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A genetic screen for increasing metabolic flux in the isoprenoid pathway of Saccharomyces cerevisiae: Isolation of SPT15 mutants using the screen.

ABSTRACT: A genetic screen to identify mutants that can increase flux in the isoprenoid pathway of yeast has been lacking. We describe a carotenoid-based visual screen built with the core carotenogenic enzymes from the red yeast Rhodosporidium toruloides. Enzymes from this yeast displayed the required, higher capacity in the carotenoid pathway. The development also included the identification of the metabolic bottlenecks, primarily phytoene dehydrogenase, that was subjected to a directed evolution strategy to yield more active mutants. To further limit phytoene pools, a less efficient version of GGPP synthase was employed. The screen was validated with a known flux increasing gene, tHMG1. New mutants in the TATA binding protein SPT15 were isolated using this screen that increased the yield of carotenoids, and an alternate isoprenoid, ?-Farnesene confirming increase in overall flux. The findings indicate the presence of previously unknown links to the isoprenoid pathway that can be uncovered using this screen.

SUBMITTER: Wadhwa M

PROVIDER: S-EPMC5779727 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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A genetic screen for increasing metabolic flux in the isoprenoid pathway of Saccharomyces cerevisiae: Isolation of SPT15 mutants using the screen.

Wadhwa M M Bachhawat A K AK

Metabolic engineering communications 20160527

A genetic screen to identify mutants that can increase flux in the isoprenoid pathway of yeast has been lacking. We describe a carotenoid-based visual screen built with the core carotenogenic enzymes from the red yeast Rhodosporidium toruloides. Enzymes from this yeast displayed the required, higher capacity in the carotenoid pathway. The development also included the identification of the metabolic bottlenecks, primarily phytoene dehydrogenase, that was subjected to a directed evolution ...[more]

PMID: 29468122

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Project description:BackgroundProduction of isoprenoids, a large and diverse class of commercially important chemicals, can be achieved through engineering metabolism in microorganisms. Several attempts have been made to reroute metabolic flux towards isoprenoid pathway in yeast. Most approaches have focused on the core isoprenoid pathway as well as on meeting the increased precursors and cofactor requirements. To identify unexplored genetic targets that positively influence the isoprenoid pathway activity, a carotenoid based genetic screen was previously developed and three novel mutants of a global TATA binding protein SPT15 was isolated for heightened isoprenoid flux in Saccharomyces cerevisiae.ResultsIn this study, we investigated how one of the three spt15 mutants, spt15_Ala101Thr, was leading to enhanced isoprenoid pathway flux in S. cerevisiae. Metabolic flux analysis of the spt15_Ala101Thr mutant initially revealed a rerouting of the central carbon metabolism for the production of the precursor acetyl-CoA through activation of pyruvate-acetaldehyde-acetate cycle in the cytoplasm due to high flux in the reaction caused by pyruvate decarboxylase (PDC). This led to alternate routes of cytosolic NADPH generation, increased mitochondrial ATP production and phosphate demand in the mutant strain. Comparison of the transcriptomics of the spt15_Ala101Thr mutant cell with SPT15WT bearing cells shows upregulation of phosphate mobilization genes and pyruvate decarboxylase 6 (PDC6). Increasing the extracellular phosphate led to an increase in the growth rate and biomass but diverted flux away from the isoprenoid pathway. PDC6 is also shown to play a critical role in isoprenoid pathway flux under phosphate limitation conditions.ConclusionThe study not only proposes a probable mechanism as to how a spt15_Ala101Thr mutant (a global TATA binding protein mutant) could increase flux towards the isoprenoid pathway, but also PDC as a new route of metabolic manipulation for increasing the isoprenoid flux in yeast.

Project description:BackgroundTo 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.ResultsAs 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.ConclusionsBy 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.

Dataset Information

A genetic screen for increasing metabolic flux in the isoprenoid pathway of Saccharomyces cerevisiae: Isolation of SPT15 mutants using the screen.

Publications

A genetic screen for increasing metabolic flux in the isoprenoid pathway of <i>Saccharomyces cerevisiae</i>: Isolation of <i>SPT15</i> mutants using the screen.

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