mitochondrial DNA loss. While single mutants grow on media requiring mitochondrial respiration, any pairwise combination of these mutations causes a respiratory-deficient phenotype. This double-mutant phenotype allowed cloning of YME6, which is identical to MMM1 and encodes an outer mitochondrial membrane protein essential for maintaining normal mitochondrial morphology. Yeast strains bearing null mutations of MMM1 have altered mitochondrial morphology and a slow growth rate on all carbon sources and quantitatively lack mitochondrial DNA. Extragenic suppressors of MMM1 deletion mutants partially restore mitochondrial morphology to the wild-type state and have a corresponding increase in growth rate and mitochondrial DNA stability. A dominant suppressor also suppresses the phenotypes caused by a point mutation in MMM1, as well as by specific mutations in YME4 and MDM10.

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A genome-scale metabolic model of Saccharomyces cerevisiae that integrates expression constraints and reaction thermodynamics

Nature Communications ◽

10.1038/s41467-021-25158-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Omid Oftadeh ◽

Pierre Salvy ◽

Maria Masid ◽

Maxime Curvat ◽

Ljubisa Miskovic ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Expression System ◽

Biomass Composition ◽

Industrial Biotechnology ◽

Overflow Metabolism ◽

Cellular Expression ◽

A Genome ◽

Wide Range ◽

Eukaryotic Organisms ◽

Genome Scale

AbstractEukaryotic organisms play an important role in industrial biotechnology, from the production of fuels and commodity chemicals to therapeutic proteins. To optimize these industrial systems, a mathematical approach can be used to integrate the description of multiple biological networks into a single model for cell analysis and engineering. One of the most accurate models of biological systems include Expression and Thermodynamics FLux (ETFL), which efficiently integrates RNA and protein synthesis with traditional genome-scale metabolic models. However, ETFL is so far only applicable for E. coli. To adapt this model for Saccharomyces cerevisiae, we developed yETFL, in which we augmented the original formulation with additional considerations for biomass composition, the compartmentalized cellular expression system, and the energetic costs of biological processes. We demonstrated the ability of yETFL to predict maximum growth rate, essential genes, and the phenotype of overflow metabolism. We envision that the presented formulation can be extended to a wide range of eukaryotic organisms to the benefit of academic and industrial research.

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