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Heterologous phosphoketolase expression redirects flux towards acetate, perturbs sugar phosphate pools and increases respiratory demand in Saccharomyces cerevisiae
Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
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2019 (English)In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 18, article id 25Article in journal (Refereed) Published
Abstract [en]

Introduction: Phosphoketolases (Xfpk) are a non-native group of enzymes in yeast, which can be expressed in combination with other metabolic enzymes to positively influence the yield of acetyl-CoA derived products by reducing carbon losses in the form of CO2. In this study, a yeast strain expressing Xfpk from Bifidobacterium breve, which was previously found to have a growth defect and to increase acetate production, was characterized.

Results: Xfpk-expression was found to increase respiration and reduce biomass yield during glucose consumption in batch and chemostat cultivations. By cultivating yeast with or without Xfpk in bioreactors at different pHs, we show that certain aspects of the negative growth effects coupled with Xfpk-expression are likely to be explained by proton decoupling. At low pH, this manifests as a reduction in biomass yield and growth rate in the ethanol phase. Secondly, we show that intracellular sugar phosphate pools are significantly altered in the Xfpk-expressing strain. In particular a decrease of the substrates xylulose-5-phosphate and fructose-6-phosphate was detected (26% and 74% of control levels) together with an increase of the products glyceraldehyde-3-phosphate and erythrose-4-phosphate (208% and 542% of control levels), clearly verifying in vivo Xfpk enzymatic activity. Lastly, RNAseq analysis shows that Xfpk expression increases transcription of genes related to the glyoxylate cycle, the TCA cycle and respiration, while expression of genes related to ethanol and acetate formation is reduced. The physiological and transcriptional changes clearly demonstrate that a heterologous phosphoketolase flux in combination with endogenous hydrolysis of acetyl-phosphate to acetate increases the cellular demand for acetate assimilation and respiratory ATP-generation, leading to carbon losses.

Conclusion: Our study shows that expression of Xfpk in yeast diverts a relatively small part of its glycolytic flux towards acetate formation, which has a significant impact on intracellular sugar phosphate levels and on cell energetics. The elevated acetate flux increases the ATP-requirement for ion homeostasis and need for respiratory assimilation, which leads to an increased production of CO2. A majority of the negative growth effects coupled to Xfpk expression could likely be counteracted by preventing acetate accumulation via direct channeling of acetyl-phosphate towards acetyl-CoA.

Place, publisher, year, edition, pages
BioMed Central, 2019. Vol. 18, article id 25
Keywords [en]
Saccharomyces cerevisiae, Phosphoketolase, Acetyl-phosphate, Acetate, Acetyl-CoA, Physiology, Sugar phosphate, RNAseq
National Category
Microbiology
Identifiers
URN: urn:nbn:se:umu:diva-156591DOI: 10.1186/s12934-019-1072-6ISI: 000457553300001PubMedID: 30709397OAI: oai:DiVA.org:umu-156591DiVA, id: diva2:1291015
Funder
Knut and Alice Wallenberg FoundationNovo Nordisk, NNF10CC1016517Swedish Foundation for Strategic Research Swedish Research CouncilSwedish Research Council FormasCarl Tryggers foundation Available from: 2019-02-22 Created: 2019-02-22 Last updated: 2019-02-22Bibliographically approved

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Moritz, Thomas

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