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Simple Summary: Pyruvate decarboxylase (PDC) is a key enzyme involved in ethanol fermentation, a process for the production of biofuels. Thermostable and oxygen-stable PDC activity is highly desirable for biotechnological applications at high temperatures. The enzymes from the thermoacidophiles Saccharolobus (formerly Sulfolobus) solfataricus (Ss, Topt = 80 °C) and Sulfolobus acidocaldarius (Sa, Topt = 80 °C) were purified and characterized, and their biophysical and biochemical properties were determined comparatively. The purified enzymes were CoA-dependent and thermostable. There was no loss of activity in the presence of oxygen. In conclusion, both thermostable SsPDC and SaPDC catalyze the CoA-dependent production of acetaldehyde from pyruvate in the presence of oxygen. Pyruvate decarboxylase (PDC) is a key enzyme involved in ethanol fermentation, and it catalyzes the decarboxylation of pyruvate to acetaldehyde and CO2. Bifunctional PORs/PDCs that also have additional pyruvate:ferredoxin oxidoreductase (POR) activity are found in hyperthermophiles, and they are mostly oxygen-sensitive and CoA-dependent. Thermostable and oxygen-stable PDC activity is highly desirable for biotechnological applications. The enzymes from the thermoacidophiles Saccharolobus (formerly Sulfolobus) solfataricus (Ss, Topt = 80 °C) and Sulfolobus acidocaldarius (Sa, Topt = 80 °C) were purified and characterized, and their biophysical and biochemical properties were determined comparatively. Both enzymes were shown to be heterodimeric, and their two subunits were determined by SDS-PAGE to be 37 ± 3 kDa and 65 ± 2 kDa, respectively. The purified enzymes from S. solfataricus and S. acidocaldarius showed both PDC and POR activities which were CoA-dependent, and they were thermostable with half-life times of 2.9 ± 1 and 1.1 ± 1 h at 80 °C, respectively. There was no loss of activity in the presence of oxygen. Optimal pH values for their PDC and POR activity were determined to be 7.9 and 8.6, respectively. In conclusion, both thermostable SsPOR/PDC and SaPOR/PDC catalyze the CoA-dependent production of acetaldehyde from pyruvate in the presence of oxygen. [ABSTRACT FROM AUTHOR]

[Display omitted] • pflA knockout enhanced ethanol tolerance and production in T. aotearoense SCUT27/ Δldh. • The ethanol/acetate ratio increased 2.16-fold when compared to the parental strain. • Oxidative decarboxylation of pyruvate via Pfor is favorable over Pfl for ethanol production. • Immobilized-cell fermentation was employed to further improve the ethanol production. • The highest ethanol production (45.01 g/L) in T. aotearoense SCUT27 was obtained. Thermophilic anaerobic bacteria have received increasing interest as a promising bioprocessing platform for bioethanol production due to their broad substrate range and higher operating temperature. However, ethanol concentration is still lower than the commercial requirements on account of the high by-product formation and low ethanol tolerance. Our previous transcriptome analysis showed the gene expression level of pyruvate formate lyase-activating protein (PflA) was significantly down-regulated when Thermoanaerobacterium aotearoense SCUT27/ Δldh was stimulated by various concentrations of ethanol. Based on this, we constructed the mutant strain T. aotearoense SCUT27/ Δldh / ΔpflA , which not only possesses higher ethanol tolerance and production but also has enhanced ethanol/acetate ratio that is important for economical ethanol production. Compared to T. aotearoense SCUT27/ Δldh , the mutant strain could resist to 5% (v/v) exogenous ethanol and produce 49 % more ethanol (38.82 g/L vs. 26.05 g/L) in 5-L bioreactor. In addition, the final concentration of ethanol reached 45.01 g/L by immobilized cell fermentation, which is the highest value obtained in T. aotearoense SCUT27. Simultaneously, the ethanol/acetate ratio of the mutant strain achieved 6.82, which was 2.16-fold higher than that of the parental strain. [ABSTRACT FROM AUTHOR]

A previously published genome‐scale metabolic model namely iFF708 was modified to depict the metabolic flux distribution within Saccharomyces cerevisiae grown under a redox potential–controlled very high‐gravity condition. The following modifications were made: electron transport chain (ETC) and oxidative phosphorylation, proton gradient and ATP transportation, and malate–aspartate shuttle. With these modifications, this model could describe the experimental data collected from the above‐mentioned ethanol fermentation. As a result, the simulation unveiled that the P/O ratio is critical under microaerobic conditions and the malate–aspartate shuttle is inactivated due to the shortage of electron transport across mitochondria. In other words, the limited supply of oxygen suppresses the functionality of oxidative phosphorylation, tricarboxylic acid (TCA) cycle, and ETC. In terms of the glycolytic pathway, fluxes coming from glucose‐6‐phosphate and pyruvate nodes are insensitive to the changes of fermentation redox potential. As the initial glucose concentration is greater than 250 g/L, the interactive effect between the initial glucose concentration and redox potential level becomes noticeable. [ABSTRACT FROM AUTHOR]

Acetobacter pasteurianus, an obligately oxidative bacterium, is the first organism shown to utilize pyruvate decarboxylase (PDC) as a central enzyme for oxidative metabolism. In plants, yeast, and other bacteria, PDC functions solely as part of the fermentative ethanol pathway. During the growth of A. pasteurianus on lactic acid, the central intermediate pyruvate is cleaved to acetaldehyde and CO2 by PDC. Acetaldehyde is subsequently oxidized to its final product, acetic acid. The presence of the PDC enzyme in A. pasteurianus was confirmed by zymograms stained for acetaldehyde production, enzyme assays using alcohol dehydrogenase as the coupling enzyme, and by cloning and characterization of the pdc operon. A. pasteurianus pdc was also expressed in recombinant Escherichia coli. The level of PDC activity was regulated in response to growth substrate, highest with lactic acid and absent with mannitol. The translated PDC sequence (548 amino acids) was most similar to that of Zymomonas mobilis, an obligately fermentative bacterium. A second operon (aldA) was also found which is transcribed divergently from pdc. This operon encodes a putative aldehyde dehydrogenase (ALD2; 357 amino acids) related to class III alcohol dehydrogenases and most similar to glutathione-dependent formaldehyde dehydrogenases from α-Proteobacteria and Anabeana azollae. [ABSTRACT FROM AUTHOR]

Background: Hydroponic systems are a convenient platform for plant cultivation when treatments are applied to the roots because they provide precise control of the composition of the growth medium, ensuring the availability of different compounds. A problem arises when axenic conditions are needed but the treatment of choice (exogenous organic acids or sugars) promote the growth of unwanted microorganisms. Moreover, axenic conditions are usually applied in liquid and semi-liquid growing systems, where oxygen availability can be compromised, if no aeration is provided. Results: The driver for the development of this hydroponic system was the application of the organic acid pyruvate to the roots of plants grown under aerated axenic conditions. No contamination was detected in the nutrient solution, even after the addition of pyruvate. The system was validated in pea plants treated with either pyruvate or herbicides inhibiting amino acid biosynthesis. The effects on ethanol fermentation were compared by analysing the enzymatic activity, protein content and transcriptional levels in plants treated with either pyruvate or herbicides. Conclusions: The developed system enables the study of the exogenous application of organic acids in the nutrient solution under axenic conditions and without oxygen limitation. This system allows the study of the effect of any type of treatments applied to roots under aerated axenic hydroponic systems at physiological and molecular levels. The role of pyruvate in the induction of fermentation by herbicides cannot be simply explained by an increase in substrate availability. [ABSTRACT FROM AUTHOR]