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

Author(s):Show less -

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]}

Affiliation

^[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

• Document type: Journal article
• Source: METABOLIC ENGINEERING; v. 13, n. 6, p. 694-703, NOV 2011.
• Web of Science Citations: 38

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

• FAPESP's process: 07/59776-7 - Yeast improvement by metabolic and evolutionary engineering
• Grantee: Andreas Karoly Gombert
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Regular Program Grants