Increased biosynthesis of acetyl-CoA in the yeast Saccharomyces cerevisiae by overexpression of a deregulated pantothenate kinase gene and engineering of the coenzyme A biosynthetic pathway.
Acetyl-CoA
Biosynthesis of coenzyme A
CAB genes
Feedback inhibition
Pantothenate kinase
Saccharomyces cerevisiae
Yeast
Journal
Applied microbiology and biotechnology
ISSN: 1432-0614
Titre abrégé: Appl Microbiol Biotechnol
Pays: Germany
ID NLM: 8406612
Informations de publication
Date de publication:
Oct 2021
Oct 2021
Historique:
received:
08
03
2021
accepted:
06
08
2021
revised:
20
07
2021
pubmed:
8
9
2021
medline:
9
10
2021
entrez:
7
9
2021
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 34491400
doi: 10.1007/s00253-021-11523-4
pii: 10.1007/s00253-021-11523-4
pmc: PMC8494682
doi:
Substances chimiques
Acetyl Coenzyme A
72-89-9
Phosphotransferases (Alcohol Group Acceptor)
EC 2.7.1.-
pantothenate kinase
EC 2.7.1.33
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
7321-7337Subventions
Organisme : Deutsche Forschungsgemeinschaft
ID : SCHU856/9-1
Commentaires et corrections
Type : ErratumIn
Informations de copyright
© 2021. The Author(s).
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