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. 2023 Apr 4:11:1159965.
doi: 10.3389/fbioe.2023.1159965. eCollection 2023.

Protein engineering of NADH pyrophosphatase for efficient biocatalytic production of reduced nicotinamide mononucleotide

Affiliations

Protein engineering of NADH pyrophosphatase for efficient biocatalytic production of reduced nicotinamide mononucleotide

Ye Liu et al. Front Bioeng Biotechnol. .

Abstract

Introduction: NADH pyrophosphatase, a hydrolase catalyzing the phosphate bond of NADH to reduced nicotinamide mononucleotide, has potential applications in the food, cosmetic and pharmaceutical industry. Methods: Here, we investigated the effects of vector screening, promoter and RBS strategies on NADH pyrophosphatase expression and protein engineering on its enzymatic activity and thermal stability. Results: In this study, we describe a NADH pyrophosphatase derived from Escherichia coli (EcNudc). Strategies focusing on expression regulation including screening vectors, optimizing promoters and ribosome binding sites were utilized to enhance the productivity of EcNudc (1.8 U/mL). Moreover, protein engineering was adopted to further improve the catalytic properties of EcNudc, achieving 3.3-fold higher activity and 3.6-fold greater thermostability at 50°C. Furthermore, fermentation for the combined mutant R148A-H149E (EcNudc-M) production in a 7 L fermenter was implemented and the enzyme activity of EcNudc-M reached 33.0 U/mL. Finally, the EcNudc-M was applied in the catalysis of NADH with the highest NMNH yield of 16.65 g/L. Discussion: In conclusion, we constructed a commercially available genetically engineered strain with high activity and thermal stability of NADH pyrophosphatase, laying a broad foundation for the biocatalytic industrial production of NMNH and expand its application range.

Keywords: NADH pyrophosphatase; expression; protein engineering; reduced nicotinamide mononucleotide; semi-rational engineering.

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Conflict of interest statement

Authors YL, GM, MH, J-SS, and Z-HX are employed by Yixing Institute of Food and Biotechnology Co., Ltd. and Seragon Biosciences. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Biosynthesis of NMNH from NADH.
FIGURE 2
FIGURE 2
Screening of EcNudc expression vectors.
FIGURE 3
FIGURE 3
Adaptability modification of promoters and RBSs. (A) Adaptability modification of promoters. (B) Adaptability modification of RBSs with strengths.
FIGURE 4
FIGURE 4
Enzyme activity of the EcNudc and its mutants under the standard reaction condition (37°C, pH 8.0).
FIGURE 5
FIGURE 5
Site-directed saturation mutagenesis of EcNudc. (A) Relative activity with saturation mutations at the R148. (B) Relative activity with saturation mutations at the H149.
FIGURE 6
FIGURE 6
Structural analysis of the activity-related residues. (A) WT. (B) Mutant (R148A). (C) Mutant (H149E). (D) Mutant (EcNudc-M).
FIGURE 7
FIGURE 7
Thermal stability of wild type and mutants. (A) Thermal stability of wild type and mutants at 40°C. (B) Thermal stability of wild type and mutants at 50°C.
FIGURE 8
FIGURE 8
Hydrogen bonds formation between E149 and N151.
FIGURE 9
FIGURE 9
Fermentation curve of EcNudc-M and SDS-PAGE analysis of recombinant EcNudc expression. (A) Fermentation curve of EcNudc-M under constant rate replenishment strategy. (B) SDS-PAGE analysis of recombinant EcNudc expression in shake flask and fermenter level. M: protein standard marker; Lane 1: The supernatant of EcNudc-M in shake flask level; Lane 2: The supernatant of EcNudc-M in the fermenter level.
FIGURE 10
FIGURE 10
Exploration of catalytic process and biosynthesis of NMNH. (A) Relative yields of NMNH at different reaction temperatures. (B) Relative yields of NMNH at different reaction pH. (C) Biotransformation curve of NMNH at different substrate concentrations.

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References

    1. AbdelRaheim S. R., Cartwright J. L., Gasmi L., McLennan A. G. (2001). The NADH diphosphatase encoded by the Saccharomyces cerevisiae NPY1 nudix hydrolase gene is located in peroxisomes. Arch. Biochem. Biophys. 388 (1), 18–24. 10.1006/abbi.2000.2268 - DOI - PubMed
    1. Abdelraheim S. R., Spiller D. G., McLennan A. G. (2003). Mammalian NADH diphosphatases of the nudix family: Cloning and characterization of the human peroxisomal NUDT12 protein. Biochem. J. 374 (2), 329–335. 10.1042/bj20030441 - DOI - PMC - PubMed
    1. Abdelraheim S. R., Spiller D. G., McLennan A. G. (2017). Mouse Nudt13 is a mitochondrial nudix hydrolase with NAD(P)H pyrophosphohydrolase activity. Protein J. 36 (5), 425–432. 10.1007/s10930-017-9734-x - DOI - PMC - PubMed
    1. Ames B. N., Dubin D. T. (1960). The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid. J. Biol. Chem. 235, 769–775. 10.1016/S0021-9258(19)67936-6 - DOI - PubMed
    1. Bienert S., Waterhouse A., de Beer T. A., Tauriello G., Studer G., Bordoli L., et al. (2017). The SWISS-MODEL Repository-new features and functionality. Nucleic Acids Res. 45 (D1), D313–d319. 10.1093/nar/gkw1132 - DOI - PMC - PubMed

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