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E-GEOD-30535 - Engineering topology and kinetics of sucrose metabolism in Saccharomyces cerevisiae for improved ethanol yield
Released on 5 September 2011, last updated on 4 May 2014
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.
transcription profiling by array
Jean-Marc Daran <firstname.lastname@example.org>, Marinka Almering, Stephan de Kok, Thiago Basso
Increasing free-energy (ATP) conservation in maltose-grown Saccharomyces cerevisiae by expression of a heterologous maltose phosphorylase. de Kok S, Yilmaz D, Suir E, Pronk JT, Daran JM, van Maris AJ. , PMID:21684346