Physiological characterization of recombinant Saccharomyces cerevisiae expressing the Aspergillus nidulans phosphoketolase pathway: validation of activity through 13C-based metabolic flux analysis

Papini, Marta; Nookaew, Intawat; Siewers, Verena; Nielsen, Jens

doi:10.1007/s00253-012-3936-0

Cited by 32 publications

(28 citation statements)

References 36 publications

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“…As a consequence, two molecules of the NADPH are being produced for each sugar molecule passing through the PPP. As was demonstrated by Papini et al the expression of genes xpkA and ack showed a functional carbon flux through the PHK pathway and as shown in this study the expression of genes xpkA , ack and ws2 resulted in a yield of FAEEs 50% higher than in a strain not expressing the PHK pathway [20]. It was also shown that the expression of xpkA , ack resulted in an improved polyhydroxybutyrate (PHB) producing strain [22].…”

Section: Discussionsupporting

confidence: 56%

“…The ws2 gene was restricted with Not I/ Sac I from pBdJ02 and then ligated into the Not I/ Sac I sites of vector pSP-GM2 to construct pSP-B2N. The construction of plasmid pMPa containing genes xpkA and ack was described previously [20]. Plasmid pMPp was constructed accordingly with the difference that it contained pta instead of ack .…”

Section: Methodsmentioning

confidence: 99%

“…The phosphoketolase (PHK) pathway was described previously as potential alternative carbon route for different industrially relevant metabolites due to the increase of acetyl-CoA production with NADPH as co-product [20-22]. The carbon flow leading towards the PHK pathway starts with the conversion of glucose to glucose-6-phosphate in the Embden-Meyerhof-Parnas (EMP) pathway followed by conversion of glucose-6-phosphate in three reactions into ribulose-5-phosphate in the oxidative part of the pentose phosphate pathway (PPP).…”

Section: Introductionmentioning

confidence: 99%

“…It has been demonstrated that S. cerevisiae has the ability to functionally express phosphotransacetylase from B. subtilis as well as phosphoketolase and acetate kinase from A. nidulans [20,27]. The fact that more NADPH is formed when glucose is metabolized via the PPP instead of the EMP pathway makes the combination of PPP and PHK pathway an interesting alternative for FAEE production.…”

Section: Introductionmentioning

confidence: 99%

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Improved production of fatty acid ethyl esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation pathway and expression of the heterologous phosphoketolase pathway

et al. 2014

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BackgroundDue to an increasing demand of transportation fuels, a lower availability of cheap crude oil and a lack of sustainability of fossil fuels, a gradual shift from petroleum based fuels towards alternative and renewable fuel resources will be required in the near future. Fatty acid ethyl esters (FAEEs) have properties similar to current crude diesel and could therefore form an important contribution to the development of sustainable transportation fuels in future. It is important to develop novel cell factories for efficient production of FAEEs and their precursors.ResultsHere, a Saccharomyces cerevisiae cell factory expressing a heterologous wax ester synthase (ws2) from Marinobacter hydrocarbonoclasticus was used to produce FAEEs from ethanol and acyl-coenzyme A (acyl-CoA). The production of acyl-CoA requires large amounts of NADPH and acetyl-CoA. Therefore, two metabolic engineering strategies for improved provision of NADPH and acetyl-CoA were evaluated. First, the ethanol degradation pathway was employed to re-channel carbon flow towards the synthesis of acetyl-CoA. Therefore, ADH2 and ALD6 encoding, respectively, alcohol dehydrogenase and acetaldehyde dehydrogenase were overexpressed together with the heterologous gene acsSEL641P encoding acetyl-CoA synthetase. The co-overexpression of ADH2, ALD6 and acsSEL641P with ws2 resulted in 408 ± 270 μg FAEE gCDW−1, a 3-fold improvement. Secondly, for the expression of the PHK pathway two genes, xpkA and ack, both descending from Aspergillus nidulans, were co-expressed together with ws2 to catalyze, respectively, the conversion of xylulose-5-phosphate to acetyl phosphate and glyceraldehyde-3-phosphate and acetyl phosphate to acetate. Alternatively, ack was substituted with pta from Bacillus subtilis, encoding phosphotransacetylase for the conversion of acetyl phosphate to acetyl-CoA. Both PHK pathways were additionally expressed in a strain with multiple chromosomally integrated ws2 gene, which resulted in respectively 5100 ± 509 and 4670 ± 379 μg FAEE gCDW−1, an up to 1.7-fold improvement.ConclusionTwo different strategies for engineering of the central carbon metabolism for efficient provision of acetyl-CoA and NADPH required for fatty acid biosynthesis and hence FAEE production were evaluated and it was found that both the ethanol degradation pathway as well as the phosphoketolase pathway improve the yield of FAEEs.

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Section: Discussionsupporting

confidence: 56%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Improved production of fatty acid ethyl esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation pathway and expression of the heterologous phosphoketolase pathway

et al. 2014

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“…The phosphoketolase pathway was originally introduced into S. cerevisiae for improved xylose utilization [63], and later was found to enhance the synthesis of acetyl-CoA as well [13,34,51] (Fig. 3).…”

Section: Phosphoketolase Pathway (Pk)mentioning

confidence: 99%

Recent advances in biosynthesis of fatty acids derived products in Saccharomyces cerevisiae via enhanced supply of precursor metabolites

Lian

Zhao

2015

Journal of Industrial Microbiology and Biotechnology

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Fatty acids or their activated forms, fatty acyl-CoAs and fatty acyl-ACPs, are important precursors to synthesize a wide variety of fuels and chemicals, including but not limited to free fatty acids (FFAs), fatty alcohols (FALs), fatty acid ethyl esters (FAEEs), and alkanes. However, Saccharomyces cerevisiae, an important cell factory, does not naturally accumulate fatty acids in large quantities. Therefore, metabolic engineering strategies were carried out to increase the glycolytic fluxes to fatty acid biosynthesis in yeast, specifically to enhance the supply of precursors, eliminate competing pathways, and bypass the host regulatory network. This review will focus on the genetic manipulation of both structural and regulatory genes in each step for fatty acids overproduction in S. cerevisiae, including from sugar to acetyl-CoA, from acetyl-CoA to malonyl-CoA, and from malonyl-CoA to fatty acyl-CoAs. The downstream pathways for the conversion of fatty acyl-CoAs to the desired products will also be discussed.

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Improved polyhydroxybutyrate production by Saccharomyces cerevisiae through the use of the phosphoketolase pathway

Kocharin

Siewers

Nielsen

2013

Biotech & Bioengineering

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The metabolic pathways of the central carbon metabolism in Saccharomyces cerevisiae are well studied and consequently S. cerevisiae has been widely evaluated as a cell factory for many industrial biological products. In this study, we investigated the effect of engineering the supply of precursor, acetyl-CoA, and cofactor, NADPH, on the biosynthesis of the bacterial biopolymer polyhydroxybutyrate (PHB), in S. cerevisiae. Supply of acetyl-CoA was engineered by over-expression of genes from the ethanol degradation pathway or by heterologous expression of the phophoketolase pathway from Aspergillus nidulans. Both strategies improved the production of PHB. Integration of gapN encoding NADP(+) -dependent glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans into the genome enabled an increased supply of NADPH resulting in a decrease in glycerol production and increased production of PHB. The strategy that resulted in the highest PHB production after 100 h was with a strain harboring the phosphoketolase pathway to supply acetyl-CoA without the need of increased NADPH production by gapN integration. The results from this study imply that during the exponential growth on glucose, the biosynthesis of PHB in S. cerevisiae is likely to be limited by the supply of NADPH whereas supply of acetyl-CoA as precursor plays a more important role in the improvement of PHB production during growth on ethanol.

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Physiological characterization of recombinant Saccharomyces cerevisiae expressing the Aspergillus nidulans phosphoketolase pathway: validation of activity through 13C-based metabolic flux analysis

Cited by 32 publications

References 36 publications

Improved production of fatty acid ethyl esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation pathway and expression of the heterologous phosphoketolase pathway

Improved production of fatty acid ethyl esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation pathway and expression of the heterologous phosphoketolase pathway

Recent advances in biosynthesis of fatty acids derived products in Saccharomyces cerevisiae via enhanced supply of precursor metabolites

Improved polyhydroxybutyrate production by Saccharomyces cerevisiae through the use of the phosphoketolase pathway

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