초록
<P><B>Abstract</B></P><P>Sucrose is a major carbon source for industrial bioethanol production by <I>Saccharomyces cerevisiae</I>. 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 <I>SUC2</I> gene enable synthesis of extracellular and intracellular invertase isoforms, sucrose hydrolysis in <I>S. cerevisiae</I> 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 <I>SUC2</I>, resulting in predominant (94%) cytosolic localization of invertase. In anaerobic sucrose-limited chemostats, this i<I>SUC2</I>-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 <I>SUC2</I> reference strain, while transcriptome analysis revealed elevated expression of <I>AGT1</I>, encoding a disaccharide-proton symporter, and other maltose-related genes. After deletion of both copies of the duplicated <I>AGT1</I>, growth characteristics reverted to that of the unevolved <I>SUC2</I> and i<I>SUC2</I> strains. This study demonstrates that engineering the topology of sucrose metabolism is an attractive strategy to improve ethanol yields in industrial processes.</P> <P><B>Highlights</B></P><P>► Yeast invertase was relocated to the cytosol by removal of N-terminal signal peptide. ► Improved sucrose uptake kinetics obtained by evolutionary engineering in chemostats. ► Strain evolved for intracellular sucrose metabolism shows deregulated <I>MAL</I> genes. ► <I>AGT1</I>-encoded proton symporter was involved in sucrose uptake by evolved yeast strain. ► The engineered yeast strain shows an 11% increase of the ethanol yield on sucrose.</P>