{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T05:19:29Z","timestamp":1787721569443,"version":"build-2784847793"},"reference-count":40,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T00:00:00Z","timestamp":1787529600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004479","name":"Jiangxi Provincial Natural Science Foundation","doi-asserted-by":"crossref","award":["20262BAC250015"],"award-info":[{"award-number":["20262BAC250015"]}],"id":[{"id":"10.13039\/501100004479","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32160219"],"award-info":[{"award-number":["32160219"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"award":["32160219"],"award-info":[{"award-number":["32160219"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>\u03b2-Nicotinamide mononucleotide (NMN) is an important intermediate in nicotinamide adenine dinucleotide (NAD+) metabolism and has attracted increasing interest as a bioactive compound and biomanufacturing product. In this study, Pseudomonas putida KT2440 was engineered for NMN production using a \u201cblock\u2013enhance\u2013transport\u201d strategy. Deletion of nicB impaired nicotinic acid degradation and resulted in the accumulation of 66.2 \u03bcM nicotinic acid in cell extracts, whereas additional deletion of the putative NMN-consuming genes pncC and ushA did not lead to detectable NMN accumulation. Coexpression of endogenous pncB and engineered Francisella tularensis nadE* enabled low-level NMN formation through a Preiss\u2013Handler pathway-based route. By contrast, overexpression of endogenous nadA, nadB, and nadC strengthened precursor supply through the NAD+ de novo biosynthetic pathway and resulted in approximately 0.17 mM NMN in cell extracts. Chromosomal integration of an engineered Salmonella enterica pnuC* transporter cassette was associated with pronounced extracellular NMN accumulation. Additional overexpression of genes involved in downstream NAD+ metabolism or phosphoribosyl pyrophosphate supply did not considerably improve production, possibly because of metabolic competition or expression burden. The best-performing strain, LW10, produced 1.28 mM extracellular NMN, corresponding to approximately 0.43 g\/L, after 96 h of shake-flask cultivation in basal salt medium containing glucose and L-aspartate. These results suggest the feasibility of NMN biosynthesis in engineered P. putida KT2440 and highlight the importance of balancing precursor supply, competing reactions, and product transport. Thus, P. putida KT2440 represents an alternative chassis for further pathway balancing and process optimization toward fermentative NMN production.<\/jats:p>","DOI":"10.3390\/microorganisms14091877","type":"journal-article","created":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T11:47:19Z","timestamp":1787572039000},"page":"1877","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Metabolic Engineering of Pseudomonas putida KT2440 for \u03b2-Nicotinamide Mononucleotide Biosynthesis from Glucose and Aspartate"],"prefix":"10.3390","volume":"14","author":[{"given":"Luna","family":"Gao","sequence":"first","affiliation":[{"name":"Jiangxi Engineering Laboratory for the Development and Utilization of Agricultural Microbial Resources, College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang 330045, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lin","family":"Wei","sequence":"additional","affiliation":[{"name":"Jiangxi Engineering Laboratory for the Development and Utilization of Agricultural Microbial Resources, College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang 330045, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Siqi","family":"Wang","sequence":"additional","affiliation":[{"name":"Jiangxi Engineering Laboratory for the Development and Utilization of Agricultural Microbial Resources, College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang 330045, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun","family":"Li","sequence":"additional","affiliation":[{"name":"College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang 330045, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingli","family":"Liu","sequence":"additional","affiliation":[{"name":"Jiangxi Engineering Laboratory for the Development and Utilization of Agricultural Microbial Resources, College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang 330045, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhao","family":"Guo","sequence":"additional","affiliation":[{"name":"Jiangxi Engineering Laboratory for the Development and Utilization of Agricultural Microbial Resources, College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang 330045, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhi-Min","family":"Li","sequence":"additional","affiliation":[{"name":"College of Chemistry and Materials, Jiangxi Agricultural University, Nanchang 330045, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7036-9282","authenticated-orcid":false,"given":"Zhimin","family":"Li","sequence":"additional","affiliation":[{"name":"Jiangxi Engineering Laboratory for the Development and Utilization of Agricultural Microbial Resources, College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang 330045, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Navas, L.E., and Carnero, A. (2022). Nicotinamide Adenine Dinucleotide (NAD) Metabolism as a Relevant Target in Cancer. Cells, 11.","DOI":"10.3390\/cells11172627"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"4985726","DOI":"10.1155\/2023\/4985726","article-title":"NAD+-Consuming Enzymes in Stem Cell Homeostasis","volume":"2023","author":"Ji","year":"2023","journal-title":"Oxid. Med. Cell. Longev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1416","DOI":"10.1016\/j.advnut.2023.08.008","article-title":"The Safety and Antiaging Effects of Nicotinamide Mononucleotide in Human Clinical Trials: An Update","volume":"14","author":"Song","year":"2023","journal-title":"Adv. Nutr."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"4759","DOI":"10.1007\/s00253-023-12612-2","article-title":"Technology and functional insights into the nicotinamide mononucleotide for human health","volume":"107","author":"Liu","year":"2023","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2151","DOI":"10.2174\/1385272827666230201103848","article-title":"Recent Advances in the Chemical Synthesis of \u03b2-Nicotinamide Mononucleotide","volume":"26","author":"Ming","year":"2022","journal-title":"Curr. Org. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1007\/s10522-025-10270-7","article-title":"Biological properties, synthetic pathways and anti-aging mechanisms of nicotinamide mononucleotide (NMN): Research progress and challenges","volume":"26","author":"Wang","year":"2025","journal-title":"Biogerontology"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"e2300748","DOI":"10.1002\/biot.202300748","article-title":"Improvement of an enzymatic cascade synthesis of nicotinamide mononucleotide via protein engineering and reaction-process reinforcement","volume":"19","author":"Peng","year":"2024","journal-title":"Biotechnol. J."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"110122","DOI":"10.1016\/j.enzmictec.2022.110122","article-title":"An artificial multi-enzyme cascade biocatalysis for biomanufacturing of nicotinamide mononucleotide from starch and nicotinamide in one-pot","volume":"162","author":"Li","year":"2023","journal-title":"Enzym. Microb. Technol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1186\/s40643-022-00514-6","article-title":"Enzymatic synthesis of high-titer nicotinamide mononucleotide with a new nicotinamide riboside kinase and an efficient ATP regeneration system","volume":"9","author":"Qian","year":"2022","journal-title":"Bioresour. Bioprocess."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"109683","DOI":"10.1016\/j.bioorg.2026.109683","article-title":"Green manufacturing synthesis of nicotinamide mononucleotide: Pathways, catalysts, and prospects","volume":"174","author":"Peng","year":"2026","journal-title":"Bioorganic Chem."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1352","DOI":"10.1021\/acssynbio.5c00038","article-title":"Microbial Production of Nicotinamide Mononucleotide: Key Enzymes Discovery, Host Cells Selection, and Pathways Design and Optimization","volume":"14","author":"Xing","year":"2025","journal-title":"ACS Synth. Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2979","DOI":"10.1021\/acssynbio.2c00100","article-title":"Systematic Engineering of Escherichia coli for Efficient Production of Nicotinamide Mononucleotide from Nicotinamide","volume":"11","author":"Huang","year":"2022","journal-title":"ACS Synth. Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3055","DOI":"10.1021\/acssynbio.1c00333","article-title":"Biosynthesis of a Therapeutically Important Nicotinamide Mononucleotide through a Phosphoribosyl Pyrophosphate Synthetase 1 and 2 Engineered Strain of Escherichia coli","volume":"10","author":"Maharjan","year":"2021","journal-title":"ACS Synth. Biol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1016\/j.ymben.2020.11.008","article-title":"Metabolic design for selective production of nicotinamide mononucleotide from glucose and nicotinamide","volume":"65","author":"Shoji","year":"2021","journal-title":"Metab. Eng."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"11200","DOI":"10.1021\/acs.jafc.5c03017","article-title":"Improving Biosynthesis Efficiency of Nicotinamide Mononucleotide by ATP Recycling Engineering and Condition Optimization","volume":"73","author":"Tang","year":"2025","journal-title":"J. Agric. Food Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"150","DOI":"10.1186\/s12934-020-01415-z","article-title":"Metabolic engineering of Escherichia coli for optimized biosynthesis of nicotinamide mononucleotide, a noncanonical redox cofactor","volume":"19","author":"Black","year":"2020","journal-title":"Microb. Cell Fact."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"3083","DOI":"10.1073\/pnas.0811718106","article-title":"Nicotinamide mononucleotide synthetase is the key enzyme for an alternative route of NAD biosynthesis in Francisella tularensis","volume":"106","author":"Sorci","year":"2009","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2425","DOI":"10.1021\/acssynbio.4c00115","article-title":"Exploring the De Novo NMN Biosynthesis as an Alternative Pathway to Enhance NMN Production","volume":"13","author":"Wang","year":"2024","journal-title":"ACS Synth. Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1058","DOI":"10.1080\/07388551.2024.2433993","article-title":"Biological production of nicotinamide mononucleotide: A review","volume":"45","author":"Cabulong","year":"2025","journal-title":"Crit. Rev. Biotechnol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e70048","DOI":"10.1111\/1751-7915.70048","article-title":"De novo biosynthesis and nicotinamide biotransformation of nicotinamide mononucleotide by engineered yeast cells","volume":"17","author":"Ren","year":"2024","journal-title":"Microb. Biotechnol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Tan, Z., Yang, Y., Wu, Y., Yan, J., Zhang, B., Hou, Y., and Jia, S. (2024). Biosynthesis of beta-nicotinamide mononucleotide from glucose via a new pathway in Bacillus subtilis. Front. Microbiol., 15.","DOI":"10.3389\/fmicb.2024.1405736"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"103025","DOI":"10.1016\/j.copbio.2023.103025","article-title":"Pseudomonas putida as a synthetic biology chassis and a metabolic engineering platform","volume":"85","year":"2024","journal-title":"Curr. Opin. Biotechnol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"e0013624","DOI":"10.1128\/jb.00136-24","article-title":"Pseudomonas putida KT2440: The long journey of a soil-dweller to become a synthetic biology chassis","volume":"206","author":"Nikel","year":"2024","journal-title":"J. Bacteriol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"142","DOI":"10.1016\/j.ymben.2018.05.005","article-title":"Pseudomonas putida as a functional chassis for industrial biocatalysis: From native biochemistry to trans-metabolism","volume":"50","author":"Nikel","year":"2018","journal-title":"Metab. Eng."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"D1558","DOI":"10.1093\/nar\/gkac1059","article-title":"SEVA 4.0: An update of the Standard European Vector Architecture database for advanced analysis and programming of bacterial phenotypes","volume":"51","author":"Fraile","year":"2023","journal-title":"Nucleic Acids Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"434","DOI":"10.1021\/acssynbio.6b00025","article-title":"Metabolic Engineering of Pseudomonas putida KT2440 for Complete Mineralization of Methyl Parathion and gamma-Hexachlorocyclohexane","volume":"5","author":"Gong","year":"2016","journal-title":"ACS Synth. Biol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1016\/0378-1119(94)90324-7","article-title":"Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: Selection of defined deletions in the chromosome of Corynebacterium glutamicum","volume":"145","author":"Schafer","year":"1994","journal-title":"Gene"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"3403","DOI":"10.1111\/1462-2920.13230","article-title":"The revisited genome of Pseudomonas putida KT2440 enlightens its value as a robust metabolic chassis","volume":"18","author":"Belda","year":"2016","journal-title":"Environ. Microbiol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1016\/j.procbio.2024.02.004","article-title":"A native phosphoribosyltransferase, PncB, is the key NMN synthase in Bacillus subtilis","volume":"139","author":"Zhang","year":"2024","journal-title":"Process Biochem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"450","DOI":"10.1002\/jsfa.12253","article-title":"Enhancing the biosynthesis of nicotinamide mononucleotide in Lactococcus lactis by heterologous expression of FtnadE","volume":"103","author":"Kong","year":"2023","journal-title":"J. Sci. Food Agric."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"138","DOI":"10.1007\/s43393-023-00172-5","article-title":"Enhancing nicotinamide mononucleotide production from glucose in Escherichia coli by genetic engineering","volume":"4","author":"Huang","year":"2024","journal-title":"Syst. Microbiol. Biomanufacturing"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"28360","DOI":"10.1021\/acs.jafc.4c10205","article-title":"High-Level Production of Nicotinamide Mononucleotide by Engineered Escherichia Coli","volume":"72","author":"Gan","year":"2024","journal-title":"J. Agric. Food Chem."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Bi, T., Wu, T., Yang, L., Xu, Y., and Mu, X. (2023). Comprehensive Transformation of Escherichia coli for Nicotinamide Mononucleotide Production. Catalysts, 13.","DOI":"10.3390\/catal13050815"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"959","DOI":"10.1016\/j.synbio.2025.04.005","article-title":"Network of nicotinamide mononucleotide biosynthesis: Substrate tracking, rate-limiting enzyme, and regulatory strategy","volume":"10","author":"Zhang","year":"2025","journal-title":"Synth. Syst. Biotechnol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1007\/s11274-022-03274-9","article-title":"Evaluation of extracellular electron transfer in Pseudomonas aeruginosa by co-expression of intermediate genes in NAD synthetase production pathway","volume":"38","author":"Ajunwa","year":"2022","journal-title":"World J. Microbiol. Biotechnol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"264","DOI":"10.1016\/j.procbio.2023.07.002","article-title":"Enhanced production of nicotinamide mononucleotide by high cell density culture of engineered Escherichia coli","volume":"131","author":"Maharjan","year":"2023","journal-title":"Process Biochem."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"129911","DOI":"10.1016\/j.biortech.2023.129911","article-title":"A holistic approach for process intensification of nicotinamide mononucleotide production via high cell density cultivation under exponential feeding strategy","volume":"390","author":"Kafle","year":"2023","journal-title":"Bioresour. Technol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"3451","DOI":"10.1021\/acssynbio.2c00350","article-title":"Yeast Cell Surface Engineering of a Nicotinamide Riboside Kinase for the Production of beta-Nicotinamide Mononucleotide via Whole-Cell Catalysis","volume":"11","author":"He","year":"2022","journal-title":"ACS Synth. Biol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"15218","DOI":"10.1021\/acssuschemeng.3c02833","article-title":"Streamlining Design, Engineering, and Applications of Nicotinamide Riboside Kinase for Sustainable Biosynthesis of Nicotinamide Mononucleotide in Flow","volume":"11","author":"Cheng","year":"2023","journal-title":"ACS Sustain. Chem. Eng."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"4735","DOI":"10.1002\/bit.27938","article-title":"Pseudomonas putida KT2440 endures temporary oxygen limitations","volume":"118","author":"Demling","year":"2021","journal-title":"Biotechnol. Bioeng."}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/9\/1877\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T04:26:07Z","timestamp":1787718367000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/9\/1877"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,24]]},"references-count":40,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["microorganisms14091877"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms14091877","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,24]]}}}