Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of S. cerevisiae micro-scale sirred bioreactors equipped with real-time measurements of growth kinetics and humidity control

ABSTRACT: Global Gene expression analysis of Saccharomyces cerevisiae grown in micro-scale sirred bioreactors equipped with real-time measurements of growth kinetics and humidity control

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Sanchita Bhattacharya

PROVIDER: E-GEOD-2605 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Differential gene expression profiles and real-time measurements of growth parameters in Saccharomyces cerevisiae grown in microliter-scale bioreactors equipped with internal stirring.

Boccazzi Paolo P Zhang Zhiyu Z Kurosawa Kazuhiko K Szita Nicolas N Bhattacharya Sanchita S Jensen Klavs F KF Sinskey Anthony J AJ

Biotechnology progress 20060501 3

Combining real-time growth kinetics measurements with global gene expression analysis of microbial cultures is of significant value for high-throughput biological research. We have performed differential gene expression analysis in the eukaryotic model Saccharomyces cerevisiae grown in galactose and glucose media in 150 muL bioreactors equipped with sensors for in situ and real-time measurements of optical density (OD), pH, and dissolved oxygen (DO). The microbioreactors were fabricated from pol ...[more]

PMID: 16739953

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Project description:Sucrose is a major carbon source for industrial bioethanol production by Saccharomyces cerevisiae. In yeasts, two modes of sucrose metabolism occur: (i) extracellular hydrolysis by invertase, followed by uptake and metabolism of glucose and fructose, and (ii) uptake via sucrose-H+ symport followed by intracellular hydrolysis and metabolism. Although alternative start codons in the SUC2 gene enable synthesis of extracellular and intracellular invertase isoforms, sucrose hydrolysis in S. cerevisiae predominantly occurs extracellularly. In anaerobic cultures, intracellular hydrolysis theoretically enables a 9 % higher ethanol yield than extracellular hydrolysis, due to energy costs of sucrose-proton symport. This prediction was tested by engineering the promoter and 5’ coding sequences of SUC2, resulting in relocation of invertase to the cytosol. In anaerobic sucrose-limited chemostats, this iSUC2-strain showed an only 4% increased ethanol yield and high residual sucrose concentrations indicated suboptimal sucrose-transport kinetics. To improve sucrose-uptake affinity, it was subjected to 95 generations of anaerobic, sucrose-limited chemostat cultivation, resulting in a 20-fold decrease of residual sucrose concentrations and a 10-fold increase of the sucrose-transport capacity. A single-cell isolate showed an 11 % higher ethanol yield on sucrose in chemostat and batch cultures than an isogenic SUC2 reference strain, while transcriptome analysis revealed elevated expression of AGT1, encoding a disaccharide-proton symporter, and other maltose-related genes. Deletion of AGT1, which had been duplicated during laboratory evolution, restored the growth characteristics of the unevolved iSUC2 strain. This study demonstrates that engineering the topology of sucrose metabolism is an attractive strategy to improve ethanol yields in industrial processes. The goal of the present study was to investigate whether a relocation of sucrose hydrolysis to the cytosol can be used to improve ethanol yields on sucrose and which additional steps may be required to improve sucrose utilization by strains that only express intracellular invertase. To this end, the SUC2 gene was modified to cause an exclusive intracellular localization. Growth and product formation by the engineered strain were compared with that of the parental strain in anaerobic sucrose-limited chemostat cultures. Subsequently, evolutionary engineering was used to improve sucrose uptake kinetics and an evolved strain was characterized for growth and product formation in chemostat cultures. Transcriptome analysis and gene deletion studies were used to identify genetic changes in the evolved strain that contribute to its improved sucrose-uptake kinetics.

Project description:The comprehensive transcriptome of Streptomyces lividans TK24 in a biolector milliliter-scale cultivation system was compared in detail against an established 1000-fold larger lab-scale bioreactor system. For main-cultures, the modified minimal medium NMMP (D'Huys et al. 2011) was applied, containing 2.07 g/L NaH2PO4 x 1 H2O, 2.6 g/L K2HPO4, 0.6 MgSO4 x 7 H2O, 3.0 g/L (NH4)2SO4, 10 g/L D-glucose and 5 g/L BactoTM Casamino acids as well as several trace elements (1 mg/L ZnSO4 x 7 H2O, 1 mg/L FeSO4 x 7 H2O, 1 mg/L MnCl2 x 4 H2O, 1 mg/L CaCl2). pH was adjusted to 6.8 using 1 M KOH or 1 M H2SO4. In all MTP BioLector (BL) cultivations and in some bioreactor experiments (when indicated) 100 mM 2-(N-morpholino)ethanesulfonic acid (MES) was added as biological inert buffer system. Except for the phosphate buffer, all other components were prepared as stock solutions and sterilized separately. For MTP based cultivations the BL device, all stock solutions kept frozen at -20 C in aliquots sufficient for 50 mL final medium, to ensure identical conditions between experimental runs. For the system comparison experiments the same medium charge was used for all cultivations. 1 mL MTP-based BL cultivations MTP cultivations were performed in 48 well FlowerPlates (m2p-labs GmbH, Baesweiler, Germany), covered by a gas-permeable sealing foil (m2p-labs GmbH, Baesweiler, Germany). 1000 L cultivation medium was inoculated to a final OD600 of 0.2. Temperature and humidity was controlled in the incubation chamber of the BL device at 30 C and 89 % respectively. All signals (BS, DO, and pH), were measured with a cycle time of around 10 min. Altogether, 96 identical parallel milliliter-scale cultivations were performed in two separate but identical BL devices. One of these devices was additionally embedded in a lab-robotic liquid handling unit in the style of (Unthan et al. 2015; Rohe et al. 2012), to provide automated hourly sampling for substrate and cell dry weight analysis. The cultivations in the second BL unit served to generate samples for the omics-based analysis, discussed in the later sections. Bioreactor cultivations were carried out in a 1.5 L lab-scale system (DasGip, Jlich, Germany), equipped with two six blade, 46 mm Rushton turbines as well as sensors for DO (Hamilton, Visiferm DO 225) and pH (MettlerToledo, 405-DPAS-SC-K8S/225/120). Temperature was controlled at 30 C; evaporation was minimized by offgas cooling at 10 C. 700 mL phosphate buffer was sterilized directly within the reactor glass vessel; all other media components were added sterile and premixed subsequently by using a 500 mL feeding flask. 0.5 mL anti-foam (AF204 (Sigma, MO, USA)) was added before DO calibration. The final 1000 mL medium was inoculated with washed mycelium to a final OD600 of 0.2. Both, aeration and agitation were set to fixed values at 0.5 vvm and 800 rpm respectively. For both techniques samples were taken during the late growth phase, two hours after the switch to process phase II.

Dataset Information

Transcription profiling of S. cerevisiae micro-scale sirred bioreactors equipped with real-time measurements of growth kinetics and humidity control

Publications

Differential gene expression profiles and real-time measurements of growth parameters in Saccharomyces cerevisiae grown in microliter-scale bioreactors equipped with internal stirring.

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