Abstract
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.
Highlights
Coenzyme A (CoA) is an indispensable and ubiquitous metabolic cofactor of numerous enzymes requiring activated carboxylic acids as CoA thioesters
We conclude that variants A22G, F103V, D114, and W331L are critical for pantothenate kinase (PanK) activity of the yeast enzyme
Similar to what has been shown for E. coli (Rock et al 2003), this work demonstrates that biochemical regulation of PanK is a key mechanism controlling the metabolic flux towards CoA and its major thioester derivative acetyl-CoA
Summary
Coenzyme A (CoA) is an indispensable and ubiquitous metabolic cofactor of numerous enzymes requiring activated carboxylic acids as CoA thioesters. Besides its enzymatic PPCDC activity, Cab physically interacts with Cab, Hal, Vhs, Cab, and Cab (but not with Cab1/PanK) and functions as a scaffold of the yeast CoA synthesizing protein complex (CoA-SPC) with a molecular weight of about 330 kDa (Olzhausen et al 2013) Of such a CoA biosynthetic complex has been recently shown for mammalian cell lines (Bakovic et al 2021). We devised a strategy to overcome the presumed PanK inhibition by CoA/acetyl-CoA and were able to identify a hyperactive and constitutive enzyme variant (W331R) Stable overexpression of this variant in combination with additional genes of CoA biosynthesis and physiological modifications of growth media allowed us to construct yeast strains in which the intracellular concentration of CoA nucleotides was increased almost 40-fold above the wild-type level
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
