Physiological characterization of recombinant Saccharomyces cerevisiae expressing the Aspergillus nidulans phosphoketolase pathway: validation of activity through 13C-based metabolic flux analysis
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Several bacterial species and filamentous fungi utilize the phosphoketolase pathway (PHK) for glucose dissimilation as an alternative to the Embden–Meyerhof–Parnas pathway. In Aspergillus nidulans, the utilization of this metabolic pathway leads to increased carbon flow towards acetate and acetyl CoA. In the first step of the PHK, the pentose phosphate pathway intermediate xylulose-5-phosphate is converted into acetylphosphate and glyceraldehyde-3-phosphate through the action of xylulose-5-phosphate phosphoketolase, and successively acetylphosphate is converted into acetate by the action of acetate kinase. In the present work, we describe a metabolic engineering strategy used to express the fungal genes of the phosphoketolase pathway in Saccharomyces cerevisiae and the effects of the expression of this recombinant route in yeast. The phenotype of the engineered yeast strain MP003 was studied during batch and chemostat cultivations, showing a reduced biomass yield and an increased acetate yield during batch cultures. To establish whether the observed effects in the recombinant strain MP003 were due directly or indirectly to the expression of the phosphoketolase pathway, we resolved the intracellular flux distribution based on 13C labeling during chemostat cultivations. From flux analysis it is possible to conclude that yeast is able to use the recombinant pathway. Our work indicates that the utilization of the phosphoketolase pathway does not interfere with glucose assimilation through the Embden–Meyerhof–Parnas pathway and that the expression of this route can contribute to increase the acetyl CoA supply, therefore holding potential for future metabolic engineering strategies having acetyl CoA as precursor for the biosynthesis of industrially relevant compounds.
KeywordsPhosphoketolase Metabolic engineering 13C-based metabolic flux analysis Acetyl CoA supply Glycolysis
The authors acknowledge the Knut and Wallenberg foundation, the Chalmers foundation, and the European Research Council. Saeed Shoaie is acknowledged for the technical support during flux calculations.
- Chung AE (1970) Pyridine nucleotide transhydrogenase from Azotobacter vinelandii. J Bacteriol 2:438–47Google Scholar
- Ciriacy M, Breitenbach I (1979) Physiological effects of seven different blocks in glycolysis in Saccharomyces cerevisiae. J Bacteriol 1:152–160Google Scholar
- Lam KB, Marmur J (1979) Isolation and characterization of Saccharomyces cerevisiae glycolytic pathway mutants. J Bacteriol 2:746–9Google Scholar
- Panagiotou G, Andersen MR, Grotkjaer T, Regueira TB, Hofmann G, Nielsen J, Olsson L (2008) Systems Analysis Unfolds the Relationship between the Phosphoketolase Pathway and Growth in Aspergillus nidulans. PLoS One 12:e3847Google Scholar
- Presecan-Siedel E, Galinier A, Longin R, Deutscher J, Danchin A, Glaser P, Martin-Verstraete I (1999) Catabolite regulation of the pta gene as part of carbon flow pathways in Bacillus subtilis. J Bacteriol 181:6889–97Google Scholar
- Ratledge C, Holdsworth JE (1985) Properties of a pentulose-5-phosphate phosphoketolase from yeasts grown on xylose. Appl Microbiol Biotechnol 22:217–221Google Scholar
- Sprague GS Jr (1977) Isolation and characterization of a Saccharomyces cerevisiae mutant deficient in pyruvate kinase activity. J Bacteriol 1:232–41Google Scholar