H+-Translocating Membrane-Bound Pyrophosphatase from Rhodospirillum rubrum Fuels Escherichia coli Cells via an Alternative Pathway for Energy Generation

Evgeniya A Malykh; Liubov I Golubeva; Ekaterina S Kovaleva; Mikhail S Shupletsov; Elena V Rodina; Sergey V Mashko; Nataliya V Stoynova

doi:10.3390/microorganisms11020294

H⁺-Translocating Membrane-Bound Pyrophosphatase from Rhodospirillum rubrum Fuels Escherichia coli Cells via an Alternative Pathway for Energy Generation

Microorganisms. 2023 Jan 23;11(2):294. doi: 10.3390/microorganisms11020294.

Authors

Evgeniya A Malykh¹, Liubov I Golubeva¹, Ekaterina S Kovaleva¹, Mikhail S Shupletsov^{1

2}, Elena V Rodina³, Sergey V Mashko^{1

4}, Nataliya V Stoynova¹

Affiliations

¹ Ajinomoto-Genetika Research Institute, 117545 Moscow, Russia.
² Computational Mathematics and Cybernetics Department, Lomonosov Moscow State University, 119991 Moscow, Russia.
³ Chemistry Department, Lomonosov Moscow State University, 119991 Moscow, Russia.
⁴ Biological Department, Lomonosov Moscow State University, 119991 Moscow, Russia.

Abstract

Inorganic pyrophosphatases (PPases) catalyze an essential reaction, namely, the hydrolysis of PP_i, which is formed in large quantities as a side product of numerous cellular reactions. In the majority of living species, PP_i hydrolysis is carried out by soluble cytoplasmic PPase (S-PPases) with the released energy dissipated in the form of heat. In Rhodospirillum rubrum, part of this energy can be conserved by proton-pumping pyrophosphatase (H⁺-PPase^Rru) in the form of a proton electrochemical gradient for further ATP synthesis. Here, the codon-harmonized gene hppa^Rru encoding H⁺-PPase^Rru was expressed in the Escherichia coli chromosome. We demonstrate, for the first time, that H⁺-PPase^Rru complements the essential native S-PPase in E. coli cells. ¹³C-MFA confirmed that replacing native PPase to H⁺-PPase^Rru leads to the re-distribution of carbon fluxes; a statistically significant 36% decrease in tricarboxylic acid (TCA) cycle fluxes was found compared with wild-type E. coli MG1655. Such a flux re-distribution can indicate the presence of an additional method for energy generation (e.g., ATP), which can be useful for the microbiological production of a number of compounds, the biosynthesis of which requires the consumption of ATP.

Keywords: 13C-MFA; ATP; H+-translocating pyrophosphatase; Rhodospirillum rubrum; inorganic pyrophosphatase; metabolic engineering; pyrophosphate hydrolysis; soluble pyrophosphatase; synthetic biology.

Grants and funding

This research received no external funding.