Re-evaluation of glycerol utilization in Saccharomyces cerevisiae: characterization of an isolate that grows on glycerol without supporting supplements

Abstract

BackgroundGlycerol has attracted attention as a carbon source for microbial production processes due to the large amounts of crude glycerol waste resulting from biodiesel production. The current knowledge about the genetics and physiology of glycerol uptake and catabolism in the versatile industrial biotechnology production host Saccharomyces cerevisiae has been mainly based on auxotrophic laboratory strains, and carried out in the presence of growth-supporting supplements such as amino acids and nucleic bases. The latter may have resulted in ambiguous conclusions concerning glycerol growth in this species. The purpose of this study was to re-evaluate growth of S. cerevisiae in synthetic glycerol medium without the addition of supplements.ResultsInitial experiments showed that prototrophic versions of the laboratory strains CEN.PK, W303, and S288c did not exhibit any growth in synthetic glycerol medium without supporting supplements. However, a screening of 52 S. cerevisiae isolates for growth in the same medium revealed a high intraspecies diversity. Within this group significant variation with respect to the lag phase and maximum specific growth rate was observed. A haploid segregant of one good glycerol grower (CBS 6412-13A) was selected for detailed analysis. Single deletions of the genes encoding for the glycerol/H+ symporter (STL1), the glycerol kinase (GUT1), and the mitochondrial FAD+-dependent glycerol 3-phosphate dehydrogenase (GUT2) abolished glycerol growth in this strain, implying that it uses the same glycerol utilization pathway as previously identified in auxotrophic laboratory strains. Segregant analysis of a cross between CBS 6412-13A and CEN.PK113-1A revealed that the glycerol growth phenotype is a quantitative trait. Genetic linkage and reciprocal hemizygosity analysis demonstrated that GUT1 CBS 6412-13A is one of the multiple genetic loci contributing to the glycerol growth phenotype.ConclusionThe S. cerevisiae intraspecies diversity with regard to glycerol growth is a valuable starting point to identify the genetic and molecular basis of this phenotype. This knowledge can be applied for further rational strain improvement with the goal of using glycerol as a carbon source in industrial biotechnology processes based on S. cerevisiae as a production organism.

Engineering yeast shikimate pathway towards production of aromatics: rational design of a chassis cell using systems and synthetic biology

“…Balancing this represents the first major challenge. The defective glycerol utilization mechanism in S. cerevisiae laboratory strains, which diminishes productivity and increases ethanol demand, may be alleviated by over-expression of STL1, GUT1 CBS6412-13A and GUT2 as described before (Swinnen et al, 2013). Improved glycerol uptake and flux into central metabolism could help to not only enhance growth but also production on glycerol.…”

“…Interestingly, further enforcing higher glycerol uptake affected pABA titer negatively and also slightly diminished the yield. This was most likely due to the impaired glycerol uptake mechanism of laboratory yeast (Swinnen et al, 2013), presumably resulting in an imbalanced metabolism of the cell if ethanol share in the carbon-source is too low.…”

Synthetic Biology and Metabolic Engineering Approaches and Its Impact on Non-Conventional Yeast and Biofuel Production

“…Thus, yeast is a good platform for developing synthetic biology techniques, and this system can be used for improving the status of biofuel production. The main disadvantage of S. cerevisiae is its inability to consume a wide range of substrates (e.g., xylose, arabinose) (Garcia Sanchez et al, 2010), and glycerol (Swinnen et al, 2013). S. cerevisiae is also not good for use in biofuel application that requires high temperatures (>34°C) (Caspeta et al, 2014).…”

Synthetic and Systems Biology Approach towards Designing Metabolic Bypass and Identifying Novel Enzymes for Cholesterol Lowering Drug Precursor (BTO) Biosynthesis from Crude Glycerol

“…Thus far a very vital question arises "why should we think for an alternative potential feedstock glycerol rather than Xylose or Arabinose?" The first and foremost reason is that different microbial regimes can uptake glycerol as carbon and energy source; mediated by the glycerol diffusion facilitator, an integral membrane protein catalyzing the rapid equilibration of concentration gradients of glycerol across the cytoplasmic membrane under specified physiological growth conditions towards high value added biomolecules generations (Beijer et al, 1993 Dharmadi et al, 2006;Ghosh et al, 2012a;Ghosh et al, 2012b;Kong et al, 2013;Lambert and Stevens, 1986;May et al, 1982;Murarka et al, 2008;Narayan et al, 2005;Swinnen et al, 2013;Taghavi et al, 2016). These glycerol utilizing microbes could be the potential hosts for chassis strain development and for functionalization of novel hypothetical biosynthetic pathways towards improving BTO productions.…”