title: Limitations in xylose-fermenting Saccharomyces cerevisiae, made evident through comprehensive metabolite profiling and thermodynamic analysis.
abstract: Little is known about how the general lack of efficiency with which recombinant Saccharomyces cerevisiae strains utilize xylose affects the yeast metabolome. Quantitative metabolomics was therefore performed for two xylose-fermenting S. cerevisiae strains, BP000 and BP10001, both engineered to produce xylose reductase (XR), NAD(+)-dependent xylitol dehydrogenase and xylulose kinase, and the corresponding wild-type strain CEN.PK 113-7D, which is not able to metabolize xylose. Contrary to BP000 expressing an NADPH-preferring XR, BP10001 expresses an NADH-preferring XR. An updated protocol of liquid chromatography/tandem mass spectrometry that was validated by applying internal (13)C-labeled metabolite standards was used to quantitatively determine intracellular pools of metabolites from the central carbon, energy, and redox metabolism and of eight amino acids. Metabolomic responses to different substrates, glucose (growth) or xylose (no growth), were analyzed for each strain. In BP000 and BP10001, flux through glycolysis was similarly reduced (∼27-fold) when xylose instead of glucose was metabolized. As a consequence, (i) most glycolytic metabolites were dramatically (≤ 120-fold) diluted and (ii) energy and anabolic reduction charges were affected due to decreased ATP/AMP ratios (3- to 4-fold) and reduced NADP(+) levels (∼3-fold), respectively. Contrary to that in BP000, the catabolic reduction charge was not altered in BP10001. This was due mainly to different utilization of NADH by XRs in BP000 (44%) and BP10001 (97%). Thermodynamic analysis complemented by enzyme kinetic considerations suggested that activities of pentose phosphate pathway enzymes and the pool of fructose-6-phosphate are potential factors limiting xylose utilization. Coenzyme and ATP pools did not rate limit flux through xylose pathway enzymes.
authors.affiliation: Graz University of Technology, Institute of Biotechnology and Biochemical Engineering, Petersgasse 12/I, A-8010 Graz, Austria. mario.klimacek@tugraz.at
authors.forename: Mario
authors.initials: M
authors.lastname: Klimacek
authors.forename: Stefan
authors.initials: S
authors.lastname: Krahulec
authors.forename: Uwe
authors.initials: U
authors.lastname: Sauer
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000449:Aldehyde Reductase / Q000235:genetics / Q000378:metabolism; D002247:Carbon Isotopes / Q000378:metabolism; D002853:Chromatography, Liquid; D050542:D-Xylulose Reductase / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D055432:Metabolomics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods; D053719:Tandem Mass Spectrometry; D013816:Thermodynamics; D014994:Xylose / Q000378:metabolism*
chemical_list: D002247:Carbon Isotopes; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D014994:Xylose; D000449:Aldehyde Reductase; D050542:D-Xylulose Reductase; D017853:Phosphotransferases (Alcohol Group Acceptor); C032700:xylulokinase