BtBs_UNIMIB_The effect of oxygen availability on recombinant protein producing S. cerevisiae
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
SUBMITTER: Laura Dato
PROVIDER: E-MEXP-3134 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:The effect of oxygen availability on recombinant protein production in Pichia pastoris
2010-06-09 | E-MEXP-2742 | biostudies-arrayexpress
Project description:We aimed to study how production of p-coumaric acid, a precursor of multiple secondary aromatic metabolites, influences the cellular metabolism of Saccharomyces cerevisiae. We evaluated the growth and p-coumaric acid production in batch and chemostat cultivations and analyzed the transcriptome and intracellular metabolome during steady state in low- and high-producers of p-coumaric acid in two strain backgrounds, S288c or CEN.PK. For analysis of the differential gene expression, we did pairwise comparisons between the optimized and non-optimized strains for p-CA production: CEN.PK strains (ST4288 and ST4408) and the S288c strains (ST4353 and ST4397). Transcriptome analysis showed that the CEN.PK strain was less affected by engineering towards higher p-CA production than the S288c strain, as the number of significantly up-/down-regulated genes was correspondingly 652 and 1927 amongst others, strain S288c had downregulations in gene sets involved in amino acid and protein biosynthesis. This suggests that CEN.PK may be a better platform strain for production of aromatic compounds than the S288c strain.
2017-11-21 | E-MTAB-6208 | biostudies-arrayexpress
Project description:To obtain insight in the genome-wide response of heterologous carotenoid production in Saccharomyces cerevisiae, we have analyzed the transcriptome of S. cerevisiae strains overexpressing carotenogenic genes from the yeast Xanthophyllomyces dendrorhous. For this purpose, two strains producing different levels of carotenoids were grown in carbon-limited continuous cultures and genome-wide expression was analyzed. The strain producing low carotenoid levels did not exhibit a clear genome-wide transcriptional response, suggesting that low carotenoid levels do not result in cellular stress. Transcriptome analysis of a strain producing high carotenoid levels resulted in specific induction of genes involved in pleiotropic drug resistance (PDR). These genes encode ATP-binding cassette (ABC) type transporters and major facilitator transporters which are involved in secretion of toxic compounds out of cells. Our results suggest that production of high amounts of carotenoids in S. cerevisiae lead to toxicity and that these cells are prone to secrete carotenoids out of the cell. Indeed, secretion of ï¢-carotene into sunflower oil was observed upon addition of this hydrophobic solvent to the growth medium. Finally, it was observed that deletion of the ABC transporter pdr10, one of the induced PDR transporters, highly decreased the transformation efficiency of an episomal vector containing carotenogenic genes. The few colored transformants that were obtained had decreased growth rates and lower carotenoid production levels compared to control strains transformed with the same carotenogenic genes. These results indicate that Pdr10 might be specifically involved in carotenoid tolerance in S. cerevisiae strains. Experiment Overall Design: The genome wide transcriptional response of S. cerevisiae cells that heterologously produce carotenoids might provide information concerning the impact of carotenoid production on yeast physiology and might identify bottlenecks relevant for the production of these compounds. DNA microarray experiments have been proven to be a powerful tool to study the genome wide transcriptional response of S. cerevisiae to changes of the physiological state and the environment (for example 3,. Genomics approaches on cells producing heterologous metabolites to study their impact on yeast physiology have not been reported yet for S. cerevisiae. Additionally, most transcriptome studies with S. cerevisiae have been performed with cells grown in shake flasks cultures. The main drawback of shake flask cultivation is that the environment is continuously changing, which may be of high influence on carotenoid production, and interpretation of transcriptome data . Chemostat cultivation offers advantages for studies with DNA microarrays because it enables cultivation of microorganisms under tightly defined environmental conditions. An interlaboratory comparison of transcriptome data obtained in chemostat cultures has indeed demonstrated that the accuracy and reproducibility of this approach are superior to those obtained in previous studies with shake-flask cultures .
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Project description:Anaerobic respiration in the metal reducer Shewanella oneidensis MR-1, unlike other bacteria, is regulated by the cAMP receptor protein, CRP. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an E. coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, DMSO, or Fe(III), whereas the deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III), and to a lesser extent with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and the cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways that include DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagella biosynthesis, and electron transport, were differentially expressed in the cyaC mutant, but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration, and may contribute to additional signaling pathways independent of CRP. To explore the roles of CyaC and CRP in the regulation of anaerobic respiration in Shewanella oneidensis MR-1, the genome-wide transcriptome profiles of ÎcyaC and Îcrp mutants were compared to those of the wild type. Given the growth deficiencies displayed by Îcrp and ÎcyaC under anaerobic conditions, all strains were cultured in chemostats under O2 limitation. Since both Îcrp and ÎcyaC strains are not impaired in aerobic respiration, oxygen-limited conditions provided the means to maintain equal growth rates (0.06 h-1) among the mutant and wild-type strains thus eliminating the effect of growth on the overall transcriptome profiles. At the same time, while growing aerobically, the cells decreased dissolved O2 concentration in the medium below the detection threshold (DOTâ¤1%) allowing for the expression of anaerobic respiratory genes. The latter was confirmed by the reduction of fumarate by the wild-type culture which produced stoichiometric amounts of succinate under O2-limited conditions. The optical density (OD600) of the wild type, ÎcyaC, and Îcrp chemostat cultures used for transcriptome profiling was 0.50, 0.45 and 0.40, respectively. All the experiments were carried out in duplicate.
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Project description:Zinc is indispensable for the catalytic activity and structural stability of many proteins, and its deficiency can have severe consequences for microbial growth in natural and industrial environments. For example, Zn depletion in wort negatively affects beer fermentation and quality. Several studies have investigated yeast adaptation to low Zn supply, but were all performed in batch cultures, where specific growth rate depends on Zn availability. The transcriptional responses to growth-rate and Zn availability are then intertwined, which obscures result interpretation. In the present study, transcriptional responses of Saccharomyces cerevisiae to Zn availability were investigated at a fixed specific growth rate under Zn limitation and excess in chemostat culture. To investigate the context-dependency of this transcriptional response, yeast was grown under several chemostat regimes resulting in various carbon (glucose), nitrogen (ammonium) and oxygen supplies. A robust set of genes that responded consistently to Zn limitation was identified and enabled the definition of a Zn-specific Zap1 regulon comprising of 26 genes and characterized by a broader ZRE consensus (MHHAACCBYNMRGGT) than so far described. Most surprising was the Zn-dependent regulation of genes involved in storage carbohydrate metabolism. Their concerted down-regulation was physiologically relevant as revealed by a substantial decrease in glycogen and trehalose cellular content under Zn limitation. An unexpectedly large amount of genes were synergistically or antagonistically regulated by oxygen and Zn availability. This combinatorial regulation suggested a more prominent involvement of Zn in mitochondrial biogenesis and function than hitherto identified. Experiment Overall Design: Zinc is indispensable for the catalytic activity and structural stability of many proteins, and its deficiency can have severe consequences for microbial growth in natural and industrial environments. For example, Zn depletion in wort negatively affects beer fermentation and quality. Several studies have investigated yeast adaptation to low Zn supply, but were all performed in batch cultures, where specific growth rate depends on Zn availability. The transcriptional responses to growth-rate and Zn availability are then intertwined, which obscures result interpretation. In the present study, transcriptional responses of Saccharomyces cerevisiae to Zn availability were investigated at a fixed specific growth rate under Zn limitation and excess in chemostat culture. To investigate the context-dependency of this transcriptional response, yeast was grown under several chemostat regimes resulting in various carbon (glucose), nitrogen (ammonium) and oxygen supplies. A robust set of genes that responded consistently to Zn limitation was identified and enabled the definition of a Zn-specific Zap1 regulon comprising of 26 genes and characterized by a broader ZRE consensus (MHHAACCBYNMRGGT) than so far described. Most surprising was the Zn-dependent regulation of genes involved in storage carbohydrate metabolism. Their concerted down-regulation was physiologically relevant as revealed by a substantial decrease in glycogen and trehalose cellular content under Zn limitation. An unexpectedly large amount of genes were synergistically or antagonistically regulated by oxygen and Zn availability. This combinatorial regulation suggested a more prominent involvement of Zn in mitochondrial biogenesis and function than hitherto identified.
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