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A water-forming NADH oxidase from Lactobacillus pentosus suitable for the regeneration of synthetic biomimetic cofactors.

ABSTRACT: The cell-free biocatalytic production of fine chemicals by oxidoreductases has continuously grown over the past years. Since especially dehydrogenases depend on the stoichiometric use of nicotinamide pyridine cofactors, an integrated efficient recycling system is crucial to allow process operation under economic conditions. Lately, the variety of cofactors for biocatalysis was broadened by the utilization of totally synthetic and cheap biomimetics. Though, to date the regeneration has been limited to chemical or electrochemical methods. Here, we report an enzymatic recycling by the flavoprotein NADH-oxidase from Lactobacillus pentosus (LpNox). Since this enzyme has not been described before, we first characterized it in regard to its optimal reaction parameters. We found that the heterologously overexpressed enzyme only contained 13% FAD. In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics. Apart from the enzymatic recycling, which gives water as a by-product by transferring four electrons onto oxygen, unbound FAD can also catalyze the oxidation of biomimetic cofactors. Here a two electron process takes place yielding H2O2 instead. The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors. With LpNox and FAD, two recycling strategies for biomimetic cofactors are described with either water or hydrogen peroxide as by-product.

SUBMITTER: Nowak C

PROVIDER: S-EPMC4584968 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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A water-forming NADH oxidase from Lactobacillus pentosus suitable for the regeneration of synthetic biomimetic cofactors.

Nowak Claudia C Beer Barbara B Pick André A Roth Teresa T Lommes Petra P Sieber Volker V

Frontiers in microbiology 20150916

The cell-free biocatalytic production of fine chemicals by oxidoreductases has continuously grown over the past years. Since especially dehydrogenases depend on the stoichiometric use of nicotinamide pyridine cofactors, an integrated efficient recycling system is crucial to allow process operation under economic conditions. Lately, the variety of cofactors for biocatalysis was broadened by the utilization of totally synthetic and cheap biomimetics. Though, to date the regeneration has been limit ...[more]

PMID: 26441891

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Project description:BACKGROUND:Water-forming NADH oxidase can oxidize cytosolic NADH to NAD(+), thus relieving cytosolic NADH accumulation in Saccharomyces cerevisiae. Previous studies of the enzyme were conducted under aerobic conditions, as O2 is the recognized electron acceptor of the enzyme. In order to extend its use in industrial production and to study its effect on anaerobes, the effects of overexpression of this oxidase in S. cerevisiae BY4741 and Clostridium acetobutylicum 428 (Cac-428) under anaerobic conditions were evaluated. RESULTS:Glucose was exhausted in the NADH oxidase-overexpressing S. cerevisiae strain (Sce-NOX) culture after 26 h, while 43.51 ± 2.18 g/L residual glucose was left in the control strain (Sce-CON) culture at this time point. After 30 h of fermentation, the concentration of ethanol produced by Sce-NOX reached 36.28 ± 1.81 g/L, an increase of 56.38 % as compared to Sce-CON (23.20 ± 1.16 g/L), while the byproduct glycerol was remarkably decreased in the culture of Sce-NOX. In the case of the C. acetobutylicum strain (Cac-NOX) overexpressing NADH oxidase, glucose consumption, cell growth rate, and the production of acetone-butanol-ethanol (ABE) all decreased, while the concentrations of acetic acid and butyric acid increased as compared to the control strain (Cac-CON). During fermentation of Cac-CON and Cac-NOX in 100-mL screw-capped bottles, the concentrations of ABE increased with increasing headspace. Additionally, several alternative electron acceptors in C. acetobutylicum fermentation were tested. Nitroblue tetrazolium and 2,6-dichloroindophenol were lethiferous to both Cac-CON and Cac-NOX. Methylene blue could relieve the effect caused by the overexpression of the NADH oxidase on the metabolic network of C. acetobutylicum strains, while cytochrome c aggravated the effect. CONCLUSIONS:The water-forming NADH oxidase could regulate the metabolism of both the S. cerevisiae and the C. acetobutylicum strains in anaerobic conditions. Thus, the recombinant S. cerevisiae strain might be useful in industrial production. Besides the recognized electron acceptor O2, methylene blue and/or the structural analogs may be the alternative elector acceptor of the NADH oxidase in anaerobic conditions.

Project description:Redox homeostasis is fundamental to the maintenance of metabolism. Redox imbalance can cause oxidative stress, which affects metabolism and growth. Water-forming NADH oxidase regulates the redox balance by oxidizing cytosolic NADH to NAD(+), which relieves cytosolic NADH accumulation through rapid glucose consumption in Saccharomyces cerevisiae, thus decreasing the production of the by product glycerol in industrial ethanol production. Here, we studied the effects of overexpression of a water-forming NADH oxidase from Lactococcus lactis on the stress response of S. cerevisiae in aerobic batch fermentation, and we constructed an interaction network of transcriptional regulation and metabolic networks to study the effects of and mechanisms underlying NADH oxidase regulation. The oxidase-overexpressing strain (NOX) showed increased glucose consumption, growth, and ethanol production, while glycerol production was remarkably lower. Glucose was exhausted by NOX at 26 h, while 18.92 ± 0.94 g/L residual glucose was left in the fermentation broth of the control strain (CON) at this time point. At 29.5 h, the ethanol concentration for NOX peaked at 35.25 ± 1.76 g/L, which was 14.37% higher than that for CON (30.82 ± 1.54 g/L). Gene expression involved in the synthesis of thiamine, which is associated with stress responses in various organisms, was increased in NOX. The transcription factor HAP4 was significantly upregulated in NOX at the late-exponential phase, indicating a diauxic shift in response to starvation. The apoptosis-inducing factor Nuc1 was downregulated while the transcription factor Sok2, which regulates the production of the small signaling molecule ammonia, was upregulated at the late-exponential phase, benefiting young cells on the rim. Reactive oxygen species production was decreased by 10% in NOX, supporting a decrease in apoptosis. The HOG pathway was not activated, although the osmotic stress was truly higher, indicating improved osmotolerance. Thus, the NADH oxidase can regulate the metabolism during aerobic fermentation in S. cerevisiae, thereby protecting cells against several stresses. Our findings indicate its suitability for use in industrial processes.

Dataset Information

A water-forming NADH oxidase from Lactobacillus pentosus suitable for the regeneration of synthetic biomimetic cofactors.

Publications

A water-forming NADH oxidase from Lactobacillus pentosus suitable for the regeneration of synthetic biomimetic cofactors.

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