β-nicotinamide mononucleotide production in Vibrio natriegens: a preliminary study

Introduction

β-nicotinamide mononucleotide (NMN) is a precursor of NAD⁺, an essential coenzyme in various metabolic processes. NAD⁺ is involved in intracellular signaling pathways, regulating mitochondrial functions and other biochemical processes.¹ In living organisms, intracellular NAD⁺ levels are directly influenced by its precursor, NMN. Recent studies reported various potential medical applications of NMN, particularly in anti-aging, cognitive-enhancing, and treatment of severe chronic diseases such as Alzheimer’s and diabetes.^2,3

There are three major ways for NMN production: chemical synthesis, enzymatic methods and fermentation methods using genetically modified microbial strains.⁴ The chemical synthesis strategies offer the advantage of low production cost; however, the resulting isomeric compounds may exhibit toxicity or lack of biological activity. The enzymatic methods enhance selectivity and reduce toxicity, but the high cost of enzymes and phosphate sources such as ATP remain a major obstacle. Recent studies indicated that the fermentation approach using metabolically engineered microbial strains represents a promising solution to overcome the limitations of the latter methods. The most effective strategy for NMN biosynthesis in microbial cells primarily relies on: nicotinamide (NAM) supplement as a substrate, activation of pentose phosphate pathway to generate the precursor PRPP from carbon sources, overexpression of nicotinamide phosphoribosyltransferase (NAMPT) to catalyze the reaction between NAM and PRPP to produce NMN, and the expression of NMN transporter.^5,6 Among these factors, NAMPT is the key enzyme that dictates NMN production efficiency.^5–7 In a pioneer study, Marinescu et al. evaluated NAMPT from various sources and found that NAMPT from Haemophilus ducreyi, when expressed in E. coli BL21(DE3) pLysS, gave the highest intracellular NMN concentration (15.42 mg/L).⁷ Subsequently, Black et al. has combined the overexpression of both NAMPT and NMN synthase from Francisella tularensis (FtNadE) with the deletion of pncC and nadR, two genes involved in the catabolism of NMN, in E. coli, and achieved an intracellular NMN concentration of 1.5 mM.⁸ In another study, NAMPTs from ten organisms have been tested for the activity when expressed in E. coli and the results revealed that NAMPTs from Chitinophaga pinensis and Sphingopyxis sp. C-1 displayed significantly higher activities compared to NAMPTs derived from other sources.⁵ Similarly, Huang et al. performed a screening of eight NAMPTs from various sources and found that NAMPT from Vibrio phage KVP40 could biosynthesize 81.3 mg/L NMN intracellularly.⁶

Recently, Vibrio natriegens has emerged as a host for industrial biotechnology due to its extremely rapid growth rate with a reported doubling time of less than 10 min, non-pathogenic nature, genetic tractability, and ability to utilize a variety of carbon sources.⁹ In numerous studies, V. natriegens was used as an alternative expression system for recombinant protein production,¹⁰ and high-value metabolites such as pyruvate,¹¹ 2,3-butanediol.¹² However, the application of V. natriegens in NMN production remains unexplored. Therefore, the present study aimed to evaluate the feasibility of NMN biosynthesis in V. natriegens.

Methods

Results and discussion

In our present study, a metabolic engineering approach ( Figure 1) combining the insertion of key enzymes in the NMN biosynthetic pathway (NMN synthase from Francisella tularensis (FtNadE) and the most promising NAMPTs from previous studies) and the inactivation of endogenous NMN-degrading enzymes (NMN amidohydrolase, encoded by pncC, and NMN adenylyltransferase, encoded by nadR) was used to evaluate the NMN production capability in V. natriegens. To this end, a mutant strain, called V54-33, has been successfully generated by two rounds of MuGENT introducing araC-T7 RNA polymerase construct and FtNadE under the control of T7 promoter, respectively, while deleting pncC and nadR genes (extended data- Supplement I).¹⁴ This allowed a tight induction of NAMPTs and FtNadE by arabinose, and thereby provided a controlled regulation for NMN biosynthesis. Of note, our previous attempts using T7 IPTG-induced system were unsuccessful because of the growth inhibition in transformants (possibly due to the leaky expression of T7lac promoter) (data not shown).

Figure 1. Biosynthesis routes of β-nicotinamide mononucleotide used in this study.

NMN: nicotinamide mononucleotide.

Subsequently, four plasmids overexpressing NAMPTs from C. pinensis (CP), Sphingopyxis sp. C-1 (SSC), H. ducreyi (HD) and Vibrio phage KVP40 (VP) under the control of T7 promoter, were successfully generated (extended data-Supplement II).¹⁴ These four enzymes have been reported as the most efficient NAMPTs in separated studies.^5–7 In the next step, the strain V54-33 has been transformed with these plasmids harboring NAMPTs. SDS-PAGE analysis ( Figure 2) clearly showed that all recombinant strains carrying NAMPT plasmids displayed an intense band at the expected size of 56 kDa after induction with arabinose, while the original V54-33 strain did not possess this band. These results demonstrated that NAMPTs from CP, HD, SSC and VP were successfully expressed in V. natriegens.

Figure 2. SDS-PAGE analysis of the expression of recombinant NAMPTs in V54-33 strain.

L: GangNam-STAIN™ Prestained Protein Ladder. CP: lysate of V54-33 strain overexpressing NAMPT from Chitinophaga pinensis. HD: lysate of V54-33 strain overexpressing NAMPT from Haemophilus ducreyi. SSC: lysate of V54-33 strain overexpressing NAMPT from Sphingopyxis sp. C-1. VP: lysate of V54-33 strain overexpressing NAMPT from Vibrio phage KVP40. CT: lysate of V54-33 strain.

These recombinant strains were then transferred into modified M9 medium to assess NMN biosynthesis. HPLC quantification revealed that three out of four transformants (CP, HD, and VP) were capable of intracellular NMN accumulation, while SSC transformants and the V54-33 strain exhibited NMN levels below the limit of detection (LOD) of the method ( Figure 3, extended data-Supplement III).¹⁴ Among them, CP transformants exhibited the highest intracellular NMN titers (44.5 μM), whereas HD transformants showed significantly lower NMN accumulation (12.1 μM, p = 0.0111) ( Figure 3, extended data- Supplement III).¹⁴ The NMN concentration of VP transformants was intermediate (23.3 μM) (extended data- Supplement III),¹⁴ which aligns with prior research by Huang et al., showing that NAMPT from VP was more efficient in NMN accumulation than NAMPT from HD.⁶ When considering the absolute NMN titers reported in E. coli, NMN accumulation in CP transformants was comparable to that reported by Marinescu et al. (15.42 mg/L),⁷ but significantly lower than those reported by Black et al. (1.5 mM)⁸ and Huang et al. (81.3 mg/L).⁶ These disparities could be explained by the difference in expression systems: (i) FtNadE was expressed on a multiple-copy plasmid in E. coli in the study by Black et al.⁸ while this gene was incorporated into the chromosome of V. natriegens as a single-copy in our study; (ii) ushA encoding 5′-phosphatase capable of dephosphorylation of NMN to nicotinamide riboside and purR regulating the synthesis of the precursor phosphoribosyl pyrophosphate (PRPP) were knocked out in the study of Huang et al.⁶ whereas these genes have not been inactivated in the present study.

Figure 3. Quantification of intracellular NMN concentration of V54-33 strains harboring pACYC-Nampt plasmids.

CP: V54-33 strain overexpressing NAMPT from Chitinophaga pinensis. HD: V54-33 strain overexpressing NAMPT from Haemophilus ducreyi. SSC: V54-33 strain overexpressing NAMPT from Sphingopyxis sp. C-1. VP: V54-33 strain overexpressing NAMPT from Vibrio phage KVP40. Control: V54-33 strain. <LOD: concentration lower than limit of detection of HPLC method. Data are expressed as mean ± S.D. (error bars). Statistical analysis of differences between groups was performed by unpaired t-test (p < 0.05).

In summary, this study demonstrated the feasibility of NMN biosynthesis in V. natriegens. Further research focusing on the activation of pentose phosphate pathway and the transporters of NMN and NAM is underway to improve NMN production in this promising host.

Ethics and consent

Ethical approval and consent were not required.