Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Jan 23:10:e00122.
doi: 10.1016/j.mec.2020.e00122. eCollection 2020 Jun.

Conversion of phosphoenolpyruvate to pyruvate in Thermoanaerobacterium saccharolyticum

Jingxuan Cui  1   2 , Marybeth I Maloney  2   3 , Daniel G Olson  2   3 , Lee R Lynd  1   2   3
Affiliations

Conversion of phosphoenolpyruvate to pyruvate in Thermoanaerobacterium saccharolyticum

Jingxuan Cui et al. Metab Eng Commun. .

Abstract

Thermoanaerobacterium saccharolyticum is an anaerobic thermophile that can ferment hemicellulose to produce biofuels, such as ethanol. It has been engineered to produce ethanol at high yield and titer. T. saccharolyticum uses the Embden-Meyerhof-Parnas (EMP) pathway for glycolysis. However, the genes and enzymes used in each step of the EMP pathway in T. saccharolyticum are not completely known. In T. saccharolyticum, both pyruvate kinase (PYK) and pyruvate phosphate dikinase (PPDK) are highly expressed based on transcriptomic and proteomic data. Both enzymes catalyze the formation of pyruvate from phosphoenolpyruvate (PEP). PYK is typically the last step of EMP glycolysis pathway while PPDK is reversible and is found mostly in C4 plants and some microorganisms. It is not clear what role PYK and PPDK play in T. saccharolyticum metabolism and fermentation pathways and whether both are necessary. In this study we deleted the ppdk gene in wild type and homoethanologen strains of T. saccharolyticum and showed that it is not essential for growth or ethanol production.

Keywords: Consolidated bioprocessing; Ethanol; Pyruvate; Pyruvate kinase; Pyruvate phosphate dikinase; Thermoanaerobacterium saccharolyticum.

PubMed Disclaimer

Conflict of interest statement

Lee R. Lynd is a co-founder of the Enchi corporation, which has a financial interest in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum.

Figures

Fig. 1
Fig. 1
Possible routes for pyruvate production from PEP in T. saccharolyticum. Metabolites are shown in black and enzymes involved are shown red. Abbreviations used are G6P: glucose 6-phosphate; PEP: phosphoenolpyruvate; PTS: phosphoenolpyruvate-dependent sugar phosphotransferase system; PPDK: pyruvate phosphate dikinase; PYK: pyruvate kinase.
Fig. 2
Fig. 2
Enzyme activities of T. saccharolyticum strains. Pyruvate kinase (PYK, shown in green) and pyruvate phosphate dikinase (PPDK, shown in grey) were measured in wild type strain (LL1305), homoethanologen strain (LL1328) and the Δppdk strains (LL1580 and LL1686). wt stands for wild type, eth stands for homoethanologen. All strains were grown in MTC-6 medium containing 5 ​g/L initial cellobiose and collected at mid-log phase of growth. Error bars represent one standard deviation (n ​= ​6).
Fig. 3
Fig. 3
Influence of PPi concentration on pyruvate kinase activity. PYK activity was measured in the cell-free extract of the homoethanologen strain of T. saccharolyticum (LL1328) with addition of 2 ​mM, 5 ​mM or 10 ​mM PPi. Relative activity was calculated based on the no-inhibitor condition. Error bars represent one standard deviation (n ​= ​2).
Fig. 4
Fig. 4
Fermentation result of T. saccharolyticum ppdk deletion strains. Data was collected from 72-h batch fermentations in MTC-6 medium containing 5 ​g/L (A) or 20 ​g/L (B) initial cellobiose. The number above the ethanol bar indicates ethanol yield, as a percentage of the theoretical maximum, which is 4 ​mol of ethanol per mole of cellobiose. wt stands for wild type background, eth stands for homoethanologen background. Error bars represent one standard deviation from three replicates.
See this image and copyright information in PMC

References

    1. Acosta H., Dubourdieu M., Quiñones W., Cáceres A., Bringaud F., Concepción J.L. Pyruvate phosphate dikinase and pyrophosphate metabolism in the glycosome of Trypanosoma cruzi epimastigotes. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2004;138(4):347–356. - PubMed
    1. Benziman M., Eizen N. Pyruvate-phosphate dikinase and the control of gluconeogenesis in Acetobacter xylinum. Journal of Biological Chemistry. 1971;246(1):57–61. - PubMed
    1. Bielen A.A., Willquist K., Engman J., Van Der Oost J., Van Niel E.W., Kengen S.W. Pyrophosphate as a central energy carrier in the hydrogen-producing extremely thermophilic Caldicellulosiruptor saccharolyticus. FEMS Microbiol Lett [Internet] 2010 Jun;307(1):48–54. http://www.ncbi.nlm.nih.gov/pubmed/20557574 Available from: - PubMed
    1. Biochem J., Kukko E. The intracellular concentration of pyrophosphate in the batch culture of Escherichia coli. 1982;349:347–349. - PubMed
    1. Bornefeld T. Is light-dependent formation of inorganic pyrophosphate in Anacystis a photosynthetic process? Arch. Microbiol. 1981;129(5):371–373.

LinkOut - more resources