Modified Escherichia coli to synthesize NMN efficiently

At the forefront of health and nutrition science, niacinamide mononucleotide (NMN) has received much attention due to its outstanding efficacy in alleviating aging and disease.

As a key precursor of nicotinamide adenine dinucleotide (NAD+), it plays a crucial role in the energy metabolism of cells and in the maintenance of REDOX homeostasis.

Studies have shown that supplementing with NMN effectively restores the level of NAD+ in the human body, which in turn activates a host of Nad-dependent enzymes such as deacetylase, which is essential for promoting cell longevity, repairing damaged DNA and enhancing the ability of cells to cope with stress.

Therefore, NMN has also become a potential nutritional supplement.

Recently, researchers from the Institute of Microbiology of the Chinese Academy of Sciences and Xinyang University of China have used genetic engineering to engineer Escherichia coli to efficiently produce NMN, and finally achieved a NMN titer of 15.66 g/L through whole cell catalysis, and reached 46.66 g/L in a 2 L bioreactor. The highest production rate reported to date was achieved, demonstrating the great potential for sustainable industrial production at NMN.

The study, titled "High-Level Production of Nicotinamide Mononucleotide by Engineered Escherichia Coli," Published in the Journal of Agricultural and Food Chemistry.

NMN biosynthetic pathway in E. coli

Among the many NMN synthesis pathways, the researchers chose the classic route of NMN synthesis with niacinamide (NAM) and phosphoribose pyrophosphate (PRPP) as precursors, catalyzed by niacinamide phosphoribosyl transferase (NAMPT).

Based on E. coli BW25113, they introduced NAMPT (CpNampt) of Chitinophaga pinensis with high enzymatic activity and PRPP synthetase (BsPRS) of Bacillus subtilis into it. The pBAD-CpNampt-BsPrs plasmid was constructed in order to achieve efficient synthesis of NMN in Escherichia coli.

The production of NMN was not detected at first, which may be due to competition inhibiting its synthesis.

After knocking out pncC, nadR, ushA, umpG, umph and pncA genes, the NMN production reached 160.66 mg/L in shaker fermentation.

The researchers found that there was still a large amount of NMN hoarding inside the cells, indicating that E. coli's own NMN transporter was inefficient. To solve this problem, they introduced a highly efficient transporter, PnuC, from Bacillus mycoides, to optimize the strain.

The modified strain NMN005-Cp successfully increased the extracellular NMN production to 416.67 mg/L.

Screening exogenous NAMPT enzymes to increase NMN production in Escherichia coli

To further increase the production of NMN, the researchers decided to screen out the highly active NAMPT enzyme.

The researchers selected 16 NAMPT enzymes from various sources to study, and found that VniNampt from Vibrio nigripulchritudo had a high affinity for PRPP and NAM by analyzing enzyme affinity and substrate affinity.

The results showed that when VniNampt was overexpressed, its catalytic efficiency of NAM to NMN was the highest.

The experimental results also strongly prove this point, when the overexpression of VniNampt in Escherichia coli, the efficiency of its catalytic NAM conversion to NMN is much higher than other enzymes, and the output is as high as 823.07 mg/L.

Moreover, the researchers believe that adequate supply of PRPP, as a key precursor of NMN synthesis, can also further enhance the production of NMN. Considering that xylose can be metabolized to PRPP through a shorter pathway than glucose, the researchers decided to strengthen the xylose utilization pathway in Escherichia coli to achieve synergistic utilization of glucose and xylose. More energy for PRPP production.

The engineered strain NMN011 was constructed after knocking out the carbon metabolite repressor (CCR) effect of ptsG gene, overexpression of glk gene restored glucose non-PTS pathway utilization, and overexpression of XylE and XylFGH.

Enhancing xylose utilization to increase NMN yield

In the whole cell catalytic experiment using xylose as the only carbon source, the engineered strain NMN011 performed well, and could completely consume 10 g/L xylose within 24 hours to produce 7.27 g/L NMN, which was the highest yield of NMN synthesized by xylose and NAM.

When using the mixed carbon source of glucose and xylose, strain NMN011 could efficiently utilize both sugars at the same time, and the NMN yield reached 15.66 g/L, indicating that the synergistic utilization of disaccharides could significantly improve the NMN yield.

In order to evaluate the industrial production potential of engineered strain NMN011, the researchers also used a batch feeding strategy for high-density fermentation, that is, NAM was continuously supplemented at a rate of 0.4-0.6 g/L/h, and conditions such as temperature, pH value and dissolved oxygen were controlled.

When glucose was the only carbon source, the NMN yield reached 26.07 g/L within 72 hours.

When xylose and glucose were added together as carbon sources, NMN production was further increased, and when 20 g/L xylose was added, NMN production reached 46.66 g/L, which is the highest yield reported so far.

This study not only provides a feasible technical scheme for the large-scale production of NMN, which helps to meet the growing market demand for NMN, so that more people can benefit from the efficacy of NMN in alleviating aging and diseases, but also provides a new idea and method for the synthesis of other high value-added compounds by microorganisms.

The researchers will continue their efforts to further optimize strain performance, improve NMN yield and production efficiency, reduce production costs, and improve the sustainability of the production process.

For example, key enzymes are optimized through protein engineering techniques to improve their activity and stability;

Explore more effective carbon source utilization strategies to reduce by-product generation;

Optimize fermentation process parameters to achieve more accurate process control.

Tag: NMN

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