{
  "abstract": "Engineering redox enzymes to utilize noncanonical coenzymes is critical for advancing in vitro enzymatic biocatalysis and in vivo bioorthogonal chemistry. However, engineered NAD(P)-dependent dehydrogenases had universally underperformed on small noncanonical coenzymes compared to their wild-type (WT) counterparts on natural NAD(P). Here we developed an Iterative (one-site) Mutagenesis and Evolutionary Strategy (IMES) to evolve a flavin-free, NAD-dependent glucose dehydrogenase (GDH) on nicotinamide mononucleotide (NMN + ). By integrating rational design, directed evolution, and a novel high-throughput screening method, we obtained the variant M3 (E44H/P191T/V187M). For the first time, an engineered dehydrogenase achieved catalytic efficiencies with NMN + that surpassed those of the WT enzyme with its natural coenzyme NAD + . Specifically, variant M3 exhibited a 3.2-fold higher specific activity and a 2.06-fold greater catalytic efficiency ( k cat / K m ) with NMN + than the WT GDH with NAD + . Furthermore, we established functional NMN-recycling cascades by coupling the NMNH-generating M3 variant with four NMNH-consuming redox enzymes, including flavin-free alcohol dehydrogenase and mannitol dehydrogenase, as well as two flavin-dependent enzymes (i.e., an oxidase and a transhydrogenase). This study establishes an NMN-based cascade redox system applicable to both in vitro biocatalysis and in vivo bioorthogonal chemistry. Replacing NAD with the smaller, lower-cost NMN coenzyme greatly reduced the economic burden of cofactor supply, paving the way for more efficient and cost-effective enzymatic biocatalysis.",
  "abstract_shown": true,
  "affiliations": [
    "State Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin, China, 300308",
    "in vitro Synthetic Biology Center, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin, China, 300308",
    "University of Chinese Academy of Sciences, 19 A Yuquan Road, Beijing, China, 100049",
    "State Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology",
    "Shanghai Jiao Tong University",
    "National Center of Technology Innovation for Synthetic Biology, 32 West 7th Avenue, Tianjin, China, 300308"
  ],
  "article_type": null,
  "arxiv": null,
  "author_total": 4,
  "authors": [
    {
      "affs": [
        0,
        1,
        2
      ],
      "name": "Zhihua Nie"
    },
    {
      "affs": [
        0,
        1,
        3,
        4
      ],
      "name": "Zhan Song"
    },
    {
      "affs": [
        0,
        1,
        2
      ],
      "name": "Zhenyu Zhai",
      "orcid": "0009-0000-3250-4252"
    },
    {
      "affs": [
        0,
        1,
        2,
        5
      ],
      "name": "Yi-Heng P. Job Zhang",
      "orcid": "0000-0002-4010-2250"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/0m77vre3v0xrs.bib",
  "cited_by": 0,
  "doi": "10.26434/chemrxiv.15009579/v1",
  "fulltext_url": null,
  "id": "0m77vre3v0xrs",
  "issns": [],
  "landing_url": "https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009579/v1",
  "language": null,
  "licence": "CC-BY-NC-ND",
  "licence_label": "CC BY-NC-ND",
  "licence_source": "journal",
  "licence_url": "https://creativecommons.org/licenses/by-nc-nd/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "openalex",
      "1312|Enzyme Catalysis and Immobilization"
    ]
  ],
  "openalex": "W7214763390",
  "pdf_url": "https://chemrxiv.org/doi/pdf/10.26434/chemrxiv.15009579/v1",
  "pmcid": null,
  "pmid": null,
  "published": "2026-09-28",
  "publisher": "American Chemical Society (ACS)",
  "ris": "https://research.draconitic.com/paper/0m77vre3v0xrs.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:17:21Z",
      "native_id": "10.26434/chemrxiv.15009579",
      "source": "chemrxiv",
      "url": "https://chemrxiv.org/doi/full/10.26434/chemrxiv.15009579/v1"
    }
  ],
  "subjects": [
    {
      "field": "biochemistry",
      "origin": "openalex:1312|Enzyme Catalysis and Immobilization",
      "subfield": "molecular-biology"
    }
  ],
  "title": "Glucose Dehydrogenase Engineered for the Noncanonical Coenzyme NMN Outperforms Its Natural NAD-Dependent Activity",
  "title_text": "Glucose Dehydrogenase Engineered for the Noncanonical Coenzyme NMN Outperforms Its Natural NAD-Dependent Activity",
  "type": "preprint",
  "updated_at": "2026-10-30T12:17:21Z",
  "url": "https://research.draconitic.com/paper/0m77vre3v0xrs",
  "venue": "ChemRxiv",
  "versions": []
}