Increased isobutanol production in Saccharomyces cerevisiae by overexpression of genes in valine metabolism

<p>Abstract</p> <p>Background</p> <p>Isobutanol can be a better biofuel than ethanol due to its higher energy density and lower hygroscopicity. Furthermore, the branched-chain structure of isobutanol gives a higher octane number than the isomeric <it>n</it>-butanol. <it>Saccharomyces cerevisiae </it>was chosen as the production host because of its relative tolerance to alcohols, robustness in industrial fermentations, and the possibility for future combination of isobutanol production with fermentation of lignocellulosic materials.</p> <p>Results</p> <p>The yield of isobutanol was improved from 0.16 to 0.97 mg per g glucose by simultaneous overexpression of biosynthetic genes <it>ILV2, ILV3</it>, and <it>ILV5 </it>in valine metabolism in anaerobic fermentation of glucose in mineral medium in <it>S. cerevisiae</it>. Isobutanol yield was further improved by twofold by the additional overexpression of <it>BAT2</it>, encoding the cytoplasmic branched-chain amino-acid aminotransferase. Overexpression of <it>ILV6</it>, encoding the regulatory subunit of Ilv2, in the <it>ILV2 ILV3 ILV5 </it>overexpression strain decreased isobutanol production yield by threefold. In aerobic cultivations in shake flasks in mineral medium, the isobutanol yield of the <it>ILV2 ILV3 ILV5 </it>overexpression strain and the reference strain were 3.86 and 0.28 mg per g glucose, respectively. They increased to 4.12 and 2.4 mg per g glucose in yeast extract/peptone/dextrose (YPD) complex medium under aerobic conditions, respectively.</p> <p>Conclusions</p> <p>Overexpression of genes <it>ILV2, ILV3, ILV5</it>, and <it>BAT2 </it>in valine metabolism led to an increase in isobutanol production in <it>S. cerevisiae</it>. Additional overexpression of <it>ILV6 </it>in the <it>ILV2 ILV3 ILV5 </it>overexpression strain had a negative effect, presumably by increasing the sensitivity of Ilv2 to valine inhibition, thus weakening the positive impact of overexpression of <it>ILV2, ILV3</it>, and <it>ILV5 </it>on isobutanol production. Aerobic cultivations of the <it>ILV2 ILV3 ILV5 </it>overexpression strain and the reference strain showed that supplying amino acids in cultivation media gave a substantial improvement in isobutanol production for the reference strain, but not for the <it>ILV2 ILV3 ILV5 </it>overexpression strain. This result implies that other constraints besides the enzyme activities for the supply of 2-ketoisovalerate may become bottlenecks for isobutanol production after <it>ILV2, ILV3</it>, and <it>ILV5 </it>have been overexpressed, which most probably includes the valine inhibition to Ilv2.</p

Competitive intra- and extracellular nutrient sensing by the transporter homologue Ssy1p

Recent studies of Saccharomyces cerevisiae revealed sensors that detect extracellular amino acids (Ssy1p) or glucose (Snf3p and Rgt2p) and are evolutionarily related to the transporters of these nutrients. An intriguing question is whether the evolutionary transformation of transporters into nontransporting sensors reflects a homeostatic capability of transporter-like sensors that could not be easily attained by other types of sensors. We previously found SSY1 mutants with an increased basal level of signaling and increased apparent affinity to sensed extracellular amino acids. On this basis, we propose and test a general model for transporter- like sensors in which occupation of a single, central ligand binding site increases the activation energy needed for the conformational shift between an outward-facing, signaling conformation and an inward-facing, nonsignaling conformation. As predicted, intracellular leucine accumulation competitively inhibits sensing of extracellular amino acids. Thus, a single sensor allows the cell to respond to changes in nutrient availability through detection of the relative concentrations of intra- and extracellular ligand

BAP2, a gene encoding a permease for branched-chain amino acids in Saccharomyces cerevisiae

AbstractTo select the gene coding for an isoleucine permease, an isoleucine dependent strain (ilv 1 chal) was transformed with a yeast genomic multicopy library, and colonies growing at a low isoleucine concentration were selected. Partial sequencing of the responsible plasmid insert revealed the presence of a previously sequenced 609 codon open reading frame of chromosome II with homology to known permeases. Deletion, extra dosage and C-terminal truncation of this gene were constructed in a strain lacking the general amino acid permease, and amino acid uptake was measured during growth in synthetic complete medium. The following observations prompted us to name the gene BAP2 (branched-chain amino acid permease). Deletion of BAP2 reduced uptake of leucine, isoleucine and valine by 25–50%, while the uptake of 8 other l-α-amino acids was unlatered or slightly increased. Introduction of BAP2 on a centromere-based vector, leading to a gene dosage of two or slightly more, caused a 50% increase in leucine uptake and a smaller increase for isoleucine and valine. However, when the 29 C-terminal codons of the plasmid-borne copy of BAP2 were substituted, the cells more than doubled the uptake of leucine, isoleucine and valine, while no or little increase in uptake was observed for the other 8 amino acids