scholarly journals Metabolic engineering of Saccharomyces cerevisiae for production of β-carotene from hydrophobic substrates

2020 ◽  
Author(s):  
Zahra Fathi ◽  
Larissa Ribeiro Ramos Tramontin ◽  
Gholamhossein Ebrahimipour ◽  
Irina Borodina ◽  
Farshad Darvishi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Olive Oil ◽  
Dry Weight ◽  
Yeast Saccharomyces Cerevisiae ◽  
Carotene Content ◽  
Yeast Peptone Dextrose ◽  
Carotenogenic Genes ◽  
Genes Encoding ◽  
Yellow Orange ◽  
Β Carotene

Abstract β-Carotene is a yellow-orange-red pigment used in food, cosmetics, and pharmacy. There is no commercial yeast-based process for β-carotene manufacturing. In this work, we engineered the baker's yeast Saccharomyces cerevisiae by expression of lipases and carotenogenic genes to enable the production of β-carotene on hydrophobic substrates. First, the extracellular lipase (LIP2) and two cell-bound lipases (LIP7 and LIP8) from oleaginous yeast Yarrowia lipolytica were expressed either individually or in combination in S. cerevisiae. The engineered strains could grow on olive oil and triolein as the sole carbon source. The strain expressing all three lipases had ∼40% lipid content per dry weight. Next, we integrated the genes encoding β-carotene biosynthetic pathway, crtI, crtYB, and crtE from Xanthophyllomyces dendrorhous. The resulting engineered strain bearing the lipases and carotenogenic genes reached a titer of 477.9 mg/L β-carotene in yeast peptone dextrose medium supplemented with 1% (v/v) olive oil, which was 12-fold higher than an analogous strain without lipases. The highest β-carotene content of 46.5 mg/g DCW was obtained on mineral medium supplemented with 1% (v/v) olive oil. The study demonstrates the potential of applying lipases and hydrophobic substrate supplementation for the production of carotenoids in S. cerevisiae.

scholarly journals High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous

2007 ◽  
Vol 73 (13) ◽  
pp. 4342-4350 ◽  
Author(s):  
René Verwaal ◽  
Jing Wang ◽  
Jean-Paul Meijnen ◽  
Hans Visser ◽  
Gerhard Sandmann ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Performance ◽  
Dry Weight ◽  
Yeast Cells ◽  
Xanthophyllomyces Dendrorhous ◽  
Biotechnological Production ◽  
Carotenoid Production ◽  
Carotenogenic Genes ◽  
Β Carotene ◽  
Ggpp Synthase

ABSTRACT To determine whether Saccharomyces cerevisiae can serve as a host for efficient carotenoid and especially β-carotene production, carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae. Because overexpression of these genes from an episomal expression vector resulted in unstable strains, the genes were integrated into genomic DNA to yield stable, carotenoid-producing S. cerevisiae cells. Furthermore, carotenoid production levels were higher in strains containing integrated carotenogenic genes. Overexpression of crtYB (which encodes a bifunctional phytoene synthase and lycopene cyclase) and crtI (phytoene desaturase) from X. dendrorhous was sufficient to enable carotenoid production. Carotenoid production levels were increased by additional overexpression of a homologous geranylgeranyl diphosphate (GGPP) synthase from S. cerevisiae that is encoded by BTS1. Combined overexpression of crtE (heterologous GGPP synthase) from X. dendrorhous with crtYB and crtI and introduction of an additional copy of a truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (tHMG1) into carotenoid-producing cells resulted in a successive increase in carotenoid production levels. The strains mentioned produced high levels of intermediates of the carotenogenic pathway and comparable low levels of the preferred end product β-carotene, as determined by high-performance liquid chromatography. We finally succeeded in constructing an S. cerevisiae strain capable of producing high levels of β-carotene, up to 5.9 mg/g (dry weight), which was accomplished by the introduction of an additional copy of crtI and tHMG1 into carotenoid-producing yeast cells. This transformant is promising for further development toward the biotechnological production of β-carotene by S. cerevisiae.

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 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 Electromagnetic Fields (0.04 to 0.39) mT effect on cellular growth cycles of Saccharomyces cerevisiae wine strains

2019 ◽  
Vol 17 (2) ◽  
pp. 196
Author(s):  
Eliseo Amado-González ◽  
Alveiro Álvarez Ovallos ◽  
Alfonso Quijano Parra
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Electromagnetic Fields ◽  
Biomass Production ◽  
Exposure Time ◽  
Culture Media ◽  
Dry Weight ◽  
Cellular Growth ◽  
Biomass Growth ◽  
Yeast Saccharomyces Cerevisiae ◽  
Growth Cycles

Low frecuency electromagnetic fields effect (EMF) on growth cycles of yeast Saccharomyces cerevisiae wine strains Rv1 and Rhône were studied.  A cylindrical coil induced magnetic fields with inductions up to 0,39 mT. Exposure time to EMF varied between (1 – 10) min at 30 °C.  The biomass growth were monitored in the reactor culture media (yeast extract + by measurement optical density from (0 to 32) h. The biomass was found by dry weight. After yeast expose to the different EMF, the number of growth cycles decreased from 4 cycles to 2 or 1. However, the biomass production increased almost 50 %.  The best biomass production was found at 0.39 mT and 10 min exposure time.  Keywords: Electromagnetic fields, Saccharomyces cerevisiae, biomass production, RV1

scholarly journals Expression of Bovine F1-ATPase with Functional Complementation in Yeast Saccharomyces cerevisiae

2005 ◽  
Vol 280 (23) ◽  
pp. 22418-22424 ◽  
Author(s):  
Neeti Puri ◽  
Jie Lai-Zhang ◽  
Scott Meier ◽  
David M. Mueller
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mass ◽  
Atp Synthase ◽  
Specific Activity ◽  
Functional Complementation ◽  
Yeast Saccharomyces Cerevisiae ◽  
F1 Atpase ◽  
Mitochondrial Atp Synthase ◽  
Genes Encoding ◽  
Molecular Machinery

The mitochondrial F1F0-ATP synthase is a multimeric enzyme complex composed of at least 16 unique peptides with an overall molecular mass of ∼600 kDa. F1-ATPase is composed of α3β3γδϵ with an overall molecular mass of 370 kDa. The genes encoding bovine F1-ATPase have been expressed in a quintuple yeast Saccharomyces cerevisiae deletion mutant (ΔαΔβΔγΔδΔϵ). This strain expressing bovine F1 is unable to grow on medium containing a non-fermentable carbon source (YPG), indicating that the enzyme is non-functional. However, daughter strains were easily selected for growth on YPG medium and these were evolved for improved growth on YPG medium. The evolution of the strains was presumably due to mutations, but mutations in the genes encoding the subunits of the bovine F1-ATPase were not required for the ability of the cell to grow on YPG medium. The bovine enzyme expressed in yeast was partially purified to a specific activity of about half of that of the enzyme purified from bovine heart mitochondria. These results indicate that the molecular machinery required for the assembly of the mitochondrial ATP synthase is conserved from bovine and yeast and suggest that yeast may be useful for the expression, mutagenesis, and analysis of the mammalian F1- or F1F0-ATP synthase.

Synthesis and expression of genes encoding tuna, pigeon, and horse cytochromes c in the yeast Saccharomyces cerevisiae

Gene ◽  
1991 ◽  
Vol 105 (1) ◽  
pp. 73-81 ◽  
Author(s):  
David R. Hickey ◽  
Krishna Jayaraman ◽  
Charles T. Goodhue ◽  
Janak Shah ◽  
Sarah A. Fingar ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Saccharomyces Cerevisiae ◽  
Expression Of Genes ◽  
Cytochromes C ◽  
Genes Encoding

scholarly journals Genetic analysis of the bipolar pattern of bud site selection in the yeast Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (4) ◽  
pp. 1857-1870 ◽  
Author(s):  
J E Zahner ◽  
H A Harkins ◽  
J R Pringle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Interaction ◽  
Previous Analysis ◽  
Intracellular Localization ◽  
Proximal Pole ◽  
Yeast Saccharomyces Cerevisiae ◽  
Novel Gene ◽  
Division Site ◽  
Genes Encoding ◽  
Bud Site

Previous analysis of the bipolar budding pattern of Saccharomyces cerevisiae has suggested that it depends on persistent positional signals that mark the region of the division site and the tip of the distal pole on a newborn daughter cell, as well as each previous division site on a mother cell. In an attempt to identify genes encoding components of these signals or proteins involved in positioning or responding to them, we identified 11 mutants with defects in bipolar but not in axial budding. Five mutants displaying a bipolar budding-specific randomization of budding pattern had mutations in four previously known genes (BUD2, BUD5, SPA2, and BNI1) and one novel gene (BUD6), respectively. As Bud2p and Bud5p are known to be required for both the axial and bipolar budding patterns, the alleles identified here probably encode proteins that have lost their ability to interact with the bipolar positional signals but have retained their ability to interact with the distinct positional signal used in axial budding. The function of Spa2p is not known, but previous work has shown that its intracellular localization is similar to that postulated for the bipolar positional signals. BNI1 was originally identified on the basis of genetic interaction with CDC12, which encodes one of the neck-filament-associated septin proteins, suggesting that these proteins may be involved in positioning the bipolar signals. One mutant with a heterogeneous budding pattern defines a second novel gene (BUD7). Two mutants budding almost exclusively from the proximal pole carry mutations in a fourth novel gene (BUD9). A bud8 bud9 double mutant also buds almost exclusively from the proximal pole, suggesting that Bud9p is involved in positioning the proximal pole signal rather than being itself a component of this signal.

scholarly journals Oxygen and haem regulate the synthesis of peroxisomal proteins: catalase A, acyl-CoA oxidase and Pex1p in the yeast Saccharomyces cerevisiae; the regulation of these proteins by oxygen is not mediated by haem

2000 ◽  
Vol 350 (1) ◽  
pp. 313-319 ◽  
Author(s):  
Marek SKONECZNY ◽  
Joanna RYTKA
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Genome ◽  
Oxygen Sensor ◽  
Transcriptional Factor ◽  
The Other ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Regulatory Factors ◽  
Genes Encoding ◽  
Acyl Coa Oxidase

Saccharomyces cerevisiae genes related to respiration are typically controlled by oxygen and haem. Usually the regulation by these factors is co-ordinated; haem is indicated as the oxygen sensor. However, the responsiveness of peroxisome functions to these regulatory factors is poorly understood. The expression of CTA1, POX1 and PEX1 genes encoding the peroxisomal proteins catalase A, acyl-CoA oxidase and Pex1p peroxin respectively was studied under various conditions: in anaerobiosis, in the absence of haem and in respiratory incompetence caused by the lack of a mitochondrial genome (ρ0). The influence of haem deficiency or ρ0 on peroxisomal morphology was also investigated. Respiratory incompetence has no effect on the expression of CTA1 and POX1, whereas in the absence of haem their expression is markedly decreased. The synthesis of Pex1p is decreased in ρ0 cells and is decreased even more in haem-deficient cells. Nevertheless, peroxisomal morphology in both these types of cell does not differ significantly from the morphology of peroxisomes in wild-type cells. The down-regulating effect of anoxia on the expression of CTA1 and POX1 is even stronger than the effect of haem deficiency and is not reversed by the addition of exogenous haem or the presence of endogenous haem. Moreover, neither of these genes responds to the known haem-controlled transcriptional factor Hap1p. In contrast with the other two genes studied, PEX1 is up-regulated in anaerobiosis. The existence of one or more novel mechanisms of regulation of peroxisomal genes by haem and oxygen, different from those already known in S. cerevisiae, is postulated.

Ribosomal protein S14 is encoded by a pair of highly conserved, adjacent genes on the X chromosome of Drosophila melanogaster

1988 ◽  
Vol 8 (10) ◽  
pp. 4314-4321
Author(s):  
S J Brown ◽  
D D Rhoads ◽  
M J Stewart ◽  
B Van Slyke ◽  
I T Chen ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drosophila Melanogaster ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Ribosomal Protein ◽  
X Chromosome ◽  
Duplicated Genes ◽  
Ribosomal Protein Genes ◽  
Yeast Saccharomyces Cerevisiae ◽  
Genes Encoding

We describe a Drosophila DNA clone of tandemly duplicated genes encoding an amino acid sequence nearly identical to human ribosomal protein S14 and yeast rp59. Despite their remarkably similar exons, the locations and sizes of introns differ radically among the Drosophila, human, and yeast (Saccharomyces cerevisiae) ribosomal protein genes. Transcripts of both Drosophila RPS14 genes were detected in embryonic and adult tissues and are the same length as mammalian S14 message. Drosophila RPS14 was mapped to region 7C5-9 on the X chromosome. This interval also encodes a previously characterized Minute locus, M(1)7C.

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