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

AbstractCoenzyme 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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Increased biosynthesis of acetyl-CoA in the yeast Saccharomyces cerevisiae by overexpression of a deregulated pantothenate kinase gene

10.1101/2021.05.25.445608 ◽

2021 ◽

Author(s):

Judith Olzhausen ◽

Mathias Grigat ◽

Larissa Seifert ◽

Tom Ulbricht ◽

Hans-Joachim Schueller

Keyword(s):

Reference Strain ◽

Coenzyme A ◽

Genetic Selection ◽

Fold Increase ◽

Temperature Sensitive ◽

Acetyl Coa ◽

Kinase Gene ◽

Yeast Saccharomyces Cerevisiae ◽

Pantothenate Kinase ◽

Selection Of

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 (4-fold 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.

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Genetic analysis of coenzyme A biosynthesis in the yeast Saccharomyces cerevisiae: identification of a conditional mutation in the pantothenate kinase gene CAB1

Current Genetics ◽

10.1007/s00294-009-0234-1 ◽

2009 ◽

Vol 55 (2) ◽

pp. 163-173 ◽

Cited By ~ 26

Author(s):

Judith Olzhausen ◽

Sabrina Schübbe ◽

Hans-Joachim Schüller

Keyword(s):

Saccharomyces Cerevisiae ◽

Genetic Analysis ◽

Coenzyme A ◽

Kinase Gene ◽

Yeast Saccharomyces Cerevisiae ◽

Pantothenate Kinase ◽

Coenzyme A Biosynthesis

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Cloning and characterization of DST2, the gene for DNA strand transfer protein beta from Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2583 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2583-2592 ◽

Cited By ~ 57

Author(s):

C C Dykstra ◽

K Kitada ◽

A B Clark ◽

R K Hamatake ◽

A Sugino

Keyword(s):

Saccharomyces Cerevisiae ◽

Intragenic Recombination ◽

Temperature Sensitive ◽

Strand Transfer ◽

Gene Encoding ◽

Yeast Saccharomyces Cerevisiae ◽

Prolonged Incubation ◽

Transfer Protein ◽

Dna Strand

The gene encoding the 180-kDa DNA strand transfer protein beta from the yeast Saccharomyces cerevisiae was identified and sequenced. This gene, DST2 (DNA strand transferase 2), was located on chromosome VII. dst2 gene disruption mutants exhibited temperature-sensitive sporulation and a 50% longer generation time during vegetative growth than did the wild type. Spontaneous mitotic recombination in the mutants was reduced severalfold for both intrachromosomal recombination and intragenic gene conversion. The mutants also had reduced levels of the intragenic recombination that is induced during meiosis. Meiotic recombinants were, however, somewhat unstable in the mutants, with a decrease in recombinants and survival upon prolonged incubation in sporulation media. spo13 or spo13 rad50 mutations did not relieve the sporulation defect of dst2 mutations. A dst1 dst2 double mutant has the same phenotype as a dst2 single mutant. All phenotypes associated with the dst2 mutations could be complemented by a plasmid containing DST2.

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A Role for Ubiquitination in Mitochondrial Inheritance in Saccharomyces cerevisiae

The Journal of Cell Biology ◽

10.1083/jcb.145.6.1199 ◽

1999 ◽

Vol 145 (6) ◽

pp. 1199-1208 ◽

Cited By ~ 129

Author(s):

Harold A. Fisk ◽

Michael P. Yaffe

Keyword(s):

Saccharomyces Cerevisiae ◽

Site Directed Mutagenesis ◽

Temperature Sensitive ◽

Mitochondrial Inheritance ◽

Wild Type ◽

Gene Encoding ◽

Yeast Saccharomyces Cerevisiae ◽

Protein Ubiquitination ◽

Ubiquitin Ligase Activity ◽

Mitochondrial Distribution

The smm1 mutation suppresses defects in mitochondrial distribution and morphology caused by the mdm1-252 mutation in the yeast Saccharomyces cerevisiae. Cells harboring only the smm1 mutation themselves display temperature-sensitive growth and aberrant mitochondrial inheritance and morphology at the nonpermissive temperature. smm1 maps to RSP5, a gene encoding an essential ubiquitin-protein ligase. The smm1 defects are suppressed by overexpression of wild-type ubiquitin but not by overexpression of mutant ubiquitin in which lysine-63 is replaced by arginine. Furthermore, overexpression of this mutant ubiquitin perturbs mitochondrial distribution and morphology in wild-type cells. Site-directed mutagenesis revealed that the ubiquitin ligase activity of Rsp5p is essential for its function in mitochondrial inheritance. A second mutation, smm2, which also suppressed mdm1-252 defects, but did not cause aberrant mitochondrial distribution and morphology, mapped to BUL1, encoding a protein interacting with Rsp5p. These results indicate that protein ubiquitination mediated by Rsp5p plays an essential role in mitochondrial inheritance, and reveal a novel function for protein ubiquitination.

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Cloning and characterization of DST2, the gene for DNA strand transfer protein beta from Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2583-2592.1991 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2583-2592 ◽

Cited By ~ 1

Author(s):

C C Dykstra ◽

K Kitada ◽

A B Clark ◽

R K Hamatake ◽

A Sugino

Keyword(s):

Saccharomyces Cerevisiae ◽

Intragenic Recombination ◽

Temperature Sensitive ◽

Strand Transfer ◽

Gene Encoding ◽

Yeast Saccharomyces Cerevisiae ◽

Prolonged Incubation ◽

Transfer Protein ◽

Dna Strand

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Isolation of a temperature-sensitive mutant with an altered tRNA nucleotidyltransferase and cloning of the gene encoding tRNA nucleotidyltransferase in the yeast Saccharomyces cerevisiae.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)46210-7 ◽

1990 ◽

Vol 265 (27) ◽

pp. 16216-16220

Author(s):

M Aebi ◽

G Kirchner ◽

J Y Chen ◽

U Vijayraghavan ◽

A Jacobson ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Temperature Sensitive ◽

Sensitive Mutant ◽

Gene Encoding ◽

Yeast Saccharomyces Cerevisiae ◽

Temperature Sensitive Mutant

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Impaired coenzyme A synthesis in fission yeast causes defective mitosis, quiescence-exit failure, histone hypoacetylation and fragile DNA

Open Biology ◽

10.1098/rsob.120117 ◽

2012 ◽

Vol 2 (9) ◽

pp. 120117 ◽

Cited By ~ 24

Author(s):

Takahiro Nakamura ◽

Tomáš Pluskal ◽

Yukinobu Nakaseko ◽

Mitsuhiro Yanagida

Keyword(s):

Histone Acetylation ◽

Fission Yeast ◽

Coenzyme A ◽

Housekeeping Gene ◽

Strand Break ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Acetyl Coa ◽

Synthetic Lethal ◽

Sh Group

Biosynthesis of coenzyme A (CoA) requires a five-step process using pantothenate and cysteine in the fission yeast Schizosaccharomyces pombe . CoA contains a thiol (SH) group, which reacts with carboxylic acid to form thioesters, giving rise to acyl-activated CoAs such as acetyl-CoA. Acetyl-CoA is essential for energy metabolism and protein acetylation, and, in higher eukaryotes, for the production of neurotransmitters. We isolated a novel S. pombe temperature-sensitive strain ppc1-537 mutated in the catalytic region of phosphopantothenoylcysteine synthetase (designated Ppc1), which is essential for CoA synthesis. The mutant becomes auxotrophic to pantothenate at permissive temperature, displaying greatly decreased levels of CoA, acetyl-CoA and histone acetylation. Moreover, ppc1-537 mutant cells failed to restore proliferation from quiescence. Ppc1 is thus the product of a super-housekeeping gene. The ppc1-537 mutant showed combined synthetic lethal defects with five of six histone deacetylase mutants, whereas sir2 deletion exceptionally rescued the ppc1-537 phenotype. In synchronous cultures, ppc1-537 cells can proceed to the S phase, but lose viability during mitosis failing in sister centromere/kinetochore segregation and nuclear division. Additionally, double-strand break repair is defective in the ppc1-537 mutant, producing fragile broken DNA, probably owing to diminished histone acetylation. The CoA-supported metabolism thus controls the state of chromosome DNA.

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The Saccharomyces cerevisiae gene SDS22 encodes a potential regulator of the mitotic function of yeast type 1 protein phosphatase.

Molecular and Cellular Biology ◽

10.1128/mcb.15.7.3777 ◽

1995 ◽

Vol 15 (7) ◽

pp. 3777-3785 ◽

Cited By ~ 58

Author(s):

S H MacKelvie ◽

P D Andrews ◽

M J Stark

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein A ◽

Catalytic Subunit ◽

Yeast Cells ◽

Regulatory Subunit ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Gene Encoding ◽

Yeast Saccharomyces Cerevisiae

In higher eukaryotes, the activity and specificity of the type 1 protein serine-threonine phosphatase (PP1) catalytic subunit is thought to be controlled by its association with a number of regulatory or targeting subunits. Here we describe the characterization of a gene encoding one such potential polypeptide in the yeast Saccharomyces cerevisiae. The gene which we have isolated (termed SDS22) encodes a product with a high degree of sequence identity to the fission yeast sds22 protein, a known regulator of the mitotic function of PP1 in Schizosaccharomyces pombe. Using two different criteria, we have demonstrated that Sds22p and the catalytic subunit of PP1 (Glc7p) interact in yeast cells. We have also generated a temperature-sensitive allele of GLC7 (glc7-12) which causes a block to the completion of mitosis at the restrictive temperature. Additional copies of SDS22 lead to allele-specific suppression of the glc7-12 mutant, strongly suggesting that the interaction between the two proteins is of functional significance. Sds22p is therefore likely to be the second example of a PP1 regulatory subunit identified in S. cerevisiae.

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