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Investigating xylose metabolism in recombinant Saccharomyces cerevisiae via 13C metabolic flux analysis.

Feng X, Zhao H - Microb. Cell Fact. (2013)

Bottom Line: Based on in silico simulations of metabolic fluxes, reducing the cell maintenance energy was found crucial to achieve the optimal xylose-based ethanol production.Specifically, we found that the high cell maintenance energy was one of the key factors involved in xylose utilization.Potential strategies to reduce the cell maintenance energy, such as adding exogenous nutrients and evolutionary adaptation, were suggested based on the in vivo and in silico flux analysis in this study.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, Institute for Genomic Biology, Urbana, USA. zhao5@illinois.edu.

ABSTRACT

Background: To engineer Saccharomyces cerevisiae for efficient xylose utilization, a fungal pathway consisting of xylose reductase, xylitol dehydrogenase, and xylulose kinase is often introduced to the host strain. Despite extensive in vitro studies on the xylose pathway, the intracellular metabolism rewiring in response to the heterologous xylose pathway remains largely unknown. In this study, we applied 13C metabolic flux analysis and stoichiometric modeling to systemically investigate the flux distributions in a series of xylose utilizing S. cerevisiae strains.

Results: As revealed by 13C metabolic flux analysis, the oxidative pentose phosphate pathway was actively used for producing NADPH required by the fungal xylose pathway during xylose utilization of recombinant S. cerevisiae strains. The TCA cycle activity was found to be tightly correlated with the requirements of maintenance energy and biomass yield. Based on in silico simulations of metabolic fluxes, reducing the cell maintenance energy was found crucial to achieve the optimal xylose-based ethanol production. The stoichiometric modeling also suggested that both the cofactor-imbalanced and cofactor-balanced pathways could lead to optimal ethanol production, by flexibly adjusting the metabolic fluxes in futile cycle. However, compared to the cofactor-imbalanced pathway, the cofactor-balanced xylose pathway can lead to optimal ethanol production in a wider range of fermentation conditions.

Conclusions: By applying 13C-MFA and in silico flux balance analysis to a series of recombinant xylose-utilizing S. cerevisiae strains, this work brings new knowledge about xylose utilization in two aspects. First, the interplays between the fungal xylose pathway and the native host metabolism were uncovered. Specifically, we found that the high cell maintenance energy was one of the key factors involved in xylose utilization. Potential strategies to reduce the cell maintenance energy, such as adding exogenous nutrients and evolutionary adaptation, were suggested based on the in vivo and in silico flux analysis in this study. In addition, the impacts of cofactor balance issues on xylose utilization were systemically investigated. The futile pathways were identified as the key factor to adapt to different degrees of cofactor imbalances and suggested as the targets for further engineering to tackle cofactor-balance issues.

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Enzyme activities of xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulose kinase (XKS) in S. cerevisiae strains. The activities of XRs with both NADPH and NADH as the cofactors were measured.
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Figure 7: Enzyme activities of xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulose kinase (XKS) in S. cerevisiae strains. The activities of XRs with both NADPH and NADH as the cofactors were measured.

Mentions: In this study, we cultured strains in the oxygen limited conditions with defined minimal medium based on several considerations. First, as reported by many groups, few S. cerevisiae strains can grow in defined minimal medium under anaerobic conditions with xylose as the sole carbon source, since the oxygen is needed to resolve the cofactor imbalance [40]. On the other hand, the oxygen-limited condition has been suggested as optimal for ethanol production by an in silico simulation [4,40]. In addition, the application of 13C-MFA strictly requires the using of defined minimal medium for culturing recombinant S. cerevisiae strains. However, many labs, including ours, often choose nutrient-rich medium (e.g. YPAX) to create yeast mutants and characterize the xylose fermentation. Compared to the previous characterization of the same strains in nutrient-rich medium (i.e. YPAX), the metabolic behaviors are significantly different in defined minimal medium in terms of growth rates and ethanol production. In order to confirm that the discoveries from the 13C-MFA in minimal medium is also applicable to the studies of xylose utilization in rich medium, we have designed another set of isotopic tracing experiments by providing 4% [1-13C] xylose into the YPAX medium. The 13C enrichments of all the amino acids detected were only 20 ~ 50% of their counterparts from the minimal medium (Figure 6), indicating that the nutrient transport rather than de novo synthesis was the major pathway for building block production. As revealed by 13C-MFA in this study, the GAM plays a pivotal role in xylose utilization, especially in the stressful cultivation conditions. With the building blocks, such as amino acids, provided by the direct uptake from the medium instead of de novo synthesis, nutrient-rich medium is less stressful than the minimal medium, leading to a lower maintenance requirement and better fermentation performance. In addition, compared to those in the nutrient-rich medium, the in vitro enzyme activities of XR and XDH activities in the minimal medium (Figure 7) were within the same level [28]. However, the activities of XKS were about two magnitudes lower (0.001 ~ 0.003 U/mg compared to 0.1 ~ 0.4 U/mg), which could be another reason for the inconsistent metabolic behaviors of the same strain in different media.


Investigating xylose metabolism in recombinant Saccharomyces cerevisiae via 13C metabolic flux analysis.

Feng X, Zhao H - Microb. Cell Fact. (2013)

Enzyme activities of xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulose kinase (XKS) in S. cerevisiae strains. The activities of XRs with both NADPH and NADH as the cofactors were measured.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842631&req=5

Figure 7: Enzyme activities of xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulose kinase (XKS) in S. cerevisiae strains. The activities of XRs with both NADPH and NADH as the cofactors were measured.
Mentions: In this study, we cultured strains in the oxygen limited conditions with defined minimal medium based on several considerations. First, as reported by many groups, few S. cerevisiae strains can grow in defined minimal medium under anaerobic conditions with xylose as the sole carbon source, since the oxygen is needed to resolve the cofactor imbalance [40]. On the other hand, the oxygen-limited condition has been suggested as optimal for ethanol production by an in silico simulation [4,40]. In addition, the application of 13C-MFA strictly requires the using of defined minimal medium for culturing recombinant S. cerevisiae strains. However, many labs, including ours, often choose nutrient-rich medium (e.g. YPAX) to create yeast mutants and characterize the xylose fermentation. Compared to the previous characterization of the same strains in nutrient-rich medium (i.e. YPAX), the metabolic behaviors are significantly different in defined minimal medium in terms of growth rates and ethanol production. In order to confirm that the discoveries from the 13C-MFA in minimal medium is also applicable to the studies of xylose utilization in rich medium, we have designed another set of isotopic tracing experiments by providing 4% [1-13C] xylose into the YPAX medium. The 13C enrichments of all the amino acids detected were only 20 ~ 50% of their counterparts from the minimal medium (Figure 6), indicating that the nutrient transport rather than de novo synthesis was the major pathway for building block production. As revealed by 13C-MFA in this study, the GAM plays a pivotal role in xylose utilization, especially in the stressful cultivation conditions. With the building blocks, such as amino acids, provided by the direct uptake from the medium instead of de novo synthesis, nutrient-rich medium is less stressful than the minimal medium, leading to a lower maintenance requirement and better fermentation performance. In addition, compared to those in the nutrient-rich medium, the in vitro enzyme activities of XR and XDH activities in the minimal medium (Figure 7) were within the same level [28]. However, the activities of XKS were about two magnitudes lower (0.001 ~ 0.003 U/mg compared to 0.1 ~ 0.4 U/mg), which could be another reason for the inconsistent metabolic behaviors of the same strain in different media.

Bottom Line: Based on in silico simulations of metabolic fluxes, reducing the cell maintenance energy was found crucial to achieve the optimal xylose-based ethanol production.Specifically, we found that the high cell maintenance energy was one of the key factors involved in xylose utilization.Potential strategies to reduce the cell maintenance energy, such as adding exogenous nutrients and evolutionary adaptation, were suggested based on the in vivo and in silico flux analysis in this study.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, Institute for Genomic Biology, Urbana, USA. zhao5@illinois.edu.

ABSTRACT

Background: To engineer Saccharomyces cerevisiae for efficient xylose utilization, a fungal pathway consisting of xylose reductase, xylitol dehydrogenase, and xylulose kinase is often introduced to the host strain. Despite extensive in vitro studies on the xylose pathway, the intracellular metabolism rewiring in response to the heterologous xylose pathway remains largely unknown. In this study, we applied 13C metabolic flux analysis and stoichiometric modeling to systemically investigate the flux distributions in a series of xylose utilizing S. cerevisiae strains.

Results: As revealed by 13C metabolic flux analysis, the oxidative pentose phosphate pathway was actively used for producing NADPH required by the fungal xylose pathway during xylose utilization of recombinant S. cerevisiae strains. The TCA cycle activity was found to be tightly correlated with the requirements of maintenance energy and biomass yield. Based on in silico simulations of metabolic fluxes, reducing the cell maintenance energy was found crucial to achieve the optimal xylose-based ethanol production. The stoichiometric modeling also suggested that both the cofactor-imbalanced and cofactor-balanced pathways could lead to optimal ethanol production, by flexibly adjusting the metabolic fluxes in futile cycle. However, compared to the cofactor-imbalanced pathway, the cofactor-balanced xylose pathway can lead to optimal ethanol production in a wider range of fermentation conditions.

Conclusions: By applying 13C-MFA and in silico flux balance analysis to a series of recombinant xylose-utilizing S. cerevisiae strains, this work brings new knowledge about xylose utilization in two aspects. First, the interplays between the fungal xylose pathway and the native host metabolism were uncovered. Specifically, we found that the high cell maintenance energy was one of the key factors involved in xylose utilization. Potential strategies to reduce the cell maintenance energy, such as adding exogenous nutrients and evolutionary adaptation, were suggested based on the in vivo and in silico flux analysis in this study. In addition, the impacts of cofactor balance issues on xylose utilization were systemically investigated. The futile pathways were identified as the key factor to adapt to different degrees of cofactor imbalances and suggested as the targets for further engineering to tackle cofactor-balance issues.

Show MeSH
Related in: MedlinePlus