Elucidation of new condition-dependent roles for fructose-1,6-bisphosphatase linked to cofactor balances.

Du Toit W P Schabort,Stephanus G Kilian,James C Du Preez,Marie-Joelle Virolle

doi:10.1371/journal.pone.0177319

Du Toit W P Schabort, Stephanus G Kilian + Show 2 more

Open Access

https://doi.org/10.1371/journal.pone.0177319

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Journal: PloS one	Publication Date: May 25, 2017
License type: CC BY 4.0

Affiliation: University of the Free State

Abstract

The cofactor balances in metabolism is of paramount importance in the design of a metabolic engineering strategy and understanding the regulation of metabolism in general. ATP, NAD+ and NADP+ balances are central players linking the various fluxes in central metabolism as well as biomass formation. NADP+ is especially important in the metabolic engineering of yeasts for xylose fermentation, since NADPH is required by most yeasts in the initial step of xylose utilisation, including the fast-growing Kluyveromyces marxianus. In this simulation study of yeast metabolism, the complex interplay between these cofactors was investigated; in particular, how they may affect the possible roles of fructose-1,6-bisphosphatase, the pentose phosphate pathway, glycerol production and the pyruvate dehydrogenase bypass. Using flux balance analysis, it was found that the potential role of fructose-1,6-bisphosphatase was highly dependent on the cofactor specificity of the oxidative pentose phosphate pathway and on the carbon source. Additionally, the excessive production of ATP under certain conditions might be involved in some of the phenomena observed, which may have been overlooked to date. Based on these findings, a strategy is proposed for the metabolic engineering of a future xylose-fermenting yeast for biofuel production.

Highlights

Differential RNA-seq transcriptomics of Kluyveromyces marxianus was performed with glucose or xylose as the carbon source under aerobic conditions [1, 2]
The overall pattern of regulation in K. marxianus grown with D-xylose as carbon source instead of glucose resembled that of glucose derepression in S. cerevisiae, including the strong up-regulation of peroxisomal metabolism [1, 2], it did not represent a complete gluconeogenic response
The experimental xylose uptake and growth rates were approximately 50% of those for glucose, the power of Flux Balance Analysis (FBA) lies in calculating fluxes relative to a reference flux; in this case, the sugar uptake flux

Summary

Introduction

Differential RNA-seq transcriptomics of Kluyveromyces marxianus was performed with glucose or xylose as the carbon source under aerobic conditions [1, 2]. It is to be expected that much of the differential response results from glucose derepression, as is the case with Saccharomyces cerevisiae in the absence of glucose, where the response is due to carbon source responsive transcription factors such as Adr and Mig. The overall pattern of regulation in K. marxianus grown with D-xylose as carbon source instead of glucose resembled that of glucose derepression in S. cerevisiae, including the strong up-regulation of peroxisomal metabolism [1, 2], it did not represent a complete gluconeogenic response. Other signals are seemingly required for up-regulation of the glyoxylate cycle, which would be required if the cells were growing on acetyl-CoA.

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