Comparative proteomics analysis of engineered Saccharomyces cerevisiae with enhanced biofuel precursor production

Abstract

The yeast Saccharomyces cerevisiae was metabolically modified for enhanced biofuel precursor production by knocking out genes encoding mitochondrial isocitrate dehydrogenase and over-expression of a heterologous ATP-citrate lyase. A comparative iTRAQ-coupled 2D LC-MS/MS analysis was performed to obtain a global overview of ubiquitous protein expression changes in S. cerevisiae engineered strains. More than 300 proteins were identified. Among these proteins, 37 were found differentially expressed in engineered strains and they were classified into specific categories based on their enzyme functions. Most of the proteins involved in glycolytic and pyruvate branch-point pathways were found to be up-regulated and the proteins involved in respiration and glyoxylate pathway were however found to be down-regulated in engineered strains. Moreover, the metabolic modification of S. cerevisiae cells resulted in a number of up-regulated proteins involved in stress response and differentially expressed proteins involved in amino acid metabolism and protein biosynthesis pathways. These LC-MS/MS based proteomics analysis results not only offered extensive information in identifying potential protein-protein interactions, signal pathways and ubiquitous cellular changes elicited by the engineered pathways, but also provided a meaningful biological information platform serving further modification of yeast cells for enhanced biofuel production.

Figure 1. Real-time RT-PCR analysis of mRNA levels of selected differentially expressed proteins among wt-pvtu, ∆idh1/2-pvtu and ∆idh1/2-acl strains.

Three independent experiments were carried out. The mRNA levels in wt-pvtu strain were taken as controls (100%) and mRNA levels in ∆idh1/2-pvtu and ∆idh1/2-acl strains were being ratio to that in the wt-pvtu strain.

Figure 2. Differential production levels of intracellular amino acids among wt-pvtu, ∆idh1/2-pvtu and ∆idh1/2-acl strains.

Three independent experiments were carried out. The amino acid levels in wt-pvtu strain were taken as controls and the amino acid levels in ∆idh1/2-pvtu and ∆idh1/2-acl strains were calculated accordingly.

Figure 3. Differential production levels of intracellular metabolites among wt-pvtu, ∆idh1/2-pvtu and ∆idh1/2-acl strains.

Three independent experiments were carried out. The metabolites production levels in wt-pvtu strain were taken as controls and the metabolites production levels in ∆idh1/2-pvtu and ∆idh1/2-acl strains were calculated accordingly.

Figure 4. Schematic diagram of metabolic pathway changes in S.

cerevisiae engineered strains. The engineered pathway in yeast with enhanced fatty acid synthesis was indicated by red dashed box. Both of the up-regulated and down regulated metabolic pathways in ∆idh1/2-acl strain were indicated by the different colored arrows. The proteins involved in other pathways that haven’t been detected from LC-MS/MS analysis were not shown in this diagram.