Recombinant strains producing nicotinamide mononucleotide and their applications

Abstract

This invention relates to the field of biotechnology, and more particularly to recombinant strains producing nicotinamide mononucleotide (NMN) and their applications. This invention enhances NMN accumulation through metabolic flux redirection and gene knockout, successfully establishing an NMN synthetic pathway in *E. coli*. Furthermore, by optimizing the NAM-NMN transport system and the PRPP synthesis pathway, the NMN yield is effectively increased to 7.0 g / L. This invention also optimizes the NMN production process, achieving a maximum NMN yield of 28.5 g / L in a fed-batch fermentation system, a significant increase compared to traditional batch fermentation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a recombinant strain producing nicotinamide mononucleotide and its application. BACKGROUND

[0002] Nicotinamide mononucleotide (NMN) is an important intermediate in the biosynthetic pathway of nicotinamide adenine dinucleotide (NAD + ) and widely exists in mammals and microorganisms. In recent years, NMN has attracted widespread attention due to its potential application in anti-aging, prevention of metabolic diseases (such as type 2 diabetes, obesity and heart failure). NAD + As a key coenzyme of cellular energy metabolism, it plays an important role in maintaining mitochondrial function, anti-inflammation, promoting cell survival and delaying the aging process, so NMN as a precursor of NAD + is considered to be a functional molecule with important clinical value.

[0003] Traditionally, the synthesis of NMN mainly relies on chemical synthesis and enzymatic synthesis, which usually requires the use of expensive raw materials such as nicotinamide riboside (NR), phosphoribosyl pyrophosphate (PRPP) and various catalysts (such as ATP, AMP, NAD + , etc.). Although chemical and enzymatic synthesis has the advantage of high efficiency, it is high in cost, complex in process and potentially harmful to the environment. Therefore, producing NMN through microbial fermentation pathway has gradually become a more economically feasible and environmentally friendly choice.

[0004] Currently, the production of NMN based on microorganisms is mainly achieved through fermentation of recombinant engineering strains (such as Escherichia coli). However, traditional batch fermentation process often faces the following problems:

[0005] 1. Low production efficiency: due to fast substrate consumption, accumulation of metabolic products and metabolic inhibition of bacterial cells, the product yield and yield of batch fermentation are relatively low. Studies have shown that through batch fermentation method, recombinant E. coli can only produce about 2.31 mM of NMN at most.

[0006] 2. Substrate and nutrient inhibition: under the condition of high concentration of substrates (such as glucose and nicotinamide), microbial metabolism will produce by-products such as acetic acid, which will inhibit cell growth and reduce the production efficiency of NMN. In addition, high concentration of NAM (nicotinamide) will also have toxic effect on cells, further inhibiting product formation.

[0007] 3. Unstable production process: In the existing batch fermentation system, it is often difficult to stably control the fermentation environment due to fluctuations in substrate supply and oxygen concentration, thereby affecting the metabolic activity of the recombinant strain and the yield of NMN.

[0008] In the current study, the method of increasing the yield of NMN by high-density cell culture and fed-batch fermentation has been proposed. For example, Shoji et al. improved the yield of NMN by modifying the recombinant E. coli and using glucose and nicotinamide as substrates in a 1L medium using fed-batch fermentation technology, achieving a maximum NMN yield of 6.79g / L. However, this method still has the following shortcomings:

[0009] 1. Low substrate utilization efficiency: Although fed-batch fermentation improves the production efficiency of NMN to some extent, the single substrate supply strategy used by it is prone to lead to accumulation of sugar substrates, which in turn produces harmful by-products such as acetic acid, ultimately inhibiting the growth of the bacteria.

[0010] 2. Nutrient and inducer selection: Shoji's method relies on expensive inducers (such as IPTG) to initiate gene expression of the strain, which significantly increases production costs in industrial-scale production. In addition, the medium formula and feeding strategy in the existing scheme are relatively single, and it is difficult to adapt to the dynamic needs of nutrients in different fermentation stages.

[0011] 3. Complex purification process: Traditional NMN purification process usually needs multi-step chromatographic separation, such as ion exchange and reverse phase high performance liquid chromatography (HPLC), which not only increases the production time and cost, but also reduces the overall yield.

[0012] In summary, the current NMN production technology has many bottlenecks in production efficiency, substrate utilization and cost control, etc. Therefore, the present invention aims to significantly improve the yield and purity of NMN, reduce production costs, and achieve industrialized economic feasibility by optimizing the high-density cell culture and linear fed-batch fermentation strategy of recombinant E. coli, as well as the simplified size exclusion chromatography purification process. SUMMARY

[0013] Therefore, the present invention provides a recombinant strain for producing nicotinamide mononucleotide and its application.

[0014] Application of gene combination in microbial fermentation production of NMN;

[0015] The gene combination includes gene A and gene B; the gene A includes niaP gene and / or SBniaP gene; the gene B includes at least one of BMpunC, SPpunC, CPpunC and SApunC;

[0016] The niaP gene is derived from Burkholderia cepacia; the SBniaP gene is derived from Steroidobacteraceae bacterium; and the BMpunC gene is derived from Bacillus mycoides.

[0017] SPpunC is derived from Streptococcus pneumoniae; CPpunC is derived from Clostridium perfringens; SApunC is derived from Streptococcus agalactiae.

[0018] In some specific embodiments, the genes niaP, SBniaP, BMpunC, SPpunC, CPpunC, and SApunC from the above sources are optimized according to the codon preference of E. coli, and the optimized sequences have:

[0019] 1) Nucleotide sequences as shown in SEQ ID NO: 1-6; or

[0020] 2) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases in the sequence shown in 1), and whose function is the same as or similar to that in 1); or

[0021] 3) A nucleotide sequence that has at least 80% homology with the sequence shown in 1) or 2).

[0022] In some embodiments, the gene combination further includes at least one of ptsG, 6-phosphoglucose dehydrogenase gene, and PRPP synthase gene;

[0023] In this invention, ptsG, gnd, and prs are endogenous genes of the microorganism. In some specific embodiments of this invention, ptsG, gnd, and prs are all derived from Escherichia coli, specifically accessed as ptsG gene (ACT42992.1), gnd gene (ACT43782.1), and prs gene (ACT43074.1).

[0024] In some embodiments, the gene combination described above further includes the ccNAMPT1 gene; the ccNAMPT1 gene has:

[0025] I) The nucleotide sequence shown in SEQ ID NO: 7; or

[0026] II) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases in the sequence shown in I), and whose function is the same as or similar to that in I); or

[0027] III) is a nucleotide sequence that has at least 80% homology with the sequence shown in I) or II).

[0028] In this invention, the ccNAMPT1 gene (WP_098192935.1) is derived from Chitinophaga caeni and optimized according to the codon bias of E. coli. The optimized nucleic acid sequence has the following characteristics:

[0029] 1) A nucleotide sequence as shown in SEQ ID NO: 7; or

[0030] 2) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases in the sequence shown in 1), and whose function is the same as or similar to that in 1); or

[0031] 3) A nucleotide sequence that has at least 80% homology with the sequence shown in 1) or 2).

[0032] In the application described in this invention, the microorganisms ferment NAM as a substrate to produce NMN. The microorganisms include *Escherichia coli*, *Saccharomyces cerevisiae*, *Pichia pastoris*, *Corynebacterium glutamicum*, *Bacillus subtilis*, or lactic acid bacteria. In a specific embodiment of this invention, the microorganism is *Escherichia coli*.

[0033] The present invention also provides a gene combination, characterized in that it includes gene A and gene B;

[0034] The gene A includes the niaP gene and / or the SBniaP gene; the gene B includes at least one of BMpunC, SPpunC, CPpunC, and SApunC.

[0035] In some specific embodiments, the gene combinations described in this invention include any of the following combinations:

[0036] Combination 1: niaP and SPpunC;

[0037] Combination 2: niaP and CPpunC;

[0038] Combination 3: niaP and SApunC;

[0039] Combination 4: niaP-B and MpunC;

[0040] Combination 5: PAniaP-CPpunC;

[0041] Combination 6: PAniaP-SApunC.

[0042] In the gene combination described in this invention, the source and specific sequence of genes A and B are as described above, and will not be repeated here.

[0043] In some embodiments, the gene combination further includes at least one of ptsG, 6-phosphoglucose dehydrogenase gene, and PRPP synthase gene;

[0044] The sources of the ptsG gene, 6-phosphogluconic acid dehydrogenase gene, and PRPP synthase gene are as described above and will not be repeated here.

[0045] In some implementations, the gene combination further includes the ccNAMPT1 gene. The source and specific sequence of the ccNAMPT1 gene are as described above.

[0046] The present invention also provides an expression module, including a promoter and at least one gene from the gene combination described in the present invention.

[0047] In the expression unit described in this invention, the initiation is selected from T7 promoter, trc, tac, lacV, pFAB46, or pFAB100, etc. In a specific embodiment of this invention, the promoter is the T7 promoter. Furthermore, the expression unit may also include regulatory elements such as enhancers, replicons, and terminators; this invention does not impose any special limitations on these.

[0048] The present invention also provides an expression vector comprising the aforementioned gene combination or the aforementioned expression unit.

[0049] In this invention, the backbone of the expression vector includes pET, pCDF, pRSF, and pGEX series vectors, specifically pET21b, pCDFDuet-1, pRSFDuet-1, pGEX-5x-1, and others. In a specific embodiment of this invention, the backbone is pET21b or pCDFDuet-1.

[0050] The present invention also provides a recombinant strain which is transformed or transfected with the expression vector, or whose genome integrates the gene combination described herein, or whose genome integrates the expression module described herein.

[0051] In this invention, the recombinant strain is an *E. coli* strain expressing ptsG, gnd, prs and any one of the gene combinations shown in combinations 1 to 6, and in which the pncC, nadR, ushA, pncB, pgi, edd and pncA genes are knocked out.

[0052] The substrate bacteria of the recombinant strains described in this invention include *Escherichia coli*, *Saccharomyces cerevisiae*, *Bacillus subtilis*, or lactic acid bacteria. In some embodiments of this invention, the substrate bacteria are *Escherichia coli*. Specifically, the *Escherichia coli* is selected from [specific strains]. In a specific embodiment, this invention uses *E. coli* BL21(DE3) as an example for the modification of genetically engineered strains.

[0053] In some specific embodiments, this invention uses E. coli BL21(DE3) as the substrate bacterium. By knocking out pncC, nadR, ushA, and pncB, and inserting the NMN synthesis-related ccNAMPT1 gene, a recombinant strain ENM02 was obtained. This improved the synthesis efficiency of nicotinamide mononucleotide (NMN) and reduced NMN degradation. Experiments showed that the NMN accumulation of the ENM02 recombinant strain reached 3.5 g / L after 48 hours of shake-flask fermentation, which was 2.6 times higher than that of the control group without gene knockout. Figure 1 ).

[0054] Furthermore, this invention uses ENM02 as the starting strain and optimizes the transport system of NMN and NAM by introducing transport proteins. Specifically, this invention compares the effects of introducing transport protein combinations from different sources on NMN yield, and screens for six transport protein combinations that can significantly increase NMN yield (combinations 1-6 as described above). Six recombinant strains, ENM04-1 to ENM04-4 and ENM04-10 to ENM04-11, were obtained, with NMN yields ranging from 3.8 to 5.2 g / L after 48 hours. Among them, ENM04-1 achieved an NMN yield of 5.2 g / L after 48 hours of shake-flask fermentation.

[0055] Furthermore, by optimizing the PRPP (ribose-phosphorylated pyrophosphate) synthesis pathway, the yield of NMN was further increased. Specifically, the following steps were taken: Using ENM04-1 (named ENM04) as the starting strain, the pgi gene (ACT45688.1) was knocked out, and the ptsG gene (ACT42992.1) was inserted at the corresponding site to increase the flow of glucose into the phosphorylation pathway. The edd gene (ACT43676.1) was knocked out, and the gnd gene (ACT43782.1) was inserted at the corresponding site to increase the flow of the pentose phosphate pathway (PPP), thereby increasing PRPP production. The pncA gene (ACT43591.1) was knocked out, and the prs gene (ACT43074.1) was inserted at the corresponding site to obtain the recombinant strain ENM05. After 48 hours of shake-flask fermentation, the NMN yield reached 7.0 g / L.

[0056] This invention also provides a method for constructing recombinant strains, comprising the following steps:

[0057] (1) Knock out at least one of pncC, nadR, ushA and pncB in the basal bacteria;

[0058] (2) Transform into the gene combinations shown in combinations 1 to 8 as described above;

[0059] (3) Replace the original pgi, edd and pncA genes of the basal bacteria with ptsG, gnd and prs genes in sequence.

[0060] The present invention also provides a method for preparing NMN, which uses NAM as a substrate and ferments the recombinant strain to obtain a product containing NMN.

[0061] The preparation method of the present invention includes: using NMA as a substrate, fermenting the recombinant strain according to any one of claims 10 or 11 or the recombinant strain constructed by the construction method of claim 12; the resulting fermentation broth is extracted and purified to obtain NMN.

[0062] In the preparation method of the present invention, the fermentation includes initial fermentation and fed-batch fermentation;

[0063] The initial fermentation includes: inoculating the recombinant strain into a fermentation medium and fermenting for 5 to 7 hours; the initial fermentation medium includes 10 to 25 g / L glucose and 1 to 10 g / L NAM.

[0064] The fed-batch fermentation process involves adding a feed solution containing 400–600 g / L glucose, 40–80 g / L yeast extract, and 40–80 g / L tryptone, followed by fermentation, induced expression, and collection of the NMN-containing product.

[0065] In this invention, the strain is seed cultured before the initial fermentation to obtain a seed culture. In some specific embodiments, the seed culture includes: inoculating the strain into LB medium and culturing at 37°C and 200 rpm for 12 hours with shaking.

[0066] In this invention, the seed liquid is subjected to initial fermentation to obtain an initial fermentation broth. The initial fermentation time is 5–7 hours, specifically 5 hours, 6 hours, 7 hours, or any value within the above two ranges. The glucose concentration in the initial fermentation culture medium can specifically be 10 g / L, 15 g / L, 20 g / L, 25 g / L, or any value within the above two ranges.

[0067] In this invention, the initial fermentation medium comprises a basal medium and nicotinamide (NAM). In some embodiments, the concentration of nicotinamide in the initial fermentation medium is 1–10 g / L. In some specific embodiments, the concentration of NAM in the fermentation medium is 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, or any value within the above two ranges.

[0068] In this invention, the initial fermentation includes cultivation at 37°C, 300 rpm, and an aeration rate of 1.5 vvm.

[0069] In this invention, fed-batch fermentation is initiated after the glucose in the fermenter is depleted (approximately 6 hours). In this invention, during the fed-batch fermentation, the glucose concentration in the feed solution is 400–600 g / L, specifically 400 g / L, 500 g / L, 600 g / L, or any value within the above two ranges. The yeast extract concentration in the feed solution is 40–80 g / L, specifically 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, or any value within the above two ranges. The trypsin concentration in the feed solution is 40–80 g / L, specifically 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, or any value within the above two ranges.

[0070] In this invention, during the fed-batch fermentation, fermentation reaches OD (dose-to-temperature). 600 When the value is between 100 and 180, induced expression is performed. In some specific embodiments, the OD... 600 The values ​​are 100, 110, 120, 130, 140, 150, 160, 170, 180, or any value within the above two ranges. In this invention, the feeding rate of the feed solution increases linearly from 10 mL / h to 20 mL / h.

[0071] This invention employs a linear fed-batch strategy, gradually replenishing high concentrations of glucose and substrate solution (nicotinamide). This effectively controls the substrate concentration, reduces the accumulation of byproducts such as acetic acid, and achieves stable production under high-density cell culture conditions. Simultaneously, precise control of dissolved oxygen and nutrient supply ultimately improves the fermentation efficiency and yield of NMN.

[0072] In this invention, the fermentation is carried out in a fermenter, but other suitable fermentation equipment in the art may also be used, and this invention does not impose any special limitations on it.

[0073] In this invention, when the OD600 reaches 150 or higher, the feeding rate is maintained at 20 mL / h, and the expression of NMN synthesis-related genes is induced by adding an inducer. In some embodiments, the inducer is lactose. In some specific embodiments, the inducer is a 1% lactose solution.

[0074] In this invention, the extraction is a separation and purification of the fermentation broth supernatant.

[0075] In this invention, the purification method includes size exclusion chromatography (SEC) purification. In some specific embodiments, the chromatographic column used for size exclusion chromatography (SEC) is a HiPrep 26 / 60 SepacrylS-200HP size exclusion column.

[0076] This invention uses *E. coli* as the substrate bacteria and achieves efficient and sustainable NMN production by increasing PRPP supply, optimizing the transport system, and reducing degradation pathways. Ultimately, this effectively improves the yield and purity of NMN, making it more suitable for industrial-scale economic production. The specific modification strategies include the following three aspects:

[0077] 1. Knock out NMN-related degradation genes to reduce product loss: In order to reduce the degradation of NMN, this invention knocks out genes related to NMN degradation and inserts genes related to NMN synthesis to prevent the synthesized NMN from being further decomposed, thereby effectively accumulating higher concentrations of NMN in the cell and significantly increasing the final yield. The cumulative amount of NMN in shake-flask fermentation reached 3.5 g / L within 48 hours.

[0078] 2. Optimizing NAM and NMN transport to enhance metabolic flow control: This invention optimizes the cellular transport system to improve the efficiency of NAM transport into the cell and promote the effective transport of NMN out of the cell. By increasing the intracellular concentration of NAM, a sufficient supply of raw materials for NMN synthesis is ensured; simultaneously, by enhancing the transport of NMN out of the cell, the accumulation pressure of NMN in the cell is reduced, thereby achieving dynamic balance and control of intracellular metabolic flow, ensuring efficient NMN production, with NMN reaching 5.2 g / L within 48 hours through shake-flask fermentation.

[0079] 3. Increase PRPP supply and improve NMN synthesis efficiency: This invention accelerates NMN synthesis by reducing PRPP consumption pathways and optimizing metabolic flow to PRPP supply, thereby increasing PRPP intracellular concentration. By regulating the PRPP generation metabolic pathway and prioritizing its use for NMN synthesis, the limitation caused by insufficient PRPP supply is overcome, and the NMN yield is improved. The cumulative NMN yield after 48 hours of shake-flask fermentation reaches 7.0 g / L.

[0080] Furthermore, this invention optimizes the preparation process of NMN, including optimizations in both the fermentation and purification processes:

[0081] (1) Fermentation process

[0082] In traditional batch fermentation, adding substrate all at once is detrimental to cell growth and metabolism, resulting in less than ideal NMN yield. This invention employs linear fed-batch fermentation, gradually adding substrate and controlling dissolved oxygen levels to ensure stable cell growth in high-density culture while reducing the accumulation of byproducts.

[0083] (2) Purification process

[0084] After fermentation, the bacterial cells are collected by centrifugation and disrupted. Following extraction and purification, NMN can be obtained. Traditional multi-step purification processes for NMN are time-consuming, costly, and yield low purification rates, making them unsuitable for large-scale production. This invention optimizes the NMN purification process by employing size exclusion chromatography (SEC) for single-step purification, increasing product purity to over 98% while maintaining a purification yield of 53.9%.

[0085] The optimized NMN process of this invention achieved a maximum NMN yield of 28.5 g / L in a fed-batch fermentation system, significantly increasing yield compared to traditional batch fermentation. This demonstrates that the present invention, through the combination of metabolic flux regulation, transport system optimization, and genetic engineering techniques, enables highly efficient NMN production on an industrial scale. It has broad applications in pharmaceuticals, health products, and bioenergy, and offers significant economic and social benefits. Attached Figure Description

[0086] Figure 1 The results of shake-flask fermentation of the NMN degradation gene knockout strain are shown.

[0087] Figure 2 The results of shake-flask fermentation of the strain overexpressing the transport protein are shown.

[0088] Figure 3 The results of shake-flask fermentation of PRPP-enhanced strains are shown.

[0089] Figure 4 The results show the comparison between fed-batch fermentation and traditional batch fermentation on NMN production.

[0090] Figure 5 The HPLC analysis results of single-step size exclusion chromatography purification of NMN are shown.

[0091] Figure 6 LC-MS analysis of purified NMN is shown. Detailed Implementation

[0092] This invention provides recombinant strains producing nicotinamide mononucleotide and their applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0093] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0094] This invention provides a method for the efficient production of nicotinamide mononucleotide (NMN) using an engineered Escherichia coli strain through high-density cell culture technology and optimized fed-batch fermentation. The method significantly improves NMN yield and production efficiency by integrating metabolic flux regulation strategies, transport system optimization, and degradation gene knockout.

[0095] In this invention, the high-yield engineered strain is constructed from recombinant E. coli BL21(DE3). An engineered strain with highly efficient PRPP synthesis capability is constructed through gene insertion and knockout, and expression regulation via the pET21b vector.

[0096] In order to improve the supply of intracellular PRPP, the present invention adopts the following strategies: (1) reduce the metabolic consumption pathway of PRPP by knocking out genes related to PRPP consumption, thereby increasing the effective utilization rate of PRPP; (2) increase the production of intracellular glucose-6-phosphate by optimizing glucose metabolism pathway, thereby increasing the flux of the pentose phosphate pathway (PPP) to increase the synthesis of PRPP.

[0097] This invention provides a method for improving the efficiency of nicotinamide (NAM) transport into cells. By enhancing transport proteins on the cell membrane, efficient NAM uptake is achieved, significantly increasing the intracellular NAM concentration and providing sufficient substrate for NMN synthesis. Furthermore, overexpression of the transport proteins improves the extracellular secretion efficiency of NMN, reducing the pressure for intracellular NMN accumulation.

[0098] To reduce NMN degradation, this invention prevents the further metabolic breakdown of synthesized NMN by knocking out genes associated with NMN degradation. The gene knockout method employs the CRISPR-Cas9 system to ensure efficient and stable NMN accumulation during the production process.

[0099] In terms of fermentation technology, this invention employs fed-batch fermentation to achieve high-density cell culture. The fermentation is divided into two stages: (1) initial batch culture, using 20 g / L glucose as the carbon source, to achieve rapid cell growth within 6 hours; (2) in the fed-batch stage, a linear feeding strategy is used to gradually add 600 g / L glucose, 60 g / L yeast extract, and 60 g / L tryptone feed solution to control the substrate concentration in the culture medium and avoid the accumulation of acetic acid. The feeding rate is 10 mL per hour, and the feeding rate is controlled by online monitoring of dissolved oxygen levels to ensure that dissolved oxygen is maintained above 40%.

[0100] During the fermentation induction phase, when the OD600 reaches 100-120, 1% lactose is added to replace IPTG as the inducer to reduce induction costs and minimize inhibition of cell growth. Optimization of fermentation temperature (controlled at 30℃ during induction) and pH (maintained at 6.8-7.0) ensures efficient expression of the recombinant strain and continuous NMN production.

[0101] After fermentation, the cell bodies were collected by centrifugation and the cells were lysed. This invention employs an ultrasonic lysis method to extract intracellular NMN. To achieve efficient purification, the NMN was isolated and purified using single-step size exclusion chromatography (SEC). A HiPrep 26 / 60 Sepacryl S-200HP column was used, with a flow rate of 0.4 mL / min, and 50 mM formic acid was used as the isocratic elution solvent. The purified NMN was verified by liquid chromatography-mass spectrometry (LC-MS), showing a purity of over 98% and an overall yield of 53.9%.

[0102] The method of this invention achieved a maximum NMN yield of 28.5 g / L in a fed-batch fermentation system, significantly increasing the yield compared to traditional batch fermentation. The successful implementation of this method demonstrates that by combining metabolic flux regulation, transport system optimization, and genetic engineering techniques, highly efficient NMN production can be achieved on an industrial scale.

[0103] The efficient NMN production method provided by this invention is suitable for large-scale industrial production and can be widely used in the fields of medicine, health products and bioenergy, with significant economic and social benefits.

[0104] The optimized niaP gene (WP_062884942.1) nucleic acid sequence SEQ ID NO: 1 is:

[0105]

[0106]

[0107] The optimized SBniaP(HKU12810.1) gene nucleic acid sequence SEQ ID NO: 2 is as follows:

[0108]

[0109]

[0110] The optimized nucleic acid sequence of BMpunC (WP_215553083.1) is SEQ ID NO: 3 as follows:

[0111]

[0112] The optimized nucleic acid sequence of SPpunC (CJB91739.1) is SEQ ID NO: 4 as follows:

[0113]

[0114] The optimized CPpunC (HFK8301865.1) nucleic acid sequence SEQ ID NO: 5 is:

[0115]

[0116]

[0117] The optimized nucleic acid sequence of SApunC (HFU9558888.1) is SEQ ID NO: 6 as follows:

[0118]

[0119] The optimized CCNAMPT1 gene (WP_098192935.1) nucleic acid sequence SEQ ID NO: 7 is as follows:

[0120]

[0121] The optimized nucleic acid sequence of PAniaP (WP_349544922.1) is SEQ ID NO: 8 as follows:

[0122]

[0123] The present invention will be further illustrated below with reference to the embodiments:

[0124] Example 1: Knockout of NMN Degradation Genes and Its Effect on NMN Production

[0125] (I) Implementation steps:

[0126] In this embodiment, gene knockout of E. coli BL21(DE3) strain improved the synthesis efficiency of nicotinamide mononucleotide (NMN) and reduced NMN degradation. The specific operation steps are as follows:

[0127] 1. Strain construction:

[0128] This invention uses E. coli BL21(DE3) as the starting strain. First, multiple genes related to NMN degradation, including pncC (ACT44369.1) and nadR, are knocked out using CRISPR-Cas9 gene editing technology.

[0129] (ACT46046.1), ushA (ACT42330.1), and pncB (ACT42830.1). The purpose of gene knockout is to reduce the degradation of NMN in cells and ensure higher NMN accumulation. The resulting strain was ENM01.

[0130] 2. Plasmid construction and transformation:

[0131] Based on the gene knockout described above, the ccNAMPT1 gene sequence, which is related to NMN synthesis, was inserted into the expression plasmid pET21b vector to enhance NMN biosynthesis. The constructed expression plasmid was introduced into the ENM01 strain under ampicillin (75 μg / mL) selection pressure to obtain ENM02. Simultaneously, the corresponding plasmid was introduced into the E. coli BL21(DE3) strain to obtain ENM03.

[0132] 3. Shake flask fermentation conditions:

[0133] The recombinant strain was inoculated into 10 mL of Luria-Bertani (LB) medium and cultured at 37 °C and 200 rpm for 12 hours with shaking to obtain the seed culture. Subsequently, 5% (v / v) of the seed culture was transferred to a 250 mL Erlenmeyer flask containing 100 mL of M9 medium (composition: 13.3 g / L KH₂PO₄, 4 g / L (NH₄)₂HPO₄, 1.2 g / L MgSO₄·7H₂O, 20 g / L glucose, and 5 g / L nicotinamide (NAM)). Fermentation experiments were conducted at 37 °C and 200 rpm for 24 and 48 hours.

[0134] 4. Shake-flask fermentation induction and sample collection:

[0135] Six hours after the start of fermentation, expression was induced by adding 1% lactose (final concentration). 10 mL samples were taken at 24 and 48 hours for subsequent NMN detection and analysis. The samples were centrifuged (8000 rpm, 4°C, 10 min) to separate the supernatant, and filtered through a 0.22 μm PTFE membrane to ensure the purity of the fermentation broth.

[0136] 5. Methods for detecting NMN concentration:

[0137] NMN concentration was quantitatively analyzed by high-performance liquid chromatography (HPLC, Younglin Inc., Korea) using an Eclipse plus C18 column (4.6 × 250 mm, 5 μm), with 10% acetonitrile as the mobile phase, isocratic elution at a flow rate of 1 mL / min, and a detection wavelength of 260 nm. Standard curves were plotted based on NMN standards of known concentrations.

[0138] (II) Experimental Results:

[0139] By knocking out the pncC, nadR, ushA, and pncB genes, this invention significantly reduced NMN degradation, thereby increasing NMN production. Under shake-flask fermentation conditions with 5 g / L NAM added, the recombinant strain with the relevant genes knocked out achieved an NMN accumulation of 3.5 g / L within 48 hours, which was 2.6 times higher than the control group without gene knockout. Figure 1 ).

[0140] Example 2: Optimization of NAM and NMN transport systems and their impact on NMN yield

[0141] (I) Implementation steps:

[0142] In this embodiment, the NMN production efficiency of recombinant E. coli is improved by optimizing the transport system of nicotinamide (NAM) and nicotinamide mononucleotide (NMN). The specific operation steps are as follows:

[0143] 1. Selection of starting strain and plasmid construction:

[0144] In this embodiment, the recombinant E. coli BL21(DE3) strain ENM02, which underwent gene knockout and metabolic flux optimization, was selected as the starting strain. Based on this, the pCDFDuet-1 expression vector was used to insert the transporter protein coding sequence, and both transporters were simultaneously expressed using homologous recombination technology.

[0145] BMpunC (WP_215553083.1) and homologs SPpunC (CJB91739.1), CPpunC (HFK8301865.1), SApunC (HFU9558888.1) and niaP (WP_062884942.1) and homologs SBniaP (HKU12810.1) and PAniaP (WP_349544922.1) were selected. After codon optimization for E. coli, plasmids were constructed according to Table 1. The constructed plasmids were introduced into the ENM02 strain by electroporation to obtain the final engineered strains ENM04-1 to ENM04-10. The specific information of the inserted genes in the strains is shown in Table 1.

[0146] Table 1. Strain Information

[0147]

[0148] 2. Screening and identification of transformed strains:

[0149] The transformed recombinant strain was inoculated onto LB agar containing streptomycin (50 μg / mL) and ampicillin (75 μg / mL) to screen for resistant colonies. Multiple colonies were selected for PCR verification and sequencing to ensure correct gene insertion and expression.

[0150] 3. Shake-flask fermentation experimental conditions:

[0151] Comparative fermentation experiments were conducted using ENM02 and ENM04 strains, respectively. The seed culture was inoculated into 250 mL Erlenmeyer flasks containing 100 mL of M9 medium (13.3 g / L KH2PO4, 4 g / L (NH4)2HPO4, 1.2 g / L MgSO4·7H2O, 20 g / L glucose, and 5 g / L NAM), and cultured by shaking at 37 °C and 200 rpm.

[0152] 4. Shake-flask fermentation induction and sample collection:

[0153] After 6 hours of fermentation, 1% lactose (final concentration) was added as an inducer. 10 mL samples were taken at 24 and 48 hours of fermentation. The supernatant was separated by centrifugation (8000 rpm, 4℃, 10 min), filtered through a 0.22 μm PTFE membrane, and then used for subsequent NMN concentration determination.

[0154] 5. Methods for detecting NMN concentration:

[0155] The concentration of NMN was determined using high-performance liquid chromatography (HPLC, Younglin Inc., Korea). An Eclipseplus C18 column (4.6 × 250 mm, 5 μm) was used, with 10% acetonitrile as the mobile phase, a flow rate of 1 mL / min, and a detection wavelength of 260 nm. The standard curve was plotted based on NMN standards of known concentrations.

[0156] (II) Experimental Results:

[0157] Experimental results showed that the NMN production of the ENM04 strain transformed with the niaP and SPpunC genes was significantly higher than that of the control strain ENM02 during 48 hours of fermentation. Figure 2 This result indicates that optimizing the transport system of NAM and NMN can effectively improve the intracellular uptake of NAM and the extracellular secretion efficiency of NMN, thereby significantly increasing the synthesis rate and cumulative yield of NMN.

[0158] in conclusion:

[0159] By introducing the niaP and SPpunC genes, this invention optimized the NAM uptake and NMN secretion system of recombinant E. coli, resulting in an NMN production of 5.2 g / L within 48 hours, a 49% increase compared to the unoptimized control group. This demonstrates the important role of transporter overexpression in improving metabolic flux efficiency and NMN production.

[0160] Example 3: Optimization of PRPP synthesis capacity and its effect on increasing NMN production

[0161] (I) Implementation steps:

[0162] This embodiment improves the NMN production efficiency of recombinant E. coli strains by further optimizing the PRPP (ribose-phosphoric acid pyrophosphate) synthesis pathway. The specific steps are as follows:

[0163] 1. Selection and genetic modification of the starting strain:

[0164] The ENM04-1 strain, incorporating the niaP and SPpunC genes, was named ENM04. Further modifications were made to the ENM04 strain to enhance PRPP synthesis. The following gene knockout and insertion were performed using CRISPR-Cas9 technology:

[0165] Knocking out the pgi gene (ACT45688.1) and inserting the ptsG gene (ACT42992.1) at the corresponding site increases glucose flux to the phosphorylation pathway. Knocking out the edd gene (ACT43676.1) and inserting the gnd gene (ACT43782.1) at the corresponding site increases pentose phosphate pathway (PPP) flux, thereby increasing PRPP production. Knocking out the pncA gene (ACT43591.1) and inserting the prs gene (ACT43074.1) at the corresponding site directly enhances the PRPP synthesis rate.

[0166] Through the above gene editing operations, the engineered strain ENM05 was finally obtained.

[0167] 2. Screening and validation of strains:

[0168] The modified strains were transferred to LB agar containing ampicillin (75 μg / mL) and streptomycin (50 μg / mL) for screening. Positive clones were selected for PCR verification, and sequencing confirmed the correct insertion of the ptsG, gnd, and prs genes, as well as the successful knockout of the pgi, edd, and pncA genes.

[0169] 3. Shake-flask fermentation experimental conditions:

[0170] Comparative fermentation experiments were conducted using strains ENM04 and ENM05. The seed culture of the strains was inoculated into 250 mL Erlenmeyer flasks containing 100 mL of M9 medium (13.3 g / L KH₂PO₄, 4 g / L (NH₄)₂HPO₄, 1.2 g / L MgSO₄·7H₂O, 20 g / L glucose, and 5 g / L nicotinamide (NAM)). The flasks were then incubated at 37 °C and 200 rpm.

[0171] 4. Shake-flask fermentation induction and sample collection:

[0172] After 6 hours of fermentation, expression was induced by adding 1% lactose (final concentration). 10 mL samples were taken at 24 and 48 hours of fermentation to detect NMN concentration. The samples were centrifuged (8000 rpm, 4℃, 10 min) to separate the supernatant, and then filtered through a 0.22 μm PTFE membrane.

[0173] 5. Methods for detecting NMN concentration:

[0174] The concentration of NMN was quantitatively analyzed using high-performance liquid chromatography (HPLC, Younglin Inc., Korea). An Eclipse plus C18 column (4.6 × 250 mm, 5 μm) was used, with 10% acetonitrile as the mobile phase, a flow rate of 1 mL / min, and a detection wavelength of 260 nm.

[0175] (II) Experimental Results:

[0176] Fermentation yield analysis of ENM04 and ENM05 strains showed that ENM05 produced significantly higher NMN yields than ENM04 at both 24 and 48 hours. Figure 3 This indicates that the combined modification of knocking out pgi, edd, and pncA genes and inserting ptsG, gnd, and prs genes effectively increased the supply of intracellular PRPP, thereby significantly improving NMN production.

[0177] in conclusion:

[0178] This embodiment effectively improved the NMN synthesis capacity of the recombinant strain by enhancing the PRPP synthesis pathway. Results showed that the genetically engineered ENM05 strain achieved a cumulative NMN yield of 7.0 g / L within 48 hours, a 34% increase compared to the unengineered control group. The PRPP optimization strategy of this invention has significant application potential in industrial-scale NMN production, effectively reducing production costs and increasing yield.

[0179] Meanwhile, this invention uses ENM04-2 and ENM04-3 as starting bacteria, respectively, and performs gene modification according to the modification method of ENM04-1 to obtain ENM05-A and ENM05-B, respectively. Shake flask fermentation and yield analysis are performed according to the above method. The results show that the modified strains have increased by 19% and 31% respectively compared with the unmodified control group.

[0180] Example 4: Optimization of fed-batch fermentation strategy and its effect on increasing NMN yield

[0181] (I) Implementation steps:

[0182] This embodiment improves the NMN production efficiency of recombinant E. coli strains by optimizing the fed-batch fermentation process. The specific operation steps are as follows:

[0183] 1. Selection of starting strain:

[0184] In this embodiment, the metabolically engineered high-efficiency NMN-producing strain ENM05 was selected for fermentation experiments. To verify the impact of the fed-batch strategy on NMN yield, traditional batch fermentation was used as a control.

[0185] 2. Fermentation conditions and culture medium preparation:

[0186] Fermentation container: 2.5L fermentation tank;

[0187] Initial working volume: 1.5L;

[0188] The basal medium (M9 medium) has the following components:

[0189] KH2PO4: 13.3 g / L;

[0190] (NH4)2HPO4: 4g / L;

[0191] MgSO4·7H2O: 1.2 g / L;

[0192] Glucose: 20g / L;

[0193] Nicotinamide (NAM): 5g / L;

[0194] Lactose, an inducer, is added at a concentration of 1% during fermentation.

[0195] The initial pH was adjusted to 6.8, the culture temperature was set to 37℃, the stirring speed was set to 300 rpm, and the aeration rate was set to 1.5 vvm.

[0196] 3. Fermentation process:

[0197] (A) Traditional batch fermentation:

[0198] Seed culture:

[0199] The ENM05 strain was inoculated into 10 mL of LB medium and cultured at 37°C with shaking at 200 rpm for 12 hours to obtain the seed culture.

[0200] Inoculate 5% (v / v) of the seed culture into a 2.5L fermenter, with an initial culture medium volume of 1.5L.

[0201] Fermentation stage:

[0202] Fermentation was carried out in a fermenter at 37°C and 300 rpm, with an initial aeration rate of 1.5 vvm.

[0203] No feeding is performed during fermentation. Glucose and NAM are added only once and supplied completely at the beginning of fermentation.

[0204] Once the glucose is depleted (approximately 12 hours), the system enters the decay phase.

[0205] Sample collection:

[0206] 10 mL samples were taken every 5 hours after fermentation began.

[0207] The sample was centrifuged (8000 rpm, 4℃, 10 min) to separate the supernatant and filtered through a 0.22 μm PTFE membrane.

[0208] (B) Feed-in batch fermentation:

[0209] Seed culture:

[0210] The ENM05 strain was inoculated into 10 mL of LB medium and cultured at 37°C with shaking at 200 rpm for 12 hours to obtain the seed culture.

[0211] Inoculate 5% (v / v) of the seed culture into a 2.5L fermenter, with an initial culture medium volume of 1.5L.

[0212] Initial fermentation stage:

[0213] The fermenter was used for cultivation at 37°C, 300 rpm, and an aeration rate of 1.5 vvm. The glucose concentration was 20 g / L, and the NAM concentration was 5 g / L.

[0214] Once the glucose in the fermenter is depleted (approximately 6 hours), the feeding phase is initiated.

[0215] Replenishment in batches:

[0216] The supplemental feeding solution consists of 600 g / L glucose, 60 g / L yeast extract, and 60 g / L trypsin.

[0217] Replenishment strategy:

[0218] Phase 1 (6-12 hours): Feeding rate is 10 mL / h to ensure rapid cell proliferation while maintaining dissolved oxygen level above 40%.

[0219] Phase 2 (12-24 hours): The feed rate was adjusted to 20 mL / h to increase the NMN synthesis rate while controlling acetic acid accumulation.

[0220] Phase 3 (after 24 hours): When OD600 reaches 150 or higher, maintain the feeding rate at 20 mL / h and induce the expression of NMN synthesis-related genes by adding 1% lactose.

[0221] Sample collection:

[0222] 10 mL samples were taken every 5 hours after fermentation began.

[0223] The sample was centrifuged (8000 rpm, 4℃, 10 minutes) to separate the supernatant, and then filtered through a 0.22 μm PTFE membrane for NMN concentration detection.

[0224] 4. Methods for detecting NMN concentration:

[0225] The concentration of NMN was quantitatively analyzed using high-performance liquid chromatography (HPLC, Younglin Inc., Korea). An Eclipse plus C18 column (4.6 × 250 mm, 5 μm) was used, with 10% acetonitrile as the mobile phase, a flow rate of 1 mL / min, and a detection wavelength of 260 nm. The concentration of NMN was quantitatively analyzed based on a standard curve.

[0226] (II) Experimental Results:

[0227] A t-test analysis of the yield data of ENM05 strain under two fermentation strategies showed that the fed-batch fermentation strategy significantly improved the yield of NMN. Within 50 hours, the yield of the fed-batch fermentation strategy was 133% higher than that of the conventional batch fermentation.

[0228] This invention also conducted fermentation experiments on strains ENM05-A and ENM05-B according to the two fermentation strategies described above. The results showed that ENM05-A and ENM05-B, using the fed-batch fermentation process of this application, significantly increased the yield of NMN, increasing it by 79% and 103% respectively compared to traditional batch fermentation within 50 hours of fermentation.

[0229] in conclusion:

[0230] This embodiment demonstrates that optimizing the fed-batch fermentation process can significantly improve the NMN production efficiency of recombinant E. coli strains. Compared with traditional batch fermentation, the fed-batch fermentation strategy effectively avoids substrate depletion and acetic acid accumulation problems, while promoting continuous cell growth and increasing the NMN synthesis rate. The fed-batch fermentation strategy of this invention has broad application prospects in industrial-scale NMN production, and can significantly reduce production costs and increase NMN yield.

[0231] Example 5: Single-step purification process of NMN and its effect on product purity

[0232] (I) Implementation steps:

[0233] This embodiment optimizes the single-step purification process of NMN to improve product purity and yield while reducing production costs. The specific operating steps are as follows:

[0234] 1. Sample preparation:

[0235] The fermentation broth of strain ENM05, obtained through a fed-batch fermentation process (Example 4), was used as the raw material. After fermentation, the broth was immediately collected for NMN extraction and purification.

[0236] The fermentation broth was first centrifuged (8000 rpm, 4°C, 20 min) to separate the supernatant and remove bacterial cells. The separated supernatant was then filtered through a 0.22 μm PTFE membrane to ensure sample purity.

[0237] 2. Ultrasonic extraction of NMN:

[0238] The fermentation supernatant, after centrifugation and filtration, was filtered through a 0.22 μm filter membrane to remove any possible residual cell debris and impurities.

[0239] 3. Size exclusion chromatography (SEC) purification:

[0240] NMN was purified in a single step using a HiPrep 26 / 60 Sepacryl S-200HP size exclusion column (Cytiva, USA).

[0241] Mobile phase: 50 mM formic acid (pH 5.0)

[0242] Flow rate: 0.4 mL / min

[0243] Washout mode: Isocratic washout

[0244] Use an automatic collector to collect the fractions at specified time intervals.

[0245] 4. HPLC analysis confirms purity:

[0246] The collected purified fractions were analyzed by high performance liquid chromatography (HPLC, Younglin Inc., Korea).

[0247] The Eclipse plus C18 column (4.6×250mm, 5μm) was used, the mobile phase was 10% acetonitrile, the flow rate was 1mL / min, and the detection wavelength was 260nm.

[0248] The purity of the target product was confirmed by comparing the retention times of known NMN standards.

[0249] 5. Calculation of purification efficiency:

[0250] The structure of the purified product was further verified using LC-MS (maXis 4G, Bruker Bioscience). The purification yield was calculated based on the mass and concentration of NMN before and after purification.

[0251] (II) Experimental Results:

[0252] After single-step size exclusion chromatography purification, the final purity of NMN reached over 98%, a significant improvement compared to the unpurified fermentation broth (initial purity of approximately 70%). Figure 5 ).

[0253] Purification yield: The total amount of NMN recovered from the fermentation broth was 53.9%.

[0254] LC-MS analysis showed that the molecular weight of purified NMN (m / z = 335 [M+H]) + Consistent with the standard product Figure 6 ).

[0255] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of a gene combination in microbial fermentation production of NMN. The gene combination is gene A and gene B; the gene A is niaP gene or PA niaP gene; the gene B is one of BM punC gene, SP punC gene, CP punC gene and SA punC gene. The niaP gene is derived from Burkholderia cepacia; the PA niaP gene is derived from Paraburkholderia acidicola; the BM punC gene is derived from Bacillus mycoides; the SP punC is derived from Streptococcus pneumoniae; the CP punC is derived from Clostridium perfringens; and the SA punC is derived from Streptococcus agalactiae. The microorganism is Escherichia coli into which ccNAMPT1 gene is inserted and pncC, nadR, ushA and pncB genes are knocked out. The nucleotide sequence of the ccNAMPT1 gene is shown in SEQ ID NO:

7.

2. The use according to claim 1, wherein The nucleotide sequence of the niaP gene is shown in SEQ ID NO: 1; The nucleotide sequence of the PA niaP gene is shown in SEQ ID NO: 8; The nucleotide sequence of the BM punC gene is shown in SEQ ID NO: 3; The nucleotide sequence of the SP punC gene is shown in SEQ ID NO: 4; The nucleotide sequence of the CP punC gene is shown in SEQ ID NO: 5; The nucleotide sequence of the SA punC gene is shown in SEQ ID NO:

6.

3. Use according to claim 1 or 2, characterized in that, The microorganism further overexpresses ptsG, gnd and prs; The ptsG, gnd and prs are endogenous genes of the microorganism.

4. A recombinant strain, characterized in that, The genome of the microorganism is integrated with the following gene combination: The gene combination is gene A and gene B; The gene A is niaP gene or PA niaP gene; the gene B is one of BM punC gene, SP punC gene, CP punC gene and SA punC gene; The niaP gene is derived from Burkholderia cepacia; the PA niaP gene is derived from Paraburkholderia acidicola; the BM punC gene is derived from Bacillus mycoides; the SP punC is derived from Streptococcus pneumoniae; the CP punC is derived from Clostridium perfringens; and the SA punC is derived from Streptococcus agalactiae. The chassis of the recombinant strain is Escherichia coli in which ccNAMPT1 gene is inserted and pncC, nadR, ushA and pncB genes are knocked out; The nucleotide sequence of the ccNAMPT1 gene is shown in SEQ ID NO:

7.

5. The recombinant strain of claim 4, wherein, The original pgi, edd and pncA genes in the chassis are replaced by ptsG, gnd and prs genes in sequence.

6. The method for constructing a recombinant strain according to claim 4 or 5, wherein, The steps are as follows: (1) Knocking out pncC, nadR, ushA and pncB of the chassis and inserting ccNAMPT1 gene; (2) Transferring the gene combination in the recombinant strain of claim 4.

7. The construction method of claim 6, wherein, The original pgi, edd and pncA genes in the chassis are replaced by ptsG, gnd and prs genes in sequence.

8. A method for the preparation of NMN, characterized in that, With NAM as substrate, the recombinant strain of any one of claims 4 or 5 or the recombinant strain constructed by the construction method of any one of claims 6-7 is fermented, and the fermentation broth is extracted and purified to obtain NMN.

9. The production method according to claim 8, characterized by, The fermentation includes initial fermentation and fed-batch fermentation: The initial fermentation includes inoculating the recombinant strain into fermentation medium and fermenting for 5-7 hours; the medium of the initial fermentation includes 10-25 g / L glucose and 1-10 g / L NAM; The fed-batch fermentation includes adding a feed solution containing 400-600 g / L glucose, 40-80 g / L yeast extract and 40-80 g / L tryptone, fermenting, inducing expression, and collecting the product containing NMN.

10. The method of claim 9, wherein, The feed rate of the feed solution is linearly increased from 10 mL / h to 20 mL / h.

11. The preparation method according to claim 8, characterized in that, The extraction includes ultrasonic disruption extraction; and the purification method includes size exclusion chromatography purification.

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