Heterologous production of extreme alkaline thermostable NAD + -dependent formate dehydrogenase with wide-range pH activity from Myceliophthora thermophila

Altaş, Nilay; Aslan, Aşkın Sevinç; Karataş, Ersin; Chronopoulou, Evangelia; Labrou, Nikolaos E.; Bi̇nay, Barış

doi:10.1016/j.procbio.2017.06.017

Cited by 34 publications

(9 citation statements)

References 46 publications

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“…At pH 9–10, Pf LeuDH and Tb LeuDH can maintain more than 85% of their original activity ( Figure 4B ). They displayed extreme alkaline pH for the conversion of formate, which was consistent with the Mt FDH from Myceliophthora thermophile ( Altaş et al, 2017 ). However, there was a significant difference compared with the Bb FDH come from Burkholderia dolosa PC543 ( Alpdagtas et al, 2018 ).…”

Section: Resultssupporting

confidence: 76%

Expression of Novel L-Leucine Dehydrogenase and High-Level Production of L-Tert-Leucine Catalyzed by Engineered Escherichia coli

Jia

Xie

Yang

et al. 2021

Front. Bioeng. Biotechnol.

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Section: Resultssupporting

confidence: 76%

Expression of Novel L-Leucine Dehydrogenase and High-Level Production of L-Tert-Leucine Catalyzed by Engineered Escherichia coli

Jia

Xie

Yang

et al. 2021

Front. Bioeng. Biotechnol.

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“…While it is known that the thermal stability of bacterial and yeast enzymes is higher than that of plant FDHs (Sadykhov et al 2006), the thermostability of GhFDH1 is higher than the FDHs isolated from some extremophilic (Altaş et al 2017) microorganisms (Choe et al 2014;Altaş et al 2017;Ding et al 2011) (Table 4). When compared with previously reported plant FDHs, and taking into consideration the T m values obtained from the residual activity and DSC measurements, it is considered that GhFDH1 isolated from cotton, which is a hot climate plant, has a higher thermostability (53.3 °C) among plant FDHs.…”

Section: Thermal Stability Analysis Of Ghfdhmentioning

confidence: 99%

Characterization of a novel thermotolerant NAD+-dependent formate dehydrogenase from hot climate plant cotton (Gossypium hirsutum L.)

Kurt-Gür

Ordu

2018

3 Biotech

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NAD-dependent formate dehydrogenases (FDH, EC 1.2.1.2), providing energy to the cell in methylotrophic microorganisms, are stress proteins in higher plants and the level of FDH expression increases under several abiotic and biotic stress conditions. They are biotechnologically important enzymes in NAD(P)H regeneration as well as CO reduction. Here, the truncated form of the cDNA was cloned into pQE-2 vector, and overexpressed in DH5α-T1 cells. Recombinant GhFDH1 was purified 26.3-fold with a yield of 87.3%. Optimum activity was observed at pH 7.0, when substrate is formate. Kinetic analyses suggest that GhFDH1 has considerably high affinity to formate (0.76 ± 0.07 mM) and NAD (0.06 ± 0.01 mM). At the same time, the affinity (1.98 ± 0.4 mM) and catalytic efficiency (0.0041) values of the enzyme for NADP show that GhFDH1 is a valuable enzyme for protein engineering studies that is trying to change the coenzyme preference from NAD to NADP which has a much higher cost than that of NAD. Improving the NADP specificity is important for NADPH regeneration which is an important coenzyme used in many biotechnological production processes. The value of GhFDH1 is 53.3 °C and the highest enzyme activity is measured at 30 °C with a half-life of 61 h. Whilst further improvements are still required, the obtained results show that GhFDH1 is a promising enzyme for NAD(P)H regeneration for its prominent thermostability and NADP specificity.

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“…Optimal growth was observed where extracellular 1.5 < pH < 4.0, resulting in the maintenance of an internal pH 6.55 ± 0.05. at the pH values mirroring cytosolic acidification and therefore could be vulnerable to formic acid-induced pH stress. Although very little is known about the pH dependence of FDH in acidophiles, similar pH-dependent activities have been reported in several neutralophiles (Schauer and Ferry, 1982;Axley et al, 1990), anaerobes (Liu and Mortenson, 1984), and fungi (Altaş et al, 2017). In addition, the presence of several acid-labile SH groups have been attributed to the circumneutral pH optima (pH 6.5-7.5) displayed by the NAD + -dependent FDH of Methylosinus trichosporium OB3b (Trotsenko and Murrell, 2008).…”

Section: Resultsmentioning

confidence: 58%

Growth on Formic Acid Is Dependent on Intracellular pH Homeostasis for the Thermoacidophilic Methanotroph Methylacidiphilum sp. RTK17.1

et al. 2021

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Members of the genus Methylacidiphilum, a clade of metabolically flexible thermoacidophilic methanotrophs from the phylum Verrucomicrobia, can utilize a variety of substrates including methane, methanol, and hydrogen for growth. However, despite sequentially oxidizing methane to carbon dioxide via methanol and formate intermediates, growth on formate as the only source of reducing equivalents (i.e., NADH) has not yet been demonstrated. In many acidophiles, the inability to grow on organic acids has presumed that diffusion of the protonated form (e.g., formic acid) into the cell is accompanied by deprotonation prompting cytosolic acidification, which leads to the denaturation of vital proteins and the collapse of the proton motive force. In this work, we used a combination of biochemical, physiological, chemostat, and transcriptomic approaches to demonstrate that Methylacidiphilum sp. RTK17.1 can utilize formate as a substrate when cells are able to maintain pH homeostasis. Our findings show that Methylacidiphilum sp. RTK17.1 grows optimally with a circumneutral intracellular pH (pH 6.52 ± 0.04) across an extracellular range of pH 1.5–3.0. In batch experiments, formic acid addition resulted in no observable cell growth and cell death due to acidification of the cytosol. Nevertheless, stable growth on formic acid as the only source of energy was demonstrated in continuous chemostat cultures (D = 0.0052 h−1, td = 133 h). During growth on formic acid, biomass yields remained nearly identical to methanol-grown chemostat cultures when normalized per mole electron equivalent. Transcriptome analysis revealed the key genes associated with stress response: methane, methanol, and formate metabolism were differentially expressed in response to growth on formic acid. Collectively, these results show formic acid represents a utilizable source of energy/carbon to the acidophilic methanotrophs within geothermal environments. Findings expand the known metabolic flexibility of verrucomicrobial methanotrophs to include organic acids and provide insight into potential survival strategies used by these species during methane starvation.

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Heterologous production of extreme alkaline thermostable NAD + -dependent formate dehydrogenase with wide-range pH activity from Myceliophthora thermophila

Cited by 34 publications

References 46 publications

Expression of Novel L-Leucine Dehydrogenase and High-Level Production of L-Tert-Leucine Catalyzed by Engineered Escherichia coli

Expression of Novel L-Leucine Dehydrogenase and High-Level Production of L-Tert-Leucine Catalyzed by Engineered Escherichia coli

Characterization of a novel thermotolerant NAD+-dependent formate dehydrogenase from hot climate plant cotton (Gossypium hirsutum L.)

Growth on Formic Acid Is Dependent on Intracellular pH Homeostasis for the Thermoacidophilic Methanotroph Methylacidiphilum sp. RTK17.1

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