Dataset Information

Improving acetyl-CoA biosynthesis in Saccharomyces cerevisiae via the overexpression of pantothenate kinase and PDH bypass.

ABSTRACT: BACKGROUND:Acetyl-CoA is an important precursor in Saccharomyces cerevisiae. Various approaches have been adopted to improve its cytosolic level previously with the emphasis on engineering the "acetyl-" part of acetyl-CoA. To the best of our knowledge, there have been no reports on engineering the "-CoA" part so far. RESULTS:In this study, we had tried to engineer S. cerevisiae from both the "-CoA" part via pantothenate kinase overexpression (PanK from S. cerevisiae, the rate-limiting enzyme for CoA synthesis) and the "acetyl-"part through PDH bypass introduction (ALD6 from S. cerevisiae and SeAcsL641P from Salmonella enteric). A naringenin-producing reporter strain had been constructed to reflect cytosolic acetyl-CoA level as acetyl-CoA is the precursor of naringenin. It was found that PanK overexpression or PDH bypass introduction alone only led to a twofold or 6.74-fold increase in naringenin titer, but the combination of both (strain CENFPAA01) had resulted in 24.4-fold increase as compared to the control (strain CENF09) in the presence of 0.5 mM substrate p-coumaric acid. The supplement of PanK substrate pantothenate resulted in another 19% increase in naringenin production. CONCLUSIONS:To greatly enhance acetyl-CoA level in yeast cytosol, it is feasible to engineer both the "acetyl-" part and the "-CoA" part simultaneously. Insufficient CoA supply might aggravate acetyl-CoA shortage and cause low yield of target product.

SUBMITTER: Liu W

PROVIDER: S-EPMC5316175 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Improving acetyl-CoA biosynthesis in <i>Saccharomyces cerevisiae</i> via the overexpression of pantothenate kinase and PDH bypass.

Liu Wenshan W Zhang Bo B Jiang Rongrong R

Biotechnology for biofuels 20170217

<h4>Background</h4>Acetyl-CoA is an important precursor in <i>Saccharomyces cerevisiae</i>. Various approaches have been adopted to improve its cytosolic level previously with the emphasis on engineering the "acetyl-" part of acetyl-CoA. To the best of our knowledge, there have been no reports on engineering the "-CoA" part so far.<h4>Results</h4>In this study, we had tried to engineer <i>S. cerevisiae</i> from both the "-CoA" part via pantothenate kinase overexpression (PanK from <i>S. cerevisi ...[more]

PMID: 28239413

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Project description:With increasing concern about the environmental impact of a petroleum based economy, focus has shifted towards greener production strategies including metabolic engineering of microbes for the conversion of plant-based feedstocks to second generation biofuels and industrial chemicals. Saccharomyces cerevisiae is an attractive host for this purpose as it has been extensively engineered for production of various fuels and chemicals. Many of the target molecules are derived from the central metabolite and molecular building block, acetyl-CoA. To date, it has been difficult to engineer S. cerevisiae to continuously convert sugars present in biomass-based feedstocks to acetyl-CoA derived products due to intrinsic physiological constraints-in respiring cells, the precursor pyruvate is directed away from the endogenous cytosolic acetyl-CoA biosynthesis pathway towards the mitochondria, and in fermenting cells pyruvate is directed towards the byproduct ethanol. In this study we incorporated an alternative mode of acetyl-CoA biosynthesis mediated by ATP citrate lyase (ACL) that may obviate such constraints.We characterized the activity of several heterologously expressed ACLs in crude cell lysates, and found that ACL from Aspergillus nidulans demonstrated the highest activity. We employed a push/pull strategy to shunt citrate towards ACL by deletion of the mitochondrial NAD(+)-dependent isocitrate dehydrogenase (IDH1) and engineering higher flux through the upper mevalonate pathway. We demonstrated that combining the two modifications increases accumulation of mevalonate pathway intermediates, and that both modifications are required to substantially increase production. Finally, we incorporated a block strategy by replacing the native ERG12 (mevalonate kinase) promoter with the copper-repressible CTR3 promoter to maximize accumulation of the commercially important molecule mevalonate.By combining the push/pull/block strategies, we significantly improved mevalonate production. We anticipate that this strategy can be used to improve the efficiency with which industrial strains of S. cerevisiae convert feedstocks to acetyl-CoA derived fuels and chemicals.

Project description:Cytosolic acetyl-coenzyme A is a precursor for many biotechnologically relevant compounds produced by Saccharomyces cerevisiae. In this yeast, cytosolic acetyl-CoA synthesis and growth strictly depend on expression of either the Acs1 or Acs2 isoenzyme of acetyl-CoA synthetase (ACS). Since hydrolysis of ATP to AMP and pyrophosphate in the ACS reaction constrains maximum yields of acetyl-CoA-derived products, this study explores replacement of ACS by two ATP-independent pathways for acetyl-CoA synthesis. After evaluating expression of different bacterial genes encoding acetylating acetaldehyde dehydrogenase (A-ALD) and pyruvate-formate lyase (PFL), acs1M-NM-^T acs2M-NM-^T S. cerevisiae strains were constructed in which A-ALD or PFL successfully replaced ACS. In A-ALD-dependent strains, aerobic growth rates of up to 0.27 h-1 were observed, while anaerobic growth rates of PFL-dependent S. cerevisiae (0.21 h-1) were stoichiometrically coupled to formate production. In glucose-limited chemostat cultures, intracellular metabolite analysis did not reveal major differences between A-ALD-dependent and reference strains. However, biomass yields on glucose of A-ALD- and PFL-dependent strains were lower than those of the reference strain. Transcriptome analysis suggested that reduced biomass yields were caused by acetaldehyde and formate in A-ALD- and PFL-dependent strains, respectively. Transcript profiles also indicated that a previously proposed role of Acs2 in histone acetylation is probably linked to cytosolic acetyl-CoA levels rather than to direct involvement of Acs2 in histone acetylation. While, for the first time, demonstrating that yeast ACS can be fully replaced by alternative reactions, this study demonstrates that further modifications are needed to achieve optimal in vivo efficiencies of the supply of acetyl-CoA as product precursor. To investigate the impact of introduced changes in native pathway of cytosolic acetyl-CoA formation in S. cerevisiae, a DNA microarray-based transcriptome analysis was performed on aerobic or anaerobic, glucose-limited chemostat cultures.

Dataset Information

Improving acetyl-CoA biosynthesis in Saccharomyces cerevisiae via the overexpression of pantothenate kinase and PDH bypass.

Publications

Improving acetyl-CoA biosynthesis in <i>Saccharomyces cerevisiae</i> via the overexpression of pantothenate kinase and PDH bypass.

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