Coenzyme A (CoA) is a ubiquitous cofactor required for numerous enzymatic carbon group transfer reactions. CoA biosynthesis requires contributions from various amino acids with pantothenate as an important intermediate which can be imported from the medium or synthesized de novo. Investigating function and expression of structural genes involved in CoA biosynthesis of the yeast Saccharomyces cerevisiae, we show that deletion of ECM31 and PAN6 results in mutants requiring pantothenate while loss of PAN5 (related to panE from E. coli) still allows prototrophic growth. A temperature-sensitive mutant defective for fatty acid synthase activity could be functionally complemented by a gene significantly similar to eukaryotic pantothenate kinases (YDR531W). Enzymatic studies and heterologous complementation of this mutation by bacterial and mammalian genes showed that YDR531W encodes a genuine pantothenate kinase (new gene designation: CAB1, "coenzyme A biosynthesis"). A G351S missense mutation within CAB1 was identified to cause the conditional phenotype of the mutant initially studied. Similar to CAB1, genes YIL083C, YKL088W, YGR277C and YDR106C responsible for late CoA biosynthesis turned out as essential. Null mutants could be complemented by their bacterial counterparts coaBC, coaD and coaE, respectively. Comparative expression analyses showed that some CoA biosynthetic genes are weakly de-repressed with ethanol as a carbon source compared with glucose.
Coenzyme A (CoA) as an essential cofactor for acyl and acetyl transfer reactions is synthesized in five enzymatic steps from pantothenate, cysteine, and ATP. In the yeast Saccharomyces cerevisiae, products of five essential genes CAB1-CAB5 (coenzyme A biosynthesis) are required to catalyze CoA biosynthesis. In addition, nonessential genes SIS2 and VHS3 similar to CAB3 are also involved. Using epitope-tagged variants of Cab3 and Cab5, we show that both proteins cofractionate upon chromatographic separation, forming a complex of about 330 kDa. We thus systematically investigated interactions among Cab proteins. Our results show that Cab2, Cab3, Cab4, and Cab5 indeed bind to each other, with Cab3 as the sole protein, which can interact with itself and other Cab proteins. Cab3 also binds to Sis2 and Vhs3 that were previously characterized as subunits of phosphopantothenoylcysteine decarboxylase. Pantothenate kinase encoded by CAB1 as the rate-limiting enzyme of CoA biosynthesis did not interact with other Cab proteins. Mapping studies revealed that the nonconserved N-terminus of Cab3 is required for dimerization and for binding of Cab2 and Cab5. Our interaction studies confirm early reports on the existence of a CoA-synthesizing protein complex (CoA-SPC) in yeast and provide precise data on protein domains involved in complex formation.
Coenzyme A (CoA) and its derivatives such as acetyl-CoA are essential metabolites for several biosynthetic reactions. In the yeast S. cerevisiae, five enzymes (encoded by essential genes CAB1-CAB5; coenzyme A biosynthesis) are required to perform CoA biosynthesis from pantothenate, cysteine, and ATP. Similar to enzymes from other eukaryotes, yeast pantothenate kinase (PanK, encoded by CAB1) turned out to be inhibited by acetyl-CoA. By genetic selection of intragenic suppressors of a temperature-sensitive cab1 mutant combined with rationale mutagenesis of the presumed acetyl-CoA binding site within PanK, we were able to identify the variant CAB1 W331R, encoding a hyperactive PanK completely insensitive to inhibition by acetyl-CoA. Using a versatile gene integration cassette containing the TPI1 promoter, we constructed strains overexpressing CAB1 W331R in combination with additional genes of CoA biosynthesis (CAB2, CAB3, HAL3, CAB4, and CAB5). In these strains, the level of CoA nucleotides was 15-fold increased, compared to a reference strain without additional CAB genes. Overexpression of wild-type CAB1 instead of CAB1 W331R turned out as substantially less effective (fourfold increase of CoA nucleotides). Supplementation of overproducing strains with additional pantothenate could further elevate the level of CoA (2.3-fold). Minor increases were observed after overexpression of FEN2 (encoding a pantothenate permease) and deletion of PCD1 (CoA-specific phosphatase). We conclude that the strategy described in this work may improve the efficiency of biotechnological applications depending on acetyl-CoA.Key points• A gene encoding a hyperactive yeast pantothenate kinase (PanK) was constructed.• Overexpression of CoA biosynthetic genes elevated CoA nucleotides 15-fold.• Supplementation with pantothenate further increased the level of CoA nucleotides.
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