Genetik und Biochemie der Coenzym A Biosynthese in der Hefe Saccharomyces cerevisiae
Biochemie
Stoffwechselphysiologie, Biochemie und Genetik der Mikroorganismen
Zusammenfassung der Projektergebnisse
Genes required for biosynthesis of coenzyme A (CoA) are indispensable for viability of all organisms and have been even identified as responsible for special human disorders. The corresponding enzymes are not only fundamental for several biochemical pathways but can also support strategies of metabolic engineering in biotechnology. CoA and it acyl thioesters fulfill fundamental functions for various anabolic and catabolic pathways such as biosynthesis of fatty acids and sterols or degradation of biomolecules finally using the citric acid cycle. In the yeast S. cerevisiae, CoA is synthesized by 5 separate CAB genes (CoA biosynthesis). Since biosynthesis of CoA in this organism is regulated by acetyl-CoA via allosteric inhibition of the initial enzyme of the pathway (pantothenate kinase. PanK, encoded by CAB1), we wished to abolish this negative control as a prerequisite for an increase of the cellular CoA level. By a combination of genetic and biochemical approaches, we were able to construct a CAB1 gene variant {CAB1 W331R), encoding a fully active, but no longer repressible PanK. Studies of yeast CAB genes and corresponding genes from other organisms showed that (I) the human PPCS gene ( ~CAB2) could functionally replace CAB2 while mutant variants from patients (containing deletion and missense mutations) were no longer functional; (II) the yeast CAB3 gene could be substantially truncated to a conserved core region without obvious loss of function; (III) the bifunctional human COASYgene (~ CAB4 + CAB5) partially complements loss of yeast CAB4 but not of CAB5. To improve our previous attempts for overproduction of CoA in yeast, we developed a strategy for stable introduction of additional CAS gene copies, activated by the strong and constitutive promoter of the glycolytic gene TPI1. Using homology-directed integration, gene cassettes containing TPI1-CAB fusions combined with LEU2 as a selection marker could be transferred into chromosomal DNA at selected positions. Since LEU2 was flanked by recombinase recognition sequences, the marker could be removed after successful transformation ("marker rescue"). This integration strategy was applied to consecutively introduce 6 additional genes of CoA biosynthesis. By a quantitative enzymatic assay we could show that the amount of CoA nucleotides synthesized in some of the resulting yeast strains was increased 15-fold. These assays also showed that the acetyl-CoA insensitive PanK variant encoded by CAB1 W331R was absolutely essential for maximum increase of CoA levels. Despite the critical role of this gene variant, the remaining CAB genes were also required to overproduce CoA. While growth of transformants in rich medium could increase CoA yield 15-fold, only a 3-fold increase was observed after cultivation in synthetic medium, indicating that the concentration of a precursor is limiting. Indeed, we could demonstrate that supplementation of this medium with pantothenate (and less efficient with ß-alanine) was able to compensate the limitations. In conclusion, by genetic strain improvement and identification of optimum conditions of nutrient supply we could substantially increase CoA biosynthesis in yeast, allowing promising biotechnological applications in the future.
Projektbezogene Publikationen (Auswahl)
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(2009) Genetic analysis of coenzyme A biosynthesis in the yeast Saccharomyces cerevisiae: Identification of a conditional mutation in the pantothenate kinase gene CAB1. Curr Genet 55: 163-173
Olzhausen J, Schübbe S, Schüller HJ
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(2013) Molecular characterization of the heteromeric coenzyme A-synthesizing protein complex (CoA-SPC) in the yeast Saccharomyces cerevisiae. FEMS Yeast Res 13: 565-573
Olzhausen J, Moritz T, Neetz T und Schüller HJ
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(2018) Mutations in PPCS, Encoding Phosphopantothenoylcysteine Synthetase, Cause Autosomal-Recessive Dilated Cardiomyopathy. Am J Hum Genet 102:1018-1030
luso A. Wiersma M, Schüller HJ, Pode-Shakked B, Marek-Yagel D, Grigat M, Schwarzmayr T, Berutti R, Alhaddad B, Kanon B, Grzeschik NA, Okun JG, Perles Z, Salem Y, Barel O, Vardi A, Rubinshtein M, Tirosh T, Dubnov-Raz G, Messias AC, Terrile C, Barshack I, Volkov A, Avivi C, Eyal E, Mastantuono E, Kumbar M, Abudi S, Braunisch M, Strom TM, Meitinger T, Hoffmann GF, Prokisch H, Haack TB, Brundel BJJM, Haas D, Sibon OCM, Anikster Y
