scholarly journals Does co-expression of Yarrowia lipolytica genes encoding Yas1p, Yas2p and Yas3p make a potential alkane-responsive biosensor in Saccharomyces cerevisiae?

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0239882
Author(s):  
Yasaman Dabirian ◽  
Christos Skrekas ◽  
Florian David ◽  
Verena Siewers
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yarrowia Lipolytica ◽  
High Throughput Screening ◽  
Screening Tools ◽  
Evolutionary Engineering ◽  
Transportation Fuels ◽  
Sensing System ◽  
Commercial Scale ◽  
Genes Encoding ◽  
Native Host

Alkane-based biofuels are desirable to produce at a commercial scale as these have properties similar to current petroleum-derived transportation fuels. Rationally engineering microorganisms to produce a desirable compound, such as alkanes, is, however, challenging. Metabolic engineers are therefore increasingly implementing evolutionary engineering approaches combined with high-throughput screening tools, including metabolite biosensors, to identify productive cells. Engineering Saccharomyces cerevisiae to produce alkanes could be facilitated by using an alkane-responsive biosensor, which can potentially be developed from the native alkane-sensing system in Yarrowia lipolytica, a well-known alkane-assimilating yeast. This putative alkane-sensing system is, at least, based on three different transcription factors (TFs) named Yas1p, Yas2p and Yas3p. Although this system is not fully elucidated in Y. lipolytica, we were interested in evaluating the possibility of translating this system into an alkane-responsive biosensor in S. cerevisiae. We evaluated the alkane-sensing system in S. cerevisiae by developing one sensor based on the native Y. lipolytica ALK1 promoter and one sensor based on the native S. cerevisiae CYC1 promoter. In both systems, we found that the TFs Yas1p, Yas2p and Yas3p do not seem to act in the same way as these have been reported to do in their native host. Additional analysis of the TFs suggests that more knowledge regarding their mechanism is needed before a potential alkane-responsive sensor based on the Y. lipolytica system can be established in S. cerevisiae.

scholarly journals Does co-expression of Yarrowia lipolytica genes encoding Yas1p, Yas2p and Yas3p make a potential alkane-responsive biosensor in Saccharomyces cerevisiae?

2020 ◽  
Author(s):  
Yasaman Dabirian ◽  
Christos Skrekas ◽  
Florian David ◽  
Verena Siewers
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yarrowia Lipolytica ◽  
High Throughput Screening ◽  
Screening Tools ◽  
Evolutionary Engineering ◽  
Transportation Fuels ◽  
Sensing System ◽  
Commercial Scale ◽  
Genes Encoding ◽  
Native Host

ABSTRACTAlkane-based biofuels are desirable to produce at a commercial scale as these have properties similar to our current petroleum-derived transportation fuels. Rationally engineering microorganisms to produce a desirable compound, such as alkanes, is, however, challenging. Metabolic engineers are therefore increasingly implementing evolutionary engineering approaches combined with high-throughput screening tools, including metabolite biosensors, to identify productive targets. Engineering Saccharomyces cerevisiae to produce alkanes can be facilitated by using an alkane-responsive biosensor, which can potentially be developed from the native alkane-sensing system in Yarrowia lipolytica, a well-known alkane-assimilating yeast. This putative alkane-sensing system is, at least, based on three different transcription factors (TFs) named Yas1p, Yas2p and Yas3p. Although this system is not fully elucidated in Y. lipolytica, we were interested in evaluating the possibility of translating this system into an alkane-responsive biosensor in S. cerevisiae. We evaluated the alkane-sensing system in S. cerevisiae by developing one sensor based on the native Y. lipolytica ALK1 promoter and one sensor based on the native S. cerevisiae CYC1 promoter. In both systems, we found that the TFs Yas1p, Yas2p and Yas3p do not seem to act in the same way as these have been reported to do in their native host. Additional analysis of the TFs suggests that more knowledge regarding their mechanism is needed before a potential alkane-responsive sensor based on the Y. lipolytica system can be established in S. cerevisiae.

scholarly journals Accelerated Bioprocess Development of Endopolygalacturonase-Production with Saccharomyces cerevisiae Using Multivariate Prediction in a 48 Mini-Bioreactor Automated Platform

2018 ◽  
Author(s):  
Annina Sawatzki ◽  
Sebastian Hans ◽  
Harini Narayanan ◽  
Benjamin Haby ◽  
Niels Krausch ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Decision Making ◽  
Process Control ◽  
High Throughput Screening ◽  
Large Scale ◽  
Screening Tools ◽  
Abnormal Behavior ◽  
Scale Production ◽  
Bioprocess Development ◽  
Fed Batch

Mini-bioreactor systems enabling automatized operation of numerous parallel cultivations have been used to accelerate and optimize bioprocess development. As implementation of fed-batch conditions, multiple options of process control and sample analysis are possible, these systems represent valuable screening tools for large-scale production. However, the dynamic behavior of cultivations has not yet been considered regarding data evaluation and decision making during high-throughput screening in mini-bioreactors. In this study, the characterization of Saccharomyces cerevisiae AH22 secreting recombinant endopolygalacturonase is performed in 48 parallel fed-batch cultivations regarding 16 experimental conditions. Automated parallel process control, frequent sampling and analysis were implemented. Data-driven multivariate methods were developed to allow for fast, automated decision making as well as online predictive data analysis regarding endopolygalacturonase production. Using dynamic process information, a cultivation with abnormal behavior could be detected by principal component analysis as well as two clusters of similarly behaving cultivations, later classified according to the feeding rate. By decision tree analysis, cultivation conditions leading to an optimal recombinant product formation could be identified automatically. The developed method is easily adaptable and suitable for automatized process development reducing the experimental times and costs.

scholarly journals Regulatory Mechanisms Controlling Expression of the DAN/TIR Mannoprotein Genes During Anaerobic Remodeling of the Cell Wall in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 157 (3) ◽  
pp. 1169-1177
Author(s):  
Natalia E Abramova ◽  
Brian D Cohen ◽  
Odeniel Sertil ◽  
Rachna Kapoor ◽  
Kelvin J A Davies ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Consensus Sequence ◽  
Ectopic Expression ◽  
Constitutive Expression ◽  
Anaerobic Growth ◽  
Zinc Cluster ◽  
Sterol Transport ◽  
Genes Encoding ◽  
Altered Regulation ◽  
Repression Domain

Abstract The DAN/TIR genes of Saccharomyces cerevisiae encode homologous mannoproteins, some of which are essential for anaerobic growth. Expression of these genes is induced during anaerobiosis and in some cases during cold shock. We show that several heme-responsive mechanisms combine to regulate DAN/TIR gene expression. The first mechanism employs two repression factors, Mox1 and Mox2, and an activation factor, Mox4 (for mannoprotein regulation by oxygen). The genes encoding these proteins were identified by selecting for recessive mutants with altered regulation of a dan1::ura3 fusion. MOX4 is identical to UPC2, encoding a binucleate zinc cluster protein controlling expression of an anaerobic sterol transport system. Mox4/Upc2 is required for expression of all the DAN/TIR genes. It appears to act through a consensus sequence termed the AR1 site, as does Mox2. The noninducible mox4Δ allele was epistatic to the constitutive mox1 and mox2 mutations, suggesting that Mox1 and Mox2 modulate activation by Mox4 in a heme-dependent fashion. Mutations in a putative repression domain in Mox4 caused constitutive expression of the DAN/TIR genes, indicating a role for this domain in heme repression. MOX4 expression is induced both in anaerobic and cold-shocked cells, so heme may also regulate DAN/TIR expression through inhibition of expression of MOX4. Indeed, ectopic expression of MOX4 in aerobic cells resulted in partially constitutive expression of DAN1. Heme also regulates expression of some of the DAN/TIR genes through the Rox7 repressor, which also controls expression of the hypoxic gene ANB1. In addition Rox1, another heme-responsive repressor, and the global repressors Tup1 and Ssn6 are also required for full aerobic repression of these genes.

scholarly journals Underproduction of the Largest Subunit of RNA Polymerase II Causes Temperature Sensitivity, Slow Growth, and Inositol Auxotrophy in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 737-747 ◽  
Author(s):  
Jacques Archambault ◽  
David B Jansma ◽  
James D Friesen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Reporter Gene ◽  
Growth Medium ◽  
Temperature Sensitivity ◽  
Slow Growth ◽  
Yeast Saccharomyces Cerevisiae ◽  
Genes Encoding

Abstract In the yeast Saccharomyces cerevisiae, mutations in genes encoding subunits of RNA polymerase II (RNAPII) often give rise to a set of pleiotropic phenotypes that includes temperature sensitivity, slow growth and inositol auxotrophy. In this study, we show that these phenotypes can be brought about by a reduction in the intracellular concentration of RNAPII. Underproduction of RNAPII was achieved by expressing the gene (RPO21), encoding the largest subunit of the enzyme, from the LEU2 promoter or a weaker derivative of it, two promoters that can be repressed by the addition of leucine to the growth medium. We found that cells that underproduced RPO21 were unable to derepress fully the expression of a reporter gene under the control of the INO1 UAS. Our results indicate that temperature sensitivity, slow growth and inositol auxotrophy is a set of phenotypes that can be caused by lowering the steady-state amount of RNAPII; these results also lead to the prediction that some of the previously identified RNAPII mutations that confer this same set of phenotypes affect the assembly/stability of the enzyme. We propose a model to explain the hypersensitivity of INO1 transcription to mutations that affect components of the RNAPII transcriptional machinery.

scholarly journals A Role for the Actin Cytoskeleton of Saccharomyces cerevisiae in Bipolar Bud-Site Selection

1997 ◽  
Vol 136 (1) ◽  
pp. 111-123 ◽  
Author(s):  
Shirley Yang ◽  
Kathryn R. Ayscough ◽  
David G. Drubin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Cytoskeleton ◽  
Site Selection ◽  
Mitotic Checkpoint ◽  
Cell Cortex ◽  
Spatial Cues ◽  
Genes Encoding ◽  
Mother And Daughter ◽  
Mother Cells ◽  
Bud Site

Saccharomyces cerevisiae cells select bud sites according to one of two predetermined patterns. MATa and MATα cells bud in an axial pattern, and MATa/α cells bud in a bipolar pattern. These budding patterns are thought to depend on the placement of spatial cues at specific sites in the cell cortex. Because cytoskeletal elements play a role in organizing the cytoplasm and establishing distinct plasma membrane domains, they are well suited for positioning bud-site selection cues. Indeed, the septin-containing neck filaments are crucial for establishing the axial budding pattern characteristic of MATa and MATα cells. In this study, we determined the budding patterns of cells carrying mutations in the actin gene or in genes encoding actin-associated proteins: MATa/α cells were defective in the bipolar budding pattern, but MATa and MATα cells still exhibit a normal axial budding pattern. We also observed that MATa/α actin cytoskeleton mutant daughter cells correctly position their first bud at the distal pole of the cell, but mother cells position their buds randomly. The actin cytoskeleton therefore functions in generation of the bipolar budding pattern and is required specifically for proper selection of bud sites in mother MATa/α cells. These observations and the results of double mutant studies support the conclusion that different rules govern bud-site selection in mother and daughter MATa/α cells. A defective bipolar budding pattern did not preclude an sla2-6 mutant from undergoing pseudohyphal growth, highlighting the central role of daughter cell bud-site selection cues in the formation of pseudohyphae. Finally, by examining the budding patterns of mad2-1 mitotic checkpoint mutants treated with benomyl to depolymerize their microtubules, we confirmed and extended previous evidence indicating that microtubules do not function in axial or bipolar bud-site selection.

scholarly journals Rsp5p, a New Link between the Actin Cytoskeleton and Endocytosis in the Yeast Saccharomyces cerevisiae

2002 ◽  
Vol 22 (20) ◽  
pp. 6946-6948 ◽  
Author(s):  
Joanna Kamińska ◽  
Beata Gajewska ◽  
Anita K. Hopper ◽  
Teresa ˙Zołądek
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Cytoskeleton ◽  
Cytoskeletal Proteins ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ww Domains ◽  
Ubiquitin Protein Ligase ◽  
Growth Defects ◽  
Genes Encoding ◽  
Cytoskeleton Dynamics

ABSTRACT Rsp5p is an ubiquitin-protein ligase of Saccharomyces cerevisiae that has been implicated in numerous processes including transcription, mitochondrial inheritance, and endocytosis. Rsp5p functions at multiple steps of endocytosis, including ubiquitination of substrates and other undefined steps. We propose that one of the roles of Rsp5p in endocytosis involves maintenance and remodeling of the actin cytoskeleton. We report the following. (i) There are genetic interactions between rsp5 and several mutant genes encoding actin cytoskeletal proteins. rsp5 arp2, rsp5 end3, and rsp5 sla2 double mutants all show synthetic growth defects. Overexpressed wild-type RSP5 or mutant rsp5 genes with lesions of some WW domains suppress growth defects of arp2 and end3 cells. The defects in endocytosis, actin cytoskeleton, and morphology of arp2 are also suppressed. (ii) Rsp5p and Sla2p colocalize in abnormal F-actin-containing clumps in arp2 and pan1 mutants. Immunoprecipitation experiments confirmed that Rsp5p and Act1p colocalize in pan1 mutants. (iii) Rsp5p and Sla2p coimmunoprecipitate and partially colocalize to punctate structures in wild-type cells. These studies provide the first evidence for an interaction of an actin cytoskeleton protein with Rsp5p. (iv) rsp5-w1 mutants are resistant to latrunculin A, a drug that sequesters actin monomers and depolymerizes actin filaments, consistent with the fact that Rsp5p is involved in actin cytoskeleton dynamics.

scholarly journals Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica

2017 ◽  
Vol 16 (1) ◽  
Author(s):  
Thomas Beneyton ◽  
Stéphane Thomas ◽  
Andrew D. Griffiths ◽  
Jean-Marc Nicaud ◽  
Antoine Drevelle ◽  
...  
Keyword(s):  
Yarrowia Lipolytica ◽  
High Throughput ◽  
High Throughput Screening ◽  
Yeast Yarrowia Lipolytica

Dominant effects of tubulin overexpression in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (3) ◽  
pp. 1049-1059
Author(s):  
D Burke ◽  
P Gasdaska ◽  
L Hartwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Loss ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Inducible Promoters ◽  
Selective Degradation ◽  
Novel Structure ◽  
Genes Encoding ◽  
Transient Overexpression ◽  
Mutant Phenotypes

The consequences of altering the levels of alpha- and beta-tubulin in Saccharomyces cerevisiae were examined by constructing fusions of the structural genes encoding the tubulins to strong galactose-inducible promoters. Overexpression of beta-tubulin (TUB2) was lethal: cells arrested in the G2 stage of the cell cycle exhibited an increased frequency of chromosome loss, were devoid of microtubules, and accumulated beta-tubulin in a novel structure. Overexpression of the major alpha-tubulin gene (TUB1) was not lethal and did not affect chromosome segregation. The rate of alpha-tubulin mRNA and protein synthesis was increased, but the protein did not accumulate. Overexpression of both alpha- and beta-tubulin together resulted in arrested cell division, and cells accumulated excess tubules that contained both alpha- and beta-tubulin. Transient overexpression of both tubulins resulted in a high frequency of chromosome loss. These data suggest that strong selective pressure exists to prevent excess accumulation of microtubules or beta-tubulin and suggest a model by which this goal may be achieved by selective degradation of unassembled alpha-tubulin. Furthermore, the phenotype of beta-tubulin overexpression is similar to the phenotype of a beta-tubulin deficiency. These results add to a number of recent studies demonstrating that mutant phenotypes generated by overexpression can be informative about the function of the gene product.

Elongation factor EF-1 alpha gene dosage alters translational fidelity in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (10) ◽  
pp. 4571-4575
Author(s):  
J M Song ◽  
S Picologlou ◽  
C M Grant ◽  
M Firoozan ◽  
M F Tuite ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Dosage ◽  
Elongation Factor ◽  
Nonsense Suppression ◽  
Translational Fidelity ◽  
Genes Encoding ◽  
Alpha Gene ◽  
Nonsense Codons

Changes in the dosage of genes encoding elongation factor EF-1 alpha were shown to cause parallel changes in the misreading of nonsense codons. Higher amounts of EF-1 alpha were correlated with increased nonsense suppression, suggesting that the level of EF-1 alpha is critically involved in translational fidelity.

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