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Engineering topology and kinetics of sucrose metabolism in Saccharomyces cerevisiae for improved ethanol yield.
Basso, Thiago O; de Kok, Stefan; Dario, Marcelo; do Espirito-Santo, Júlio Cézar A; Müller, Gabriela; Schlölg, Paulo S; Silva, Carlos P; Tonso, Aldo; Daran, Jean-Marc; Gombert, Andreas K; van Maris, Antonius J A; Pronk, Jack T; Stambuk, Boris U.
Affiliation
  • Basso TO; Department of Biotechnology, Delft University of Technology, Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands.
Metab Eng ; 13(6): 694-703, 2011 Nov.
Article in En | MEDLINE | ID: mdl-21963484
ABSTRACT
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-proton 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 predominant (94%) cytosolic localization of invertase. 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 90 generations of laboratory evolution in 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 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. After deletion of both copies of the duplicated AGT1, growth characteristics reverted to that of the unevolved SUC2 and iSUC2 strains. This study demonstrates that engineering the topology of sucrose metabolism is an attractive strategy to improve ethanol yields in industrial processes.
Subject(s)

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Saccharomyces cerevisiae / Sucrose / Saccharomyces cerevisiae Proteins / Beta-Fructofuranosidase / Ethanol Type of study: Prognostic_studies Language: En Journal: Metab Eng Journal subject: ENGENHARIA BIOMEDICA / METABOLISMO Year: 2011 Document type: Article Affiliation country: Netherlands

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Saccharomyces cerevisiae / Sucrose / Saccharomyces cerevisiae Proteins / Beta-Fructofuranosidase / Ethanol Type of study: Prognostic_studies Language: En Journal: Metab Eng Journal subject: ENGENHARIA BIOMEDICA / METABOLISMO Year: 2011 Document type: Article Affiliation country: Netherlands