Single cell and in vivo analyses elucidate the effect of xylC lactonase during production of D-xylonate in Saccharomyces cerevisiae

Yvonne Nygård (Corresponding Author), Hannu Maaheimo, Dominik Mojzita, Mervi Toivari, Marilyn Wiebe, Orna Resnekov, C. Gustavo Pesce, Laura Ruohonen, Merja Penttilä

Research output: Contribution to journalArticleScientificpeer-review

13 Citations (Scopus)

Abstract

D-xylonate is a potential platform chemical which can be produced by engineered Saccharomyces cerevisiae strains. In order to address production constraints in more detail, we analysed the role of lactone ring opening in single cells and populations. Both D-xylono-γ-lactone and D-xylonate were produced when the Caulobacter crescentus xylB (D-xylose dehydrogenase) was expressed in S. cerevisiae, with or without co-expression of xylC (D-xylonolactonase), as seen by 1H NMR. XylC facilitated rapid opening of the lactone and more D-xylonate was initially produced than in its absence. Using in vivo 1H NMR analysis of cell extracts, culture media and intact cells we observed that the lactone and linear forms of D-xylonic acid were produced, accumulated intracellularly, and partially exported within 15-60min of D-xylose provision.During single-cell analysis of cells expressing the pH sensitive fluorescent probe pHluorin, pHluorin fluorescence was gradually lost from the cells during D-xylonate production, as expected for cells with decreasing intracellular pH. However, in the presence of D-xylose, only 9% of cells expressing xylB lost pHluorin fluorescence within 4.5. h, whereas 99% of cells co-expressing xylB and xylC lost fluorescence, a large proportion of which also lost vitality, during this interval. Loss of vitality in the presence of D-xylose was correlated to the extracellular pH, but fluorescence was lost from xylB and xylC expressing cells regardless of the extracellular condition.

Original languageEnglish
Pages (from-to)238-247
JournalMetabolic Engineering
Volume25
DOIs
Publication statusPublished - 1 Jan 2014
MoE publication typeA1 Journal article-refereed

Fingerprint

Xylose
Lactones
Yeast
Saccharomyces cerevisiae
Fluorescence
Cells
Nuclear magnetic resonance
Fluorescent Dyes
Culture Media
Caulobacter crescentus
Single-Cell Analysis
Acids
Cell Extracts
PHluorin
Cell Culture Techniques

Keywords

  • D-xylonic acid
  • Saccharomyces cerevisiae
  • Single-cell analysis

Cite this

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title = "Single cell and in vivo analyses elucidate the effect of xylC lactonase during production of D-xylonate in Saccharomyces cerevisiae",
abstract = "D-xylonate is a potential platform chemical which can be produced by engineered Saccharomyces cerevisiae strains. In order to address production constraints in more detail, we analysed the role of lactone ring opening in single cells and populations. Both D-xylono-γ-lactone and D-xylonate were produced when the Caulobacter crescentus xylB (D-xylose dehydrogenase) was expressed in S. cerevisiae, with or without co-expression of xylC (D-xylonolactonase), as seen by 1H NMR. XylC facilitated rapid opening of the lactone and more D-xylonate was initially produced than in its absence. Using in vivo 1H NMR analysis of cell extracts, culture media and intact cells we observed that the lactone and linear forms of D-xylonic acid were produced, accumulated intracellularly, and partially exported within 15-60min of D-xylose provision.During single-cell analysis of cells expressing the pH sensitive fluorescent probe pHluorin, pHluorin fluorescence was gradually lost from the cells during D-xylonate production, as expected for cells with decreasing intracellular pH. However, in the presence of D-xylose, only 9{\%} of cells expressing xylB lost pHluorin fluorescence within 4.5. h, whereas 99{\%} of cells co-expressing xylB and xylC lost fluorescence, a large proportion of which also lost vitality, during this interval. Loss of vitality in the presence of D-xylose was correlated to the extracellular pH, but fluorescence was lost from xylB and xylC expressing cells regardless of the extracellular condition.",
keywords = "D-xylonic acid, Saccharomyces cerevisiae, Single-cell analysis",
author = "Yvonne Nyg{\aa}rd and Hannu Maaheimo and Dominik Mojzita and Mervi Toivari and Marilyn Wiebe and Orna Resnekov and {Gustavo Pesce}, C. and Laura Ruohonen and Merja Penttil{\"a}",
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Single cell and in vivo analyses elucidate the effect of xylC lactonase during production of D-xylonate in Saccharomyces cerevisiae. / Nygård, Yvonne (Corresponding Author); Maaheimo, Hannu; Mojzita, Dominik; Toivari, Mervi; Wiebe, Marilyn; Resnekov, Orna; Gustavo Pesce, C.; Ruohonen, Laura; Penttilä, Merja.

In: Metabolic Engineering, Vol. 25, 01.01.2014, p. 238-247.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Single cell and in vivo analyses elucidate the effect of xylC lactonase during production of D-xylonate in Saccharomyces cerevisiae

AU - Nygård, Yvonne

AU - Maaheimo, Hannu

AU - Mojzita, Dominik

AU - Toivari, Mervi

AU - Wiebe, Marilyn

AU - Resnekov, Orna

AU - Gustavo Pesce, C.

AU - Ruohonen, Laura

AU - Penttilä, Merja

N1 - CA2: BA3111 CA2: BA3112 CA2: BA3114 CA2: BA311 ISI: BIOTECHNOLOGY & APPLIED MICROBIOLOGY

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N2 - D-xylonate is a potential platform chemical which can be produced by engineered Saccharomyces cerevisiae strains. In order to address production constraints in more detail, we analysed the role of lactone ring opening in single cells and populations. Both D-xylono-γ-lactone and D-xylonate were produced when the Caulobacter crescentus xylB (D-xylose dehydrogenase) was expressed in S. cerevisiae, with or without co-expression of xylC (D-xylonolactonase), as seen by 1H NMR. XylC facilitated rapid opening of the lactone and more D-xylonate was initially produced than in its absence. Using in vivo 1H NMR analysis of cell extracts, culture media and intact cells we observed that the lactone and linear forms of D-xylonic acid were produced, accumulated intracellularly, and partially exported within 15-60min of D-xylose provision.During single-cell analysis of cells expressing the pH sensitive fluorescent probe pHluorin, pHluorin fluorescence was gradually lost from the cells during D-xylonate production, as expected for cells with decreasing intracellular pH. However, in the presence of D-xylose, only 9% of cells expressing xylB lost pHluorin fluorescence within 4.5. h, whereas 99% of cells co-expressing xylB and xylC lost fluorescence, a large proportion of which also lost vitality, during this interval. Loss of vitality in the presence of D-xylose was correlated to the extracellular pH, but fluorescence was lost from xylB and xylC expressing cells regardless of the extracellular condition.

AB - D-xylonate is a potential platform chemical which can be produced by engineered Saccharomyces cerevisiae strains. In order to address production constraints in more detail, we analysed the role of lactone ring opening in single cells and populations. Both D-xylono-γ-lactone and D-xylonate were produced when the Caulobacter crescentus xylB (D-xylose dehydrogenase) was expressed in S. cerevisiae, with or without co-expression of xylC (D-xylonolactonase), as seen by 1H NMR. XylC facilitated rapid opening of the lactone and more D-xylonate was initially produced than in its absence. Using in vivo 1H NMR analysis of cell extracts, culture media and intact cells we observed that the lactone and linear forms of D-xylonic acid were produced, accumulated intracellularly, and partially exported within 15-60min of D-xylose provision.During single-cell analysis of cells expressing the pH sensitive fluorescent probe pHluorin, pHluorin fluorescence was gradually lost from the cells during D-xylonate production, as expected for cells with decreasing intracellular pH. However, in the presence of D-xylose, only 9% of cells expressing xylB lost pHluorin fluorescence within 4.5. h, whereas 99% of cells co-expressing xylB and xylC lost fluorescence, a large proportion of which also lost vitality, during this interval. Loss of vitality in the presence of D-xylose was correlated to the extracellular pH, but fluorescence was lost from xylB and xylC expressing cells regardless of the extracellular condition.

KW - D-xylonic acid

KW - Saccharomyces cerevisiae

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