Enzymatic synthesis of high-titer nicotinamide mononucleotide with a new nicotinamide riboside kinase and an efficient ATP regeneration system

Abstract

Background

β-Nicotinamide mononucleotide (NMN) is the direct precursor of nicotinamide coenzymes such as NAD⁺ and NADP⁺, which are widely applied in industrial biocatalysis especially involving cofactor-dependent oxidoreductases. Moreover, NMN is a promising candidate for medical uses since it is considered to be beneficial for improving health of aged people who usually suffer from an insufficient level of NAD⁺. To date, various methods have been developed for the synthesis of NMN. Chemical phosphorylation of nicotinamide riboside (NR) to NMN depends on excessive phosphine oxychloride and delicate temperature control, while fermentation of NMN is limited by low product titers, making it unsuitable for industrial-scale NMN production. As a result, the more efficient synthesis process of NMN is still challenging.

Aim

This work attempted to construct an eco-friendly and cost-effective biocatalytic process for transforming the chemically synthesized NR into the highly value-added NMN.

Results

A new nicotinamide riboside kinase (Klm-NRK) was identified from Kluyveromyces marxianus. The specific activity of purified Klm-NRK was 7.9 U·mg^–1 protein, ranking the highest record among the reported NRKs. The optimal pH of Klm-NRK was 7.0 in potassium phosphate buffer. The purified Klm-NRK retained a half activity after 7.29 h at 50 °C. The catalytic efficiencies (k_cat/K_M) toward ATP and nicotinamide riboside (NR) were 57.4 s⁻¹·mM⁻¹ and 84.4 s⁻¹·mM⁻¹, respectively. In the presence of an external ATP regeneration system (AcK/AcP), as much as 100 g·L^–1 of NR could be completely phosphorylated to NMN in 8 h with Klm-NRK, achieving a molar isolation yield of 84.2% and a space–time yield of 281 g·L⁻¹·day⁻¹. These inspiring results indicated that Klm-NRK is a promising biocatalyst which provides an efficient approach for the bio-manufacturing of NMN in a high titer.

Keywords

Enzymatic phosphorylation / Nicotinamide riboside / Nicotinamide riboside kinase / β-Nicotinamide mononucleotide (NMN) / ATP regeneration system

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Funding

National Key R & D Program of China(2019YFA09005000)

National Natural Science Foundation of China(21776085)

Fundamental Research Funds for the Central Universities(22221818014)