Recombinant strain for producing nicotinamide mononucleotide and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a recombinant strain for producing nicotinamide mononucleotide and application of the recombinant strain. According to the method, accumulation of the NMN is improved through metabolic flux redirection and gene knockout, a synthesis path of the NMN is successfully established in escherichia coli, and further, the yield of the NMN is effectively improved and reaches 7.0 g / L by optimizing a transport system of NAM-NMN and a PRPP synthesis path. The invention also optimizes the production process of the NMN, realizes the highest yield of the NMN of 28.5 g / L in a fed-batch fermentation system, and obviously improves the yield compared with the traditional batch fermentation.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant strain producing nicotinamide mononucleotide and an application thereof. Background Art

[0002] Nicotinamide Mononucleotide (NMN) is a nicotinamide adenine dinucleotide (NAD + ) is an important intermediate in the biosynthesis pathway and is widely present in mammals and microorganisms. In recent years, NMN has attracted widespread attention due to its potential applications in anti-aging and prevention of metabolic diseases (such as type 2 diabetes, obesity, and heart failure). + As a key coenzyme in cellular energy metabolism, NMN plays an important role in maintaining mitochondrial function, anti-inflammation, promoting cell survival and delaying aging. + It 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 require the use of expensive raw materials such as nicotinamide riboside (NR), phosphoribosyl pyrophosphate (PRPP) and a variety of catalysts (such as ATP, AMP, NAD + Although chemical and enzymatic synthesis have the advantages of high efficiency, they are costly, complex and potentially harmful to the environment. Therefore, the production of NMN through microbial fermentation has gradually become a more economically feasible and environmentally friendly option.

[0004] Currently, microbial-based NMN production is mainly achieved through fermentation methods using recombinant engineered strains (such as Escherichia coli). However, traditional batch fermentation processes often face the following problems:

[0005] 1. Low production efficiency: Due to the rapid consumption of substrates, accumulation of metabolites, and inhibition of bacterial metabolism, the product yield and output of batch fermentation are relatively low. Studies have shown that through batch fermentation methods, recombinant E. coli can only produce a maximum of about 2.31mM NMN.

[0006] 2. Substrate and nutrient inhibition: Under conditions of high substrate concentrations (such as glucose and nicotinamide), microbial metabolism will produce byproducts such as acetic acid, thereby inhibiting cell growth and reducing the production efficiency of NMN. In addition, excessive concentrations of NAM (nicotinamide) will also have a toxic effect on cells, further inhibiting product production.

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

[0008] In current research, methods to increase the production of NMN by high-density cell culture and fed-batch fermentation have been proposed. For example, Shoji et al. used fed-batch fermentation technology to transform recombinant E. coli and used glucose and nicotinamide as substrates in 1L of culture medium to achieve a maximum NMN production of 6.79 g / L. However, this method still has the following shortcomings:

[0009] 1. Low substrate utilization efficiency: Although fed-batch fermentation has improved the production efficiency of NMN to a certain extent, the single substrate supply strategy it adopts easily leads to the accumulation of sugar substrates, followed by the production of harmful byproducts such as acetic acid, which ultimately inhibits bacterial growth.

[0010] 2. Selection of nutrients and inducers: The method of Shoji et al. relies on expensive inducers (such as IPTG) to initiate gene expression in strains, which will significantly increase production costs in industrial-scale production. In addition, the culture medium formula and feeding strategy in the existing scheme are relatively simple, which is difficult to adapt to the dynamic demand for nutrients in different fermentation stages.

[0011] 3. Complex purification process: The traditional NMN purification process usually requires multi-step chromatographic separation, such as ion exchange and reversed-phase high-performance liquid chromatography (HPLC), which not only increases 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. Therefore, the present invention aims to significantly improve the yield and purity of NMN and reduce production costs by optimizing the high-density cell culture and linear fed-batch fermentation strategy of recombinant E. coli, as well as a simplified size exclusion chromatography purification process, thereby achieving the economic feasibility of industrialization. Summary of the invention

[0013] In view of this, the present invention provides a recombinant strain producing nicotinamide mononucleotide and its application.

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

[0015] The gene combination includes gene A and gene B; gene A includes niaP gene and / or SBniaP gene; 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] ; The SPpunC is derived from Streptococcus pneumoniae; the CPpunC is derived from Clostridium perfringens; and the 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) the nucleotide sequence shown in SEQ ID NO: 1 to 6; or

[0020] 2) A nucleotide sequence obtained by replacing, deleting or adding one or more bases in the sequence shown in 1), and having the same or similar function as 1); or

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

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

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

[0024] In some embodiments, the aforementioned gene combination further comprises 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 replacing, deleting or adding one or more bases in the sequence shown in I), and having the same or similar functions as I); or

[0027] III) A nucleotide sequence having at least 80% homology with the sequence shown in I) or II).

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

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

[0030] 2) A nucleotide sequence obtained by replacing, deleting or adding one or more bases in the sequence shown in 1), and having the same or similar function as 1); or

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

[0032] In the application of the present invention, the microorganism ferments NAM with NAM as a substrate to produce NMN. The microorganism includes Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Corynebacterium glutamicum, Bacillus subtilis or lactic acid bacteria. In a specific embodiment of the present 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 of the source and / or the SBniaP gene of the source; the gene B includes at least one of BMpunC, SPpunC, CPpunC, and SApunC.

[0035] In some specific embodiments, the gene combination of the present invention includes 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 of the present invention, the sources and specific sequences of genes A and B are as described above and will not be repeated here.

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

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

[0045] In some embodiments, the gene combination further comprises the ccNAMPT1 gene, wherein the source and specific sequence of the ccNAMPT1 gene are as described above.

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

[0047] In the expression unit of the present invention, the promoter is selected from T7 promoter, trc, tac, lacV, pFAB46, or pFAB100. In a specific embodiment of the present invention, the promoter is T7 promoter. On this basis, the expression unit may also include regulatory elements such as enhancers, replicons, terminators, etc., which are not particularly limited in the present invention.

[0048] The present invention also provides an expression vector, which comprises the gene combination or the expression unit.

[0049] In the present invention, the backbone of the expression vector includes pET, pCDFpRSF, and pGEX series vectors, specifically pET21b, pCDFDuet-1, pRSFDute-1, and pGEX-5x-1, including but not limited to these. In a specific embodiment of the present invention, the backbone is pET21b and pCDFDuet-1.

[0050] The present invention also provides a recombinant strain, which is transformed or transfected with the expression vector, or has the gene combination integrated into its genome, or has the expression module integrated into its genome.

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

[0052] The base bacteria of the recombinant strain of the present invention include Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis or lactic acid bacteria. In some embodiments of the present invention, the base bacteria is Escherichia coli. Specifically, the Escherichia coli is selected from. In a specific embodiment, the present invention uses E. coli BL21 (DE3) as an example to transform the genetically engineered strain.

[0053] In some specific embodiments, the present invention uses E. coli BL21 (DE3) as the base strain, knocks out pncC, nadR, ushA and pncB, and inserts the ccNAMPT1 gene related to NMN synthesis to obtain the recombinant strain ENM02, which improves the synthesis efficiency of nicotinamide mononucleotide (NMN) and reduces the degradation of NMN. The experiment showed that the NMN accumulation of the ENM02 recombinant strain after 48 hours of shake flask fermentation reached 3.5g / L, which was 2.6 times higher than that of the control group without gene knockout ( Figure 1 ).

[0054] Furthermore, the present invention uses ENM02 as the starting strain, and optimizes the transport system of NAM) and NMN by introducing transport proteins. The present invention specifically compares the effects of introducing transport protein combinations from different sources on NMN production, and screens out 6 transport protein combinations that can significantly increase NMN production (combinations 1 to 6 as described above), and obtains 6 recombinant bacteria ENM04-1 to ENM04-4 and ENM04-10 to ENM04-11, respectively, with NMN production of 3.8 to 5.2 g / L in 48 hours. Among them, the NMN production of ENM04-1 after 48 hours of shake flask fermentation reached 5.2 g / L.

[0055] Furthermore, by optimizing the synthesis pathway of PRPP (phosphoribosyl pyrophosphate), the yield of NMN is further increased. The specific operations include: the present invention uses ENM04-1 (named ENM04) as the starting bacteria, knocks out the pgi gene (ACT45688.1), and inserts the ptsG gene (ACT42992.1) at the corresponding site to increase the flow of glucose to the phosphorylation pathway, knocks out the edd gene (ACT43676.1), and inserts the gnd gene (ACT43782.1) at the corresponding site to increase the flow of the pentose phosphate pathway (PPP), thereby increasing the generation of PRPP. Knock out the pncA gene (ACT43591.1), insert the prs gene (ACT43074.1) at the corresponding site, and obtain the recombinant strain ENM05. The NMN yield reaches 7.0g / L after 48 hours of shake flask fermentation.

[0056] The present invention also provides a method for constructing a recombinant strain, comprising the following steps:

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

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

[0059] (3) The original pgi, edd and pncA genes of the chassis bacteria were replaced with ptsG, gnd and prs genes respectively.

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

[0061] The preparation method of the present invention comprises: using NMA as a substrate, fermenting the recombinant strain described in any one of claims 10 or 11 or the recombinant strain constructed by the construction method of claim 12; extracting and purifying the obtained fermentation liquid 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 comprises: inoculating the recombinant strain into a fermentation medium and fermenting for 5 to 7 hours; the medium of the initial fermentation comprises 10 to 25 g / L glucose and 1 to 10 g / L NAM.

[0064] 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 trypsin, fermenting, inducing expression, and collecting NMN-containing products.

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

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

[0067] In the present invention, the fermentation medium of the initial fermentation includes a basal medium and nicotinamide (NAM). In some embodiments, the concentration of nicotinamide in the fermentation medium of the initial fermentation is 1 to 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 numerical ranges.

[0068] In the present invention, the initial fermentation includes: culturing at 37°C, 300rpm, and a ventilation volume of 1.5vvm.

[0069] In the present invention, when the glucose in the fermentation tank is exhausted (about 6 hours), the fed batch fermentation is started. In the present invention, in the fed batch fermentation, the concentration of glucose in the feed solution is 400-600g / L, specifically 400g / L, 500g / L, 600g / L, or any value within the above two numerical ranges. The concentration of yeast extract in the feed solution is 40-80g / L, specifically 40g / L, 50g / L, 60g / L, 70g / L, 80g / L, or any value within the above two numerical ranges. In the feed solution, the concentration of trypsin is 40-80g / L, specifically 40g / L, 50g / L, 60g / L, 70g / L, 80g / L, or any value within the above two numerical ranges.

[0070] In the present invention, in the fed-batch fermentation, fermentation to OD 600 When the OD value is 100-180, the expression is induced. 600 The value is 100, 110, 120, 130, 140, 150, 160, 170, 180, or any value within the above two numerical ranges. In the present invention, the feeding rate of the feeding solution increases linearly from 10 mL / h to 20 mL / h.

[0071] The present invention adopts a linear feeding strategy, gradually supplements high concentrations of glucose and substrate solution (nicotinamide), reasonably controls the substrate concentration, reduces the accumulation of byproducts such as acetic acid, and achieves stable production under high-density cell culture conditions. At the same time, the precise control of dissolved oxygen and nutrient supply ultimately improves the fermentation efficiency and yield of NMN.

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

[0073] In the present invention, when OD600 reaches 150 or more, the feed 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 1% lactose solution.

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

[0075] In the present invention, the purification method includes size exclusion chromatography (SEC) purification. In some specific embodiments, the chromatographic column of the size exclusion chromatography (SEC) is specifically a HiPrep 26 / 60 Sepacryl S-200HP size exclusion chromatography column.

[0076] The present invention uses Escherichia coli as the base bacteria, and realizes the efficient and sustainable production of NMN by increasing the supply of PRPP, optimizing the transport system and reducing the degradation pathway, and finally effectively improves the yield and purity of NMN, making it more suitable for industrial economic production. The specific transformation strategy includes the following three aspects:

[0077] 1. Knock out NMN-related degradation genes and reduce product loss: In order to reduce the degradation of NMN, the present 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, significantly improving the final yield. The cumulative amount of NMN fermented in a shake flask within 48 hours reaches 3.5 g / L.

[0078] 2. Optimize NAM and NMN transport and enhance metabolic flow control: The present invention improves the transport efficiency of NAM into the cell and promotes the effective transport of NMN to the extracellular space by optimizing the cell transport system. By increasing the concentration of NAM in the cell, the raw material supply for NMN synthesis is ensured to be sufficient; at the same time, by enhancing the transport of NMN to the extracellular space, the accumulation pressure of NMN in the cell is reduced, thereby achieving dynamic balance and control of the intracellular metabolic flow, ensuring efficient production of NMN, and the shake flask fermentation of NMN reaches 5.2g / L within 48 hours.

[0079] 3. Increase the supply of PRPP and improve the efficiency of NMN synthesis: The present invention reduces the consumption path of PRPP and optimizes the metabolic flow to the supply of PRPP, thereby increasing the intracellular concentration of PRPP and accelerating the synthesis of NMN. By regulating the metabolic pathway of PRPP generation, it is preferentially used for the synthesis of NMN, overcoming the limitations caused by insufficient PRPP supply and improving the yield of NMN. The cumulative yield of NMN in 48-hour shake flask fermentation reaches 7.0g / L.

[0080] Furthermore, the present invention also optimizes the preparation process of NMN, including the optimization of the fermentation process and the purification process:

[0081] (1) Fermentation process

[0082] In traditional batch fermentation, adding substrates at one time is not conducive to cell growth and metabolism, and the NMN production is not ideal. The present invention adopts linear fed-batch fermentation to ensure the stable growth of cells in high-density culture by gradually supplementing substrates and controlling dissolved oxygen levels, while reducing the accumulation of by-products.

[0083] (2) Purification process

[0084] After the fermentation is completed, the bacteria are collected by centrifugation and the cells are broken. After extraction and purification, NMN can be obtained. The traditional multi-step purification process of NMN is time-consuming and costly, and the purification yield is low, which makes it difficult to meet the economic requirements of large-scale production. The present invention optimizes the purification process of NMN, uses size exclusion chromatography (SEC) for single-step purification of NMN, increases the product purity to more than 98%, and maintains a purification yield of 53.9%.

[0085] Through the NMN process optimized by the present invention, a maximum NMN yield of 28.5 g / L was achieved in the fed-batch fermentation system, which is significantly higher than the yield of traditional batch fermentation. It shows that the present invention can achieve efficient production of NMN on an industrial scale through the combination of metabolic flow regulation, transport system optimization and genetic engineering technology. It can be widely used in the fields of medicine, health products and bioenergy, and has significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 Shows the shake flask fermentation results of the NMN degradation gene knockout strain;

[0087] Figure 2 The results of shake flask fermentation of the transporter overexpressing strain are shown;

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

[0089] Figure 4 The results of the comparison between fed-batch fermentation and traditional batch fermentation on NMN production are shown;

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

[0091] Figure 6 Figure 4 shows LC-MS analysis of purified NMN. DETAILED DESCRIPTION

[0092] The present invention provides a recombinant strain producing nicotinamide mononucleotide and its application. . Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

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

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

[0095] In the present invention, the high-yield engineering strain is constructed from recombinant E. coli BL21 (DE3). The engineering strain with high-efficiency PRPP synthesis ability is constructed by gene insertion and knockout strains and expression regulation by pET21b vector.

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

[0097] The present invention provides a method for improving the efficiency of nicotinamide (NAM) transport into cells. By enhancing the transport protein on the cell membrane, the efficient uptake of NAM is achieved, the concentration of NAM in the cell is significantly increased, and sufficient substrate is provided for the synthesis of NMN. In addition, by overexpressing the transport protein, the extracellular secretion efficiency of NMN is improved, and the accumulation pressure of NMN in the cell is reduced.

[0098] In order to reduce the degradation of NMN, the present invention prevents the synthesized NMN from being further metabolized and decomposed by knocking out genes related to NMN degradation. The gene knockout method uses the CRISPR-Cas9 system to ensure that the strain maintains efficient and stable NMN accumulation during the production process.

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

[0100] During the induction phase of fermentation, when OD600 reached 100-120, 1% lactose was added instead of IPTG as an inducer to reduce the induction cost and reduce the inhibition of cell growth. By optimizing the fermentation temperature (the temperature during the induction period was controlled at 30°C) and pH (maintained at 6.8-7.0), the efficient expression of the recombinant strain and the continuous production of NMN were ensured.

[0101] After the fermentation is completed, the bacteria are collected by centrifugation and the cells are broken. The present invention uses an ultrasonic lysis method to extract intracellular NMN. In order to achieve efficient purification, the separation and purification of the NMN is carried out by single-step size exclusion chromatography (SEC). The chromatographic column used is HiPrep 26 / 60Sepacryl S-200HP, the flow rate is set to 0.4mL / min, and 50mM formic acid is used as the isocratic elution solvent. The purified NMN was verified by liquid chromatography-mass spectrometry (LC-MS), and the purity reached more than 98%, and the overall yield was 53.9%.

[0102] By the method of the present invention, a maximum NMN yield of 28.5 g / L was achieved in a fed-batch fermentation system, which was significantly higher than that of conventional batch fermentation. The successful implementation of the method shows that efficient production of NMN can be achieved on an industrial scale through the combination of metabolic flux regulation, transport system optimization and genetic engineering technology.

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

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

[0105]

[0106]

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

[0108]

[0109]

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

[0111]

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

[0113]

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

[0115]

[0116]

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

[0118]

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

[0120]

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

[0122]

[0123] The present invention will be further described below in conjunction with embodiments:

[0124] Example 1: Knockout of NMN degradation genes and its effect on NMN production

[0125] (I) Implementation steps:

[0126] In this example, the gene of E. coli BL21 (DE3) strain was knocked out to improve the synthesis efficiency of nicotinamide mononucleotide (NMN) and reduce the degradation of NMN. The specific operation steps are as follows:

[0127] 1. Strain construction:

[0128] The present invention uses E. coli BL21 (DE3) as the starting strain, firstly, the CRISPR-Cas9 gene editing technology is used to knock out multiple genes related to NMN degradation, including pncC (ACT44369.1), nadR

[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. Strain ENM01 was obtained.

[0130] 2. Plasmid construction and transformation:

[0131] Based on the above knockout genes, the expression plasmid pET21b vector was used to insert the ccNAMPT1 gene sequence related to NMN synthesis to enhance the biosynthesis of NMN. The constructed expression plasmid was introduced into the ENM01 strain under the selection pressure of ampicillin (75 μg / mL) to obtain ENM02. At the same time, 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 Luria-Bertani (LB) medium and cultured at 37°C and 200 rpm for 12 hours as seed solution. Subsequently, 5% (volume ratio) of the seed solution was transferred into a 250 mL Erlenmeyer flask containing 100 mL of M9 medium (composition: 13.3 g / L KH2PO4, 4 g / L (NH 4 ) 2 HPO 4 、1.2g / L MgSO 4 7H 2 The fermentation experiments were carried out at 37°C and 200 rpm for 24 and 48 hours.

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

[0135] 6 hours after the start of fermentation, 1% lactose (final concentration) was added to induce expression. 10 mL of samples were taken at 24 hours and 48 hours for subsequent NMN detection and analysis. The samples were centrifuged (8000 rpm, 4 ° C, 10 minutes) to separate the supernatant and filtered with a 0.22 μm PTFE filter to ensure the purity of the fermentation broth.

[0136] 5. Detection method of NMN concentration:

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

[0138] (II) Experimental results:

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

[0140] Example 2: Optimization of NAM and NMN transport systems and their effects on NMN production

[0141] (I) Implementation steps:

[0142] In this example, the transport system of nicotinamide (NAM) and nicotinamide mononucleotide (NMN) was optimized to improve the NMN production efficiency of recombinant E. coli. The specific steps are as follows:

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

[0144] This embodiment uses the E. coli BL21 (DE3) recombinant strain ENM02 with gene knockout and metabolic flux optimization as the starting strain. On this basis, the pCDFDuet-1 expression vector is used to insert the transporter coding sequence and express the two transporters simultaneously through homologous recombination technology.

[0145] BMpunC (WP_215553083.1) and homologous enzymes SPpunC (CJB91739.1), CPpunC (HFK8301865.1), SApunC (HFU9558888.1) and niaP (WP_062884942.1) and homologous enzymes SBniaP (HKU12810.1), PAniaP (WP_349544922.1) were selected, and after Escherichia coli codon optimization, 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, respectively. 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 on LB solid medium 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 the correct insertion and expression of the gene.

[0150] 3. Shake flask fermentation experimental conditions:

[0151] ENM02 and ENM04 strains were selected for comparative fermentation experiments. The seed solution was inoculated into a 250 mL triangular flask containing 100 mL M9 medium (13.3 g / L KH 2 PO 4 , 4g / L(NH 4 ) 2 HPO 4 、1.2g / L MgSO 4 7H 2 O, 20 g / L glucose and 5 g / L NAM) and cultured in shaking flasks 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 of samples were taken at 24 hours and 48 hours of fermentation. The samples were centrifuged (8000 rpm, 4°C, 10 minutes) to separate the supernatant, filtered with a 0.22 μm PTFE filter, and then the NMN concentration was detected.

[0154] 5. Detection method of NMN concentration:

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

[0156] (II) Experimental results:

[0157] The 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 ( Figure 2). This result shows 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 niaP and SPpunC genes, the present invention optimizes the NAM uptake and NMN secretion system of recombinant E. coli, so that the NMN production reaches 5.2 g / L within 48 hours, which is 49% higher than that of the unoptimized control group. This proves the important role of overexpression of transporters in improving metabolic flow efficiency and NMN production.

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

[0161] (I) Implementation steps:

[0162] This example further optimizes the synthesis pathway of PRPP (phosphoribosyl pyrophosphate) to improve the NMN production efficiency of the E. coli recombinant strain. The specific steps are as follows:

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

[0164] ENM04-1 with the niaP and SPpunC genes introduced was named ENM04. Based on the ENM04 strain, it was further modified to enhance the ability to synthesize PRPP. The following gene knockouts and insertions were performed using CRISPR-Cas9 technology:

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

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

[0167] 2. Screening and verification of strains:

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

[0169] 3. Shake flask fermentation experimental conditions:

[0170] ENM04 and ENM05 strains were selected for comparative fermentation experiments. The strain seed solution was inoculated into a 250 mL triangular flask containing 100 mL M9 medium (13.3 g / L KH 2 PO 4 , 4g / L(NH 4 ) 2 HPO 4 、1.2g / L MgSO 4 7H 2 O, 20 g / L glucose and 5 g / L nicotinamide (NAM). The shake flask was cultured at 37°C and 200 rpm.

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

[0172] After 6 hours of fermentation, 1% lactose (final concentration) was added to induce expression. 10 mL of samples were taken at 24 hours and 48 hours of fermentation to detect NMN concentration. The samples were centrifuged (8000 rpm, 4°C, 10 minutes) to separate the supernatant and filtered with a 0.22 μm PTFE filter.

[0173] 5. Detection method of NMN concentration:

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

[0175] (II) Experimental results:

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

[0177] in conclusion:

[0178] This example effectively improves the NMN synthesis capacity of the recombinant strain by enhancing the PRPP synthesis pathway. The results show that the cumulative NMN production of the genetically engineered ENM05 strain within 48 hours reached 7.0 g / L, which is 34% higher than that of the unmodified control group. The PRPP optimization strategy of the present invention has significant application potential in industrial-scale NMN production, which can effectively reduce production costs and increase yields.

[0179] At the same time, the present invention uses ENM04-2 and ENM04-3 as starting bacteria, and performs genetic 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 are 19% and 31% higher than the unmodified control group, respectively.

[0180] Example 4: Optimization of fed-batch fermentation strategy and improvement of NMN production

[0181] (I) Implementation steps:

[0182] This example optimizes the fed-batch fermentation process to improve the NMN production efficiency of the E. coli recombinant strain. The specific steps are as follows:

[0183] 1. Selection of starting strains:

[0184] In this example, the metabolically engineered efficient NMN production strain ENM05 was selected for fermentation experiments. To verify the effect of the fed-batch strategy on NMN production, traditional batch fermentation was used as a control.

[0185] 2. Fermentation conditions and medium preparation:

[0186] Fermentation container: 2.5L fermentation tank;

[0187] Initial working volume: 1.5L;

[0188] Basic culture medium (M9 medium), the composition is as follows:

[0189] KH 2 PO 4 : 13.3g / L;

[0190] (NH 4 ) 2 HPO 4 :4g / L;

[0191] MgSO 4 7H 2 O: 1.2 g / L;

[0192] Glucose: 20g / L;

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

[0194] Inducer lactose: added at a concentration of 1% during the fermentation process.

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

[0196] 3. Fermentation process:

[0197] (A) Traditional batch fermentation:

[0198] Seed cultivation:

[0199] The ENM05 strain was inoculated into 10 mL of LB medium and cultured at 37° C. and 200 rpm for 12 hours to serve as a seed solution.

[0200] 5% (volume ratio) of the seed solution was inoculated into a 2.5L fermenter, and the initial culture medium volume was 1.5L.

[0201] Fermentation stage:

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

[0203] No feeding operation was performed during the fermentation process. Glucose and NAM were added once and fully supplied at the beginning of the fermentation.

[0204] When glucose is exhausted (about 12 hours), the system enters the decay phase.

[0205] Sample collection:

[0206] After the start of fermentation, 10 mL of samples were taken every 5 h.

[0207] The sample was centrifuged (8000 rpm, 4°C, 10 minutes) to separate the supernatant, which was then filtered through a 0.22 μm PTFE filter membrane.

[0208] (B) Fed-batch fermentation:

[0209] Seed cultivation:

[0210] The ENM05 strain was inoculated into 10 mL of LB medium and cultured at 37° C. and 200 rpm for 12 hours to serve as a seed solution.

[0211] 5% (volume ratio) of the seed solution was inoculated into a 2.5L fermenter, and the initial culture medium volume was 1.5L.

[0212] Initial fermentation stage:

[0213] The fermenter was cultured at 37°C, 300 rpm, and 1.5 vvm. The glucose concentration was 20 g / L and the NAM concentration was 5 g / L.

[0214] When the glucose in the fermenter was exhausted (approximately 6 hours), the fed-batch phase was initiated.

[0215] Fed-batch phase:

[0216] Feed solution composition: 600 g / L glucose, 60 g / L yeast extract, 60 g / L tryptone.

[0217] Feeding strategy:

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

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

[0220] Phase 3 (24 hours later): When OD600 reached above 150, the feeding rate was maintained at 20 mL / h, and the expression of NMN synthesis-related genes was induced by adding 1% lactose.

[0221] Sample collection:

[0222] After the start of fermentation, 10 mL of samples were taken every 5 h.

[0223] The sample was centrifuged (8000 rpm, 4°C, 10 min) to separate the supernatant, which was then filtered through a 0.22 μm PTFE filter membrane for NMN concentration detection.

[0224] 4. Detection method of NMN concentration:

[0225] High performance liquid chromatography (HPLC, Younglin Inc. Korea) was used to quantify the NMN concentration. An Eclipse plus C18 column (4.6×250 mm, 5 μm) was used, the mobile phase was 10% acetonitrile, the flow rate was 1 mL / min, and the detection wavelength was 260 nm. The NMN concentration was quantitatively analyzed according to the standard curve.

[0226] (II) Experimental results:

[0227] The t-test analysis of the yield data of the ENM05 strain under the two fermentation strategies showed that the fed-batch fermentation strategy significantly increased the yield of NMN. Within 50 hours, the yield of the fed-batch fermentation strategy increased by 133% compared with the traditional batch fermentation.

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

[0229] in conclusion:

[0230] This example shows that by optimizing the fed-batch fermentation process, the NMN production efficiency of the recombinant strain of E. coli can be significantly improved. Compared with traditional batch fermentation, the fed-batch fermentation strategy effectively avoids the problems of substrate depletion and acetic acid accumulation, while promoting the continuous growth of cells and the increase in the synthesis rate of NMN. The fed-batch fermentation strategy of the present invention has broad application prospects in industrial-scale NMN production, which can significantly reduce production costs and increase NMN production.

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

[0232] (I) Implementation steps:

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

[0234] 1. Sample preparation:

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

[0236] The fermentation broth was first centrifuged (8000 rpm, 4°C, 20 minutes) to separate the supernatant to remove the bacteria. The separated supernatant was filtered with a 0.22 μm PTFE filter membrane to ensure the purity of the sample.

[0237] 2. Ultrasonication to extract NMN:

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

[0239] 3. Size Exclusion Chromatography (SEC) Purification:

[0240] A HiPrep 26 / 60 Sepacryl S-200HP size exclusion chromatography column (Cytiva, USA) was used for single-step purification of NMN.

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

[0242] Flow rate: 0.4mL / min

[0243] Elution mode: isocratic elution

[0244] Fractions were collected at designated time intervals using an automated collector.

[0245] 4. HPLC analysis to confirm purity:

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

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

[0248] Compare the retention time of known NMN standards to confirm the purity of the target product.

[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 more than 98%, which was significantly higher than that of the unpurified fermentation broth (initial purity was about 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] + ) is consistent with the standard ( Figure 6 ).

[0255] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of overexpression gene combination in microbial fermentation production of NMN; 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; The niaP gene is derived from Burkholderia cepacia; the SBniaP gene is derived from Steroidobacteraceae bacterium; the BMpunC gene is derived from Bacillus mycoides; the SPpunC is derived from Streptococcus pneumoniae; the CPpunC is derived from Clostridium perfringens; and the SApunC is derived from Streptococcus agalactiae.

2. The use according to claim 1, characterized in that: The niaP, SBniaP, BMpunC, SPpunC, CPpunC, and SApunC genes have the following characteristics: 1) the nucleotide sequence shown in SEQ ID NO: 1 to 6; or 2) A nucleotide sequence obtained by replacing, deleting or adding one or more bases in the sequence shown in 1), and having the same or similar function as 1); or 3) A nucleotide sequence having at least 80% homology with the sequence shown in 1) or 2).

3. The use according to claim 1 or 2, characterized in that: The gene combination further includes at least one of ptsG, 6-phosphogluconate dehydrogenase gene, and PRPP synthase gene; The ptsG, 6-phosphogluconate dehydrogenase gene and PRPP synthase gene are endogenous genes of the microorganism.

4. The use according to any one of claims 1 to 3, characterized in that: The gene combination also includes the ccNAMPT1 gene; the ccNAMPT1 gene has: I) the nucleotide sequence shown in SEQ ID NO: 7; or II), a nucleotide sequence obtained by replacing, deleting or adding one or more bases in the sequence shown in I), and having the same or similar functions as I); or III) A nucleotide sequence having at least 80% homology with the sequence shown in I) or II).

5. The use according to any one of claims 1 to 4, characterized in that: The microorganisms include Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Corynebacterium glutamicum, Bacillus subtilis or lactic acid bacteria.

6. A gene combination, characterized in that Includes gene A and gene B; The gene A includes the niaP gene of the source and / or the SBniaP gene of the source; the gene B includes at least one of BMpunC, SPpunC, CPpunC, and SApunC.

7. The gene combination according to claim 6, characterized in that: Also includes at least one of the following a) to b); a) at least one of ptsG, 6-phosphogluconate dehydrogenase gene, and PRPP synthase gene; b) ccNAMPT1 gene.

8. An expression module, characterized in that It comprises a promoter and at least one gene in the gene combination according to any one of claims 6 to 7.

9. An expression vector comprising the gene combination according to any one of claims 6 to 7 or the expression module according to claim 8.

10. A recombinant strain, characterized in that It is transformed or transfected with the expression vector according to claim 9, or the gene combination according to claim 6 or 7 is integrated into its genome, or the expression module according to claim 8 is integrated into its genome.

11. The recombinant strain according to claim 10, characterized in that The invention relates to an Escherichia coli which expresses ptsG, gnd, prs and the gene combination according to any one of claims 5 to 6, and in which pncC, nadR, ushA, pncB, pgi, edd and pncA genes are knocked out.

12. The method for constructing the recombinant strain according to claim 10 or 11, characterized in that: The steps include: (1) knocking out at least one of pncC, nadR, ushA and pncB in the bottom plate bacteria and inserting the ccNAMPT1 gene; (2) Gene A and gene B are introduced into the gene combination described in claim 5; (3) The original pgi, edd and pncA genes of the chassis bacteria were replaced with ptsG, gnd and prs genes respectively.

13. A method for preparing NMN, characterized in that: Using NAM as a substrate, the recombinant strain described in any one of claims 10 or 11 or the recombinant strain constructed by the construction method of claim 12 is fermented, and the fermentation broth is extracted and purified to obtain NMN.

14. The preparation method according to claim 13, characterized in that: The fermentation includes initial fermentation and fed-batch fermentation: The initial fermentation comprises: inoculating the recombinant strain into a fermentation medium and fermenting for 5 to 7 hours; the medium for the initial fermentation comprises 10 to 25 g / L glucose and 1 to 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 trypsin, fermenting, inducing expression, and collecting NMN-containing products.

15. The preparation method according to claim 14, characterized in that: The feeding rate of the feeding solution increased linearly from 10 mL / h to 20 mL / h.

16. The preparation method according to claim 13, characterized in that: The extraction includes ultrasonic extraction; and the purification method includes size exclusion chromatography purification.

Images