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(Referência obtida automaticamente do Web of Science, por meio da informação sobre o financiamento pela FAPESP e o número do processo correspondente, incluída na publicação pelos autores.)

Engineering topology and kinetics of sucrose metabolism in Saccharomyces cerevisiae for improved ethanol yield

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Autor(es):
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Basso, Thiago O. [1, 2, 3, 4] ; de Kok, Stefan [2, 4] ; Dario, Marcelo [5, 3] ; do Espirito-Santo, Julio Cezar A. [5] ; Mueller, Gabriela [5] ; Schloelg, Paulo S. [5] ; Silva, Carlos P. [5] ; Tonso, Aldo [1] ; Daran, Jean-Marc [2, 4] ; Gombert, Andreas K. [1] ; van Maris, Antonius J. A. [2, 4] ; Pronk, Jack T. [2, 4] ; Stambuk, Boris U. [5, 3]
Número total de Autores: 13
Afiliação do(s) autor(es):
[1] Univ Sao Paulo, Dept Chem Engn, Sao Paulo - Brazil
[2] Delft Univ Technol, Dept Biotechnol, Delft - Netherlands
[3] Univ Sao Paulo, PPG Interunidades Biotecnol, Sao Paulo - Brazil
[4] Kluyver Ctr Genom Ind Fermentat, Delft - Netherlands
[5] Univ Fed Santa Catarina, Dept Bioquim, Florianopolis, SC - Brazil
Número total de Afiliações: 5
Tipo de documento: Artigo Científico
Fonte: METABOLIC ENGINEERING; v. 13, n. 6, p. 694-703, NOV 2011.
Citações Web of Science: 38
Assunto(s):Bioetanol   Fermentação   Glucose
Resumo

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. (C) 2011 Elsevier Inc. All rights reserved. (AU)

Processo FAPESP: 07/59776-7 - Engenharia evolutiva de leveduras
Beneficiário:Andreas Karoly Gombert
Modalidade de apoio: Auxílio à Pesquisa - Programa BIOEN - Regular