阅读说明：本技术 生产β-烟酰胺核糖的重组微生物及其构建方法和应用 (Recombinant microorganism for producing beta-nicotinamide ribose and construction method and application thereof ) 是由 李芳� 万丽花 胡志浩 于 2021-06-23 设计创作，主要内容包括：本发明提供了一种生产β-烟酰胺核糖(NR)的重组微生物及其构建方法与应用,属于生物技术领域。本发明首次发现核糖核苷水解酶RihA或RihB在调节β-烟酰胺核糖产量中的应用。基于上述发现,本发明制备了一种生产β-烟酰胺核糖的重组微生物,所述的重组微生物包含以下一个或几个特征：(1)编码核糖核苷水解酶的基因缺失、失活或活力降低,或核糖核苷水解酶的含量或活力降低；(2)过表达水解磷酸基团或者水解核苷的编码基因。采用本发明所述的重组微生物以β-烟酰胺单核苷酸(NMN)为底物生产NR,可有效提高NR的产量,减缓NR的降解,降低反应的副产物,可用于β-烟酰胺核糖的大规模工业化生产。(The invention provides a recombinant microorganism for producing beta-Nicotinamide Riboside (NR) and a construction method and application thereof, belonging to the technical field of biology. The invention discovers the application of the ribonucleoside hydrolase RihA or RihB in regulating the yield of beta-nicotinamide ribose for the first time. Based on the above findings, the present invention provides a recombinant microorganism producing β -nicotinamide riboside, said recombinant microorganism comprising one or more of the following characteristics: (1) a gene encoding a ribonucleoside hydrolase is deleted, inactivated or reduced in activity, or the content or activity of the ribonucleoside hydrolase is reduced; (2) overexpresses a gene encoding a hydrolyzed phosphate group or a hydrolyzed nucleoside. The recombinant microorganism of the invention uses beta-Nicotinamide Mononucleotide (NMN) as a substrate to produce NR, can effectively improve the yield of NR, slow down the degradation of NR and reduce reaction byproducts, and can be used for large-scale industrial production of beta-nicotinamide ribose.)

1. Use of a ribonucleoside hydrolase or a gene encoding therefor for modulating the production of β -nicotinamide ribose: the ribonucleoside hydrolase is one or more of RihA, RihB or RihC.

2. Use according to claim 1, characterized in that: (ii) a decrease in the content or activity of said ribonucleoside hydrolase and/or a deletion, inactivation or reduction in the activity of a gene encoding said ribonucleoside hydrolase to increase the yield of β -nicotinamide ribose;

or: an increased amount or activity of said ribonucleoside hydrolase and/or an increased copy number or activity of a gene encoding said ribonucleoside hydrolase to reduce the amount of β -nicotinamide ribose produced.

3. The application of the ribonucleotide hydrolase or the coding gene thereof in preparing microorganisms for producing beta-nicotinamide ribose with high yield or low yield is characterized in that: the ribonucleoside hydrolase is one or more of RihA, RihB or RihC.

4. A recombinant microorganism producing β -nicotinamide riboside, characterized by: the recombinant microorganism comprises the following characteristics:

(1) a gene encoding a ribonucleoside hydrolase is deleted, inactivated or reduced in activity, or the content or activity of the ribonucleoside hydrolase is reduced;

(2) overexpresses a gene encoding a phosphohydrolase or a nucleotidase.

5. The recombinant microorganism according to claim 4, wherein:

the gene for coding the ribonucleotide hydrolase is one or more of xapA, deoD, rihA, rihB, rihC, deoA, udP or cdh;

the coding gene of the phosphohydrolase or the nucleotidase is one or more of phoA, aphA, nadN or ushA.

6. The recombinant microorganism according to claim 5, wherein:

the gene for coding the ribonucleotide hydrolase is one or more of xapA, deoD, rihA, rihC or cdh;

the coding gene of the phosphohydrolase or the nucleotidase is ushA.

7. The recombinant microorganism according to claim 6, wherein: the microorganism is escherichia coli, bacillus, corynebacterium glutamicum, salmonella or yeast.

8. Use of a recombinant microorganism of any one of claims 4-7 for the production of β -nicotinamide ribose.

9. A method for producing β -nicotinamide riboside, comprising: the method comprises the fermentation production of beta-nicotinamide ribose by using the recombinant microorganism of any one of claims 4 to 7 or the catalytic substrate production of beta-nicotinamide ribose by using the recombinant microorganism whole-cell product of any one of claims 4 to 7.

10. The method of claim 9, wherein: the substrate is NAM or NMN.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a recombinant microorganism for producing beta-nicotinamide ribose, and a construction method and application thereof.

Background

beta-Nicotinamide Riboside (NR), a derivative of vitamin B3, is a precursor for the synthesis of Nicotinamide Adenine Dinucleotide (NAD) and Nicotinamide Adenine Dinucleotide Phosphate (NADP), and the like. NR is phosphorylated by nicotinamide nucleotide kinase (NRKs) and then converted to NMN and then further to NAD. It has been shown that NR supplementation increases NAD levels and activity in yeast, mammals and human cells cultured in vitro. NAD is always in a dynamic balance of consumption and generation in vivo, and the NAD serving as an important signal molecule in cells regulates some NAD-dependent deacetylases, including Poly ADP Ribose Polymerases (PARPs) and sirtuins longevity protein family sirtl-7 (homologous gene of Sir 2), and is widely involved in important vital activities of growth and development, apoptosis, inflammation, tumor, angiogenesis, neurodegenerative diseases, cell energy metabolism and the like of organisms. Another study has shown that NR can be present in milk as an important dietary source of NAD, but NMN has not been found in the diet and its presence in serum remains controversial, therefore researchers have suggested that NR may be an important presence of NAD precursors and thus can regulate NAD levels by diet.

The prior method for producing NR is mainly a chemical synthesis method, such as reaction realized by utilizing trimethylsilyl trifluoromethanesulfonate (TMSOTf), although the method has high yield, even if the selectivity of product configuration is high, the generation of alpha-nicotinamide ribose without any purpose is difficult to avoid, and meanwhile, residual trifluoromethanesulfonic acid ions can influence the product to be eaten as food, so that the industrial production cannot be realized efficiently; tin tetrachloride is used as a catalyst to replace trimethylsilyl trifluoromethanesulfonate, although the yield is high and the operation is simple, the stereoselectivity cannot be realized to obtain the beta-configuration nicotinamide ribose, and the final product cannot meet the related quality requirements. Therefore, the chemical synthesis method has the problems of long process reaction time, difficult detection, poor quality controllability, high product purification difficulty and the like.

NR is produced by a recombinant microorganism fermentation method, mainly by utilizing a biosynthesis way of a microorganism strain, so that the difficulty brought by using a chemical reagent to the later purification is avoided, and the method has the advantages of green product, low production cost and high benefit by taking sugar as a raw material, and greatly reduces the production cost of the fermentation method by adopting the existing genetic engineering breeding technology and high-yield optimization control technology; the enzyme catalysis method for producing NR is to utilize enzyme with specific function, NMN as substrate, and catalyze reaction to generate NR under specific condition, the whole reaction condition is mild, the conversion efficiency is high, and the method is more beneficial to improving the purity and content of NR products.

At present, articles and patents for producing NR by using a recombinant microbial fermentation method have been reported, but the fermentation unit of NR in the reports is very low, for example, in the CN201680078334.8 patent, by over-expressing NaMN amidase NadE from different sources in different strains (escherichia coli, bacillus subtilis and yeast), NR is produced by using a microbial self-metabolic pathway, and the yield is only about 0.1-100 mg/L. In these reports, the metabolic pathways of NMN and NR in microorganisms have not been fully elucidated, and only some catalytic genes are simply overexpressed, without removing the degradation or utilization genes, which is the main reason for the low NR production.

Nucleoside hydrolase (nucleoside hydrolase), an enzyme that decomposes nucleosides to form nitrogenous bases and pentose sugars. For example, Escherichia coli synthesizes several nucleoside hydrolase, such as RihA, RihB and RihC (Petersen 2001), and the three genes, rihA, rihB and rihC, respectively, encode proteins with amino acid sequences of 311aa, 313aa and 301aa, and it was found that RhiA, RihB and RihC can hydrolyze various nucleosides to generate corresponding purine or pyrimidine and ribose. Studies have shown that RhiA, RihB, and RihC can hydrolyze uridine and cytidine, but no related studies have shown that RhiA, RihB, and RihC are involved in the synthesis and catabolism of NR.

NR is unstable at neutral pH and high temperature, and the water solubility of NR is extremely high, so that the separation and purification of NR from a fermentation broth is very difficult. Therefore, there is a need to develop a method for producing NR, which has a high yield and is easy to operate and can be applied to industrial production.

Disclosure of Invention

Aiming at the defects, the invention provides a recombinant microorganism for producing beta-nicotinamide ribose and a construction method thereof, and a fermentation process control scheme of the recombinant microorganism is established, so that the recombinant microorganism can be used for high-efficiency industrial production of NR, or the recombinant microorganism is used as a host strain to express specific enzyme or enzyme combination, so that NR is catalytically accumulated in vitro, and the purposes of green, environmental protection and low cost NR production are achieved.

The terms:

1. NR: unless otherwise specified, the English abbreviation "NR" in the present invention refers to beta-nicotinamide riboside, the molecular formula of which is C₁₁H₁₆N₂O₅The structural formula is shown as the following formula (1).

2. Recombinant microorganisms: unless otherwise specified, the Chinese name "recombinant microorganism" in the present invention refers to a microorganism which has been artificially treated; the human treatment includes but is not limited to: gene knockout, gene suppression, gene silencing, gene insertion, gene mutation, exogenous expression or overexpression genes and other technical means capable of realizing gene operation in the field.

3. Host bacteria: unless otherwise specified, the Chinese name "host bacterium" in the present invention refers to a microorganism to be artificially treated.

4. Seed liquid: if not specifically stated, the Chinese name "seed solution" in the present invention refers to a microbial culture solution with a certain amount of microorganisms, and excellent activity and quality, which is obtained by activating microorganisms through a test tube slant, performing flat or shake culture or performing a seed tank step-by-step expansion culture.

5. High yield: unless otherwise specified, "high yield" in the present invention means that the beta-nicotinamide riboside is produced in a higher amount than that of the parent microorganism.

6. Low yield: unless otherwise specified, "low yield" in the present invention means that the amount of beta-nicotinamide riboside produced is lower than that of the parent microorganism.

7. Exogenous expression or overexpression: as used with respect to the present invention, is to be broadly construed to include any increase in the expression of one or more proteins (including one or more nucleic acids encoding one or more proteins) of a parent microorganism as compared to the expression level of said protein (including nucleic acid expression) under the same conditions. Should not be taken to mean that the protein (or nucleic acid) is expressed at any particular level.

8. Parent microorganism: is a microorganism from which the microorganism of the present invention is derived. The microorganism of the present invention may be produced by any method such as artificial or natural selection, mutation or gene recombination. The parent microorganism can be a naturally occurring microorganism (i.e., a wild-type microorganism) or a microorganism that has been previously modified but does not produce or overproduces beta-nicotinamide ribose.

9. Carrier: it should be taken broadly to include any nucleic acid (including DNA and RNA) suitable for use as a vehicle for transferring genetic material into a cell, including plasmids, viruses (including phages), cosmids, and artificial chromosomes. The vector may include one or more regulatory elements, an origin of replication, a multiple cloning site, and/or a selectable marker.

10. Convention for protein and gene names: the English protein names related in the invention are all capital letters; the English gene names related in the invention are all in lower case italics.

In order to achieve the above object, the technical solution of the present invention is as follows:

in one aspect, the invention provides the use of a ribonucleoside hydrolase, which is one or more of RihA, RihB or RihC, or a gene encoding it, for modulating the production of β -nicotinamide ribose.

Specifically, the content or activity of the ribonucleoside hydrolase is reduced, and/or the gene encoding the ribonucleoside hydrolase is deleted, inactivated or reduced in activity, to increase the yield of β -nicotinamide ribose;

or: an increased amount or activity of said ribonucleoside hydrolase and/or an increased copy number or activity of a gene encoding said ribonucleoside hydrolase to reduce the amount of β -nicotinamide ribose produced.

In another aspect, the present invention provides a use of a ribonucleoside hydrolase, which is one or more of RihA, RihB or RihC, or a gene encoding the same, in a microorganism producing β -nicotinamide ribose with high or low yield.

In yet another aspect, the present invention provides a recombinant microorganism producing β -nicotinamide riboside, said recombinant microorganism comprising the following characteristics:

(1) a gene encoding a ribonucleoside hydrolase is deleted, inactivated or reduced in activity, or the content or activity of the ribonucleoside hydrolase is reduced;

(2) overexpresses a gene encoding a phosphohydrolase or a nucleotidase.

Specifically, the gene encoding the ribonucleoside hydrolase is one or more of xapA, deoD, rihA, rihB, rihC, deoA, udP or cdh.

Further specifically, the gene encoding ribonucleoside hydrolase is one or more of xapA, deoD, rihA, rihC or cdh.

Specifically, the coding gene of the phosphohydrolase or the nucleotidase is one or more of phoA, aphA, nadN or ushA.

More specifically, the coding gene of the phosphohydrolase or the nucleotidase is ushA, and the protease coded by ushA is 5' -nucleotidase.

Specifically, the microorganisms include, but are not limited to, Escherichia coli (Escherichia coli), Bacillus (Bacillus), Corynebacterium glutamicum (Corynebacterium glutamicum), Salmonella (Salmonella), and Yeast (Yeast).

In still another aspect, the present invention also provides the use of the above recombinant microorganism for the production of β -nicotinamide riboside.

In yet another aspect, the present invention provides a method for producing β -nicotinamide riboside, which comprises the steps of producing β -nicotinamide riboside by fermentation using the above recombinant microorganism or producing β -nicotinamide riboside by using a whole-cell product catalytic substrate of the above recombinant microorganism; the whole cell products include but are not limited to: a culture medium, a cell lysate, a supernatant fraction of the cell lysate, a precipitate fraction of the cell lysate, and the like. It is to be noted that new technologies for the manipulation of whole-cell products and their specific product types in future technological developments are also within the scope of the present invention as claimed above without departing from the inventive aspects of the present invention.

Specifically, the substrate is NAM or NMN.

Specifically, the method comprises the following steps:

activating the recombinant microorganism and preparing a seed solution;

adopting shake flask fermentation: inoculating into fermentation medium according to 8-12% inoculum size, culturing at 37 deg.C with shaking table at 250rpm for 3-4h, adding IPTG with final concentration of 1mM, simultaneously adding Nicotinamide (NAM) as substrate with final concentration of 300mg/L, adjusting shaking table temperature to 34 deg.C, and fermenting for 18-22 h.

Or fermenting by adopting a fermentation tank: inoculating 8-12% of the strain into fermentation medium, culturing at 37 deg.C, maintaining pH at 6.9 and dissolved oxygen at 30-45%, starting coupling feeding when dissolved oxygen is higher than 40%, culturing for 7-9h, maintaining OD600 to 10-30, maintaining temperature at 37 deg.C, adding IPTG with final concentration of 0.4mmol/L, inducing at 0.3-0.5 g/L.h^-1The substrate Nicotinamide (NAM) is fed-batch at the rate of (1), and the fermentation period is 18-20 h.

It should be noted that new techniques for manipulating microorganisms in future technical developments without departing from the scope of the present invention should also be within the scope of the present invention as defined in the above claims.

In further detail, in shake flask fermentation,

the inoculation amount is 10 percent.

The fermentation medium comprises the following components: 20-40g of glucose per liter, 220mL of 5N-5 times of saline solution 180-; wherein the salt solution with the volume of 5N-5 times is 75.6g of disodium hydrogen phosphate dodecahydrate, 15g of potassium dihydrogen phosphate, 2.5g of sodium chloride and 25g of ammonium chloride, and the volume is fixed to 1L by using ionized water; the TM3 solution is 2.0g of zinc chloride tetrahydrate, 2.0g of calcium chloride hexahydrate, 2.0g of sodium molybdate dihydrate, 1.9g of copper sulfate pentahydrate, 0.5g of boric acid, 100mL of hydrochloric acid, and the volume of deionized water is up to 1L.

The fermentation period is 20 h.

More specifically, the fermentation medium comprises the following components: 30g of glucose, 200mL of 5N-5 times of salt solution, 1mL of TM2 solution, 10mg of ferric citrate, 246mg of magnesium sulfate heptahydrate, 111mg of calcium chloride and 1 mu g of thiamine in each liter of culture medium, and the volume is fixed to 1L by sterile deionized water.

Further specifically, in the fermentation in a fermenter,

the inoculation amount is 10 percent.

The fermentation medium is a semisynthetic medium, 4-6g of ammonium sulfate, 1-3g of sodium chloride, 3-5g of monopotassium phosphate, 1-3g of magnesium sulfate heptahydrate, 10-20g of glucose, 0.05-0.15g of calcium chloride, 0.005-0.015g of zinc chloride, 30.5-1.5 mL of TM, 90-100mg of ferric citrate, 2-8g of corn steep liquor, 3-3 mg of VB 12, 35-45mg of NA and 0.5-1.5g of foam killer, and deionized water is used for fixing the volume; the feed medium is 550g of glucose containing 450-; the TM3 solution contains 2.0g of zinc chloride tetrahydrate, 2.0g of calcium chloride hexahydrate, 2.0g of sodium molybdate dihydrate, 1.9g of copper sulfate pentahydrate, 0.5g of boric acid and 100mL of hydrochloric acid per liter, and the volume of deionized water is fixed to 1L.

The fermentation period is 19 hours.

More specifically, the fermentation medium is a semisynthetic medium, and each liter of the medium contains 5g of ammonium sulfate, 2g of sodium chloride, 4g of monopotassium phosphate, 2g of magnesium sulfate heptahydrate, 15g of glucose, 0.105g of calcium chloride, 0.01g of zinc chloride, TM 31 mL, 94mg of ferric citrate, 5g of corn steep liquor, 12.5 mg of VBE, 40mg of NA and 1g of natural enemy, and the volume is determined by deionized water; the feed medium contained 500g of glucose per liter and was adjusted to pH 6.9.

Compared with the prior art, the invention has the advantages that:

(1) the invention discovers for the first time that the ribonucleoside hydrolase RihA, RihB or RihC can be used as a key enzyme in the degradation and metabolism process of the beta-nicotinamide ribose and can be used for regulating the yield of the beta-nicotinamide ribose.

(2) Based on the discovery, the recombinant microorganism of the invention is used for producing the beta-nicotinamide ribose, has higher yield, simple operation, short fermentation period and high production intensity, is beneficial to the separation and purification of subsequent products, and can be used for the large-scale industrial production of the beta-nicotinamide ribose.

(3) The construction method of the recombinant microorganism provided by the invention is a directed rational strain construction method, and is more efficient, convenient and highly operable compared with the traditional mutagenesis method.

Deposit description

And (3) classification and naming: escherichia coli

Latin name: escherichia coli

The biological material of the reference: SHQ05C

The preservation organization: china general microbiological culture Collection center

The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)

And (4) storage address: xilu No. 1 Hospital No. 3, the institute of microbiology, China academy of sciences, Beijing, Chaoyang

The preservation date is as follows: 2021, 05 month, 28 days

Registration number of the preservation center: CGMCC No. 22633.

Drawings

FIG. 1 is a schematic diagram of the metabolic pathway of E.coli NR.

FIG. 2 is an HPLC chromatogram for detection of NR and each byproduct.

FIG. 3 is a graph showing the reaction progress of SHA04K/pHQ07 enzyme solution.

FIG. 4 is a graph showing the comparison of reactions for expressing UshA enzyme solutions in SHQ01C, SHQ02C, and SHA04K hosts.

FIG. 5 is a graph showing the formation of NR by the reaction of SHQ05C with SHA04K expressing UshA enzyme.

FIG. 6 is a graph showing the formation of NAM in the reaction solution of SHQ05C and SHA04K expressing UshA enzyme.

FIG. 7 is a graph of NR and NAM content detected by SHQ05C/pHF57 fermentation.

Wherein: NA: nicotinic acid; NR: beta-nicotinamide riboside; NMN: beta-nicotinamide mononucleotide; NAM: nicotinamide; asp: aspartic acid; QA: quinolinic acid; NaMN: nicotinic acid mononucleotide; NaAD: nicotinic acid adenine dinucleotide; NAD: nicotinamide adenine dinucleotide; PRPP: phosphoribosyl pyrophosphate; NadC: quinolinate phosphoribosyltransferase; NrdA, NrdB: nucleoside diphosphate reductases; NadD: nicotinic acid mononucleotide adenyl transferase; NadE: an NAD synthase; NadR: nicotinamide nucleotide adenylyl transferase; PncA: a nicotinamide enzyme; PncB: nicotinic acid phosphoribosyltransferase; PncC: NMN deamidase; and (5) DeoD: purine nucleoside phosphorylase I; XapA: purine nucleoside phosphorylase II; cdh: CDP-diacylglycerol diphosphatase; UshA: 5' -nucleotidase; RihA/RihC: ribonucleoside hydrolase

Detailed Description

The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.

Description of the principle: principle of NR metabolic pathway

In microorganisms, NAD⁺(nicotinamide adenine dinucleotide) there are three synthetic pathways: a salvage pathway, a de novo synthesis pathway and a P-H pathway (also called a nicotinic acid salvage pathway).

As shown in FIG. 1, in the case of E.coli, the de novo synthetic pathway involves three major enzymes: quinolinate phosphoribosyltransferase (NadC, EC 2.4.2.19), nicotinic acid mononucleotide adenylyltransferase (NadD, EC 2.7.7.18), NAD⁺Synthetase (NadE, EC 6.3.1.5), NadC transfers the phosphoribosyl moiety from phosphoribosyl pyrophosphate to quinolinate nitrogen and catalyzes the subsequent decarboxylation of the intermediate to produce nicotinic acid mononucleotide (NaMN), NadD adenylates NaMN with Adenosine Triphosphate (ATP) to produce nicotinic acid dinucleotide (NaAD), NadD is also capable of adenylating NMN, but has lower activity, NAD, than when NaMN is used as a substrate⁺The last step of biosynthesis is catalyzed by NadE, NadE LiAmination of NaAD to NAD with ammonia or glutamine as amino donor⁺。

In addition to the de novo synthetic pathway, NAD⁺There are also various salvage pathways, such as NMN, NR, Nicotinamide (NAM) or Nicotinic Acid (NA) salvage pathways: NR is phosphorylated to NMN by nicotinamide riboside kinase (e.coli NadR, EC 2.7.1.22) or degraded in a reversible reaction to NAM and ribose phosphate by purine nucleoside phosphorylase (e.coli DeoD, b.subtilis DeoD, PupG, Pdp, EC 2.4.2.1); NAM can be phosphoribosylated to NMN by DeoD or deamidated to NA by nicotinamidase (PncA, EC 3.5.1.19); NA or NAM is converted into NaMN or NMN, respectively, by nicotinic acid phosphoribosyltransferase (e.coli PncB, b.subtilis YueK EC 6.3.4.21), NAM can also be converted into NMN by exogenous nicotinamide phosphoribosyltransferase (Nampt), extracellular NMN is dephosphorylated to NR by periplasmic acid phosphatase (e.coli UshA, b.subtilis YfkN, EC 3.1.3.5), and extracellular NR can be introduced into the cell by NR transporter (e.coli PnuC, b.subtilis NupG).

The invention also verifies that the ribonucleotide hydrolase RihA with pyrimidine specificity and the nonspecific ribonucleotide hydrolase RihC have the degradation effect on NR for the first time through experiments.

Experimental method 1: gene knockout method

The invention adopts a Datsenko method to knock out microbial genes, and roughly comprises the steps of replacing gene sequences with selectable antibiotic drug-resistant genes generated by PCR (polymerase chain reaction) by using primers with 36-nt homologous extension based on a Red recombination system, thereby achieving the purpose of gene knock-out. Specific methods for gene knock-out employed in the present invention are described in the literature: K.A.Datsenko, B.L.Wanner.one-step inactivation of chromogenes in Escherichia coli K-12using PCR products of the National Academy Sciences of the USA,2000,97(12): 6640-. Corresponding gene knockout primers are described in the literature: baba T, Ara T, Hasegawa M, et al.2006.construction of Escherichia coli K-12in-frame, single gene knockouts. the Keio collection. mol Syst Biol [ J ],2:20060008, see page 9, paragraph 2 and supplementary data Table 2.

Experimental method 2: shake flask fermentation verification of capacity of recombinant strains to produce NR

1. Reagent:

(1) fermentation medium: 30g of glucose, 200mL of 5N-5 times of salt solution, 1mL of TM2 solution, 10mg of ferric citrate, 246mg of magnesium sulfate heptahydrate, 111mg of calcium chloride and 1 mu g of thiamine in each liter of culture medium, and the volume is fixed to 1L by sterile deionized water.

Wherein the 5N-5 times of salt solution is 75.6g of disodium hydrogen phosphate dodecahydrate per liter, 15g of potassium dihydrogen phosphate per liter, 2.5g of sodium chloride per liter and 25g of ammonium chloride per liter, and the volume is fixed to 1L by using ionic water; the TM3 solution is 2.0g of zinc chloride tetrahydrate, 2.0g of calcium chloride hexahydrate, 2.0g of sodium molybdate dihydrate, 1.9g of copper sulfate pentahydrate, 0.5g of boric acid, 100mL of hydrochloric acid, and the volume of deionized water is up to 1L.

Sterilizing the above solution with high pressure steam at 121 deg.C for 20-30 min.

(2) LB culture medium: each liter of the culture medium contains 5g of yeast powder, 10g of sodium chloride, 10g of peptone and deionized water to a volume of 1L (American J. Shamebrook. Huangpetang, molecular cloning guide 2002,1595).

Sterilizing the above solution with high pressure steam at 121 deg.C for 20-30 min.

2. The instrument comprises the following steps: constant temperature shaking incubator.

3. The method comprises the following steps:

the shake flask fermentation process was as follows: (1) inoculating the recombinant microorganism strains into 4mL LB culture medium containing antibiotics, and placing the culture medium in a shaker at 37 ℃ for 250rpm culture; (2) transferring 200 mu L of the seed liquid after 16h culture to 2mL LB liquid culture medium containing antibiotics, and culturing for 4h at 37 ℃ by a shaker at 250 rpm; (3) transferring 2mL of the secondary seed liquid into a shake flask filled with 18mL of fermentation medium, placing the shake flask in a 37 ℃ shaking table, and culturing for 3-4h at 250 rpm; (4) adding IPTG to a final concentration of 1mM, adding 300mg/L substrate Nicotinamide (NAM), adjusting the temperature of a shaking table to 34 ℃, continuously culturing for about 20h, sampling, and carrying out HPLC detection, wherein the detection method is detailed in experiment method 5.

Experimental method 3: shake flask expression and collection of enzyme solution

1. Reagent:

the transcription expression of the coding gene of the enzyme in the host cell uses LB culture medium, each liter of culture medium contains 5g of yeast powder, 10g of sodium chloride, 10g of peptone and deionized water with constant volume of 1L.

Sterilizing the above solution with high pressure steam at 121 deg.C for 20-30 min.

2. The instrument comprises the following steps: constant temperature shaking incubator.

3. The method comprises the following steps:

the specific expression process is as follows: (1) activating seeds, picking single clone from a streak plate to a shake tube filled with 4mL LB, and culturing overnight at 37 ℃; (2) inoculating 1% of the inoculum size to a 250mL shake flask filled with 50mL LB culture medium, and culturing at 37 ℃ for 2.5-3h until the OD600 is 0.6-0.8; (3) adding 100 μ L of 200mM IPTG to a final concentration of 0.4mM in a shake flask, adjusting the temperature to 34 deg.C, and culturing at 250rpm for 20 h; (4) collecting thalli the next day, 7800rpm 10min, discarding supernatant, weighing thalli precipitate 0.1g, resuspending with 2mL of 2mM PBS (pH7.0) buffer solution, performing cell disruption by 40% power ultrasound for 6min (ultrasound for 2s pause for 4s), centrifuging disrupted solution after ultrasound for 7800rpm 10min, and collecting supernatant, i.e. crude enzyme solution.

Experimental method 4: production of NR by fermentation of recombinant strains in a fermenter

1. Reagent

(1) Fermentation medium: the culture medium is a semi-synthetic culture medium, and each liter of the culture medium contains 5g of ammonium sulfate, 2g of sodium chloride, 4g of potassium dihydrogen phosphate, 2g of magnesium sulfate heptahydrate, 15g of glucose, 0.105g of calcium chloride, 0.01g of zinc chloride, TM 31 mL, 94mg of ferric citrate, 5g of corn steep liquor, 12.5 mg of VBE, 40mg of NA and 1g of natural sodium hypochlorite, and the volume is determined by deionized water.

The TM3 solution is 2.0g of zinc chloride tetrahydrate, 2.0g of calcium chloride hexahydrate, 2.0g of sodium molybdate dihydrate, 1.9g of copper sulfate pentahydrate, 0.5g of boric acid, 100mL of hydrochloric acid, and the volume of deionized water is up to 1L.

(2) A supplemented medium: the pH was adjusted to 6.9 with ammonia, containing 500g of glucose per liter.

2. Instrument for measuring the position of a moving object

Constant temperature shaking incubator, fermentation cylinder.

3. Method of producing a composite material

The fermentation process is as follows: (1) activating seeds, inoculating 0.25% of the seed glycerin tube into a 250mL shake flask containing 30mL LB, 37 deg.CCulturing for 16h until OD600 is 3-4; (2) inoculating 1% of the inoculum size to a 500mL seed shake flask filled with 100mL LB culture medium, and culturing at 37 ℃ for 4h until OD600 is 1-2; (3) inoculating 10% of the strain into 5L fermentation tank containing 2L semisynthetic medium, culturing at 37 deg.C, adjusting pH to 6.9 with ammonia water, coupling at dissolved oxygen rotation speed, maintaining dissolved oxygen at 30%, and starting coupling feeding when dissolved oxygen is higher than 40% to maintain dissolved oxygen at 30-45%. Fermenting for 8h until OD600 is 10-30 deg.C, maintaining the temperature at 37 deg.C, adding IPTG to final concentration of 0.4mmol/L, inducing at 0.3-0.5 g/L.h^-1The substrate Nicotinamide (NAM) is added in a flowing manner at the speed of (1), after fermentation is carried out for 19 hours, a sample is taken for HPLC detection, and the detection method is detailed in an experimental method 5.

Experimental method 5: HPLC determination of NR and related by-products in fermentation broths

Precisely absorbing 800 μ L fermentation liquid (or 200 μ L reaction liquid) and 800 μ L acetonitrile, vortex shaking for 5min, centrifuging at 12000rpm for 2min, collecting supernatant, passing through 0.22 μm organic filter membrane, and detecting by HPLC. The HPLC parameters were as follows: agilent SB Aq 4.6 x 150mM 5 μm is adopted, the mobile phase is methanol and 10mM ammonium acetate (pH 5.0), the methanol proportion is maintained at 1% in 0.01-4.4 minutes, the flow rate is 0.8mL/min, the methanol proportion is increased from 1% to 7% in 4.4-5.4 minutes, the flow rate is 0.8mL/min, the methanol proportion is increased from 7% to 18% in 5.4-6.5 minutes, the flow rate is 1.2mL/min, the methanol proportion is decreased from 18% to 2% in 6.5-6.6 minutes, the methanol proportion is maintained at 2% in 6.6-12 minutes, the flow rate is 1.2mL/min, and the wavelength is detected by an ultraviolet detector to be 260 nm; the loading amount of the fermentation liquid is 2 mu L, and the column temperature is 30 ℃. The NMN peak time was 2.348 min, the orotic acid peak time was 2.471 min, the NR peak time was 3.074 min, the NA peak time was 3.915 min, the NAD peak time was 8.347 min, and the NAM peak time was 10.505 min. The HPLC profile is shown in FIG. 2.

Experimental method 6: plasmid and Strain information

TABLE 1 plasmid, Strain information

plasmid/Strain	Detailed information
		pHQ01	pETrc-rihA
pHQ02	pETrc-rihB
		pHQ03	pETrc-rihC
pHQ04	pETrc-deoA
		pHQ05	pETrc-udP
pHQ06	pETrc-ppnN
		pHQ07	pETrc-ushA
pHA171	pET3.1-ushA
		pHA172	pUC19-P3.1-ushA
pHF57	pHD03-ushA
		SHA04K	W3110△ushA△cdh△pncC△nadR△pncA△xapA△deoD::KanFRT
SHQ01C	SHA04△rihA::CmFRT
		SHQ02C	SHA04△rihC::CmFRT
SHQ05C	SHQ01C△rihC::CmFRT

Example 1: screening phosphohydrolase or nucleotidase encoding gene and constructing expression plasmid to produce NR

1. Phosphohydrolase or nucleotidase screening: the catalytic generation of NR by NMN is mainly realized by hydrolyzing a nucleotide side chain phosphate group, and coding genes with a hydrolyzed phosphate group or hydrolyzed nucleoside in Escherichia coli include phoA, yfbT, yniC, appA, aphA, nadN, ushA and the like. Through constructing an overexpression plasmid and carrying out in-vitro catalysis by taking NMN as a reaction substrate, a gene capable of catalyzing the NMN to generate NR is screened. Specifically, genes phoA, yfbT, yniC, appA, aphA, nadN and ushA are respectively constructed on a pETrc vector (wherein the pETrc vector takes pET30a (Wuhan vast Ling Biotech Co., Ltd., product number P0031) as a template, a T7 promoter is replaced by a trc promoter, and the sequence is shown in the following table 3), the constructed plasmid is transformed into a BL21(DE3) host, and overexpression is carried out according to the enzyme liquid expression preparation method of the experimental method 3. The enzyme catalysis reaction system is as follows: a reaction system (0.1M PBS (pH7.0)) containing 10g/L NMN and 200. mu.L enzyme solution in a water bath kettle at 37 ℃ reacts for 3h, and the HPLC method of the experimental method 5 is used for detection, so that the results are shown in Table 2, the enzyme solution expressed by the genes phoA, aphA, nadN and ushA has the function of catalyzing the NMN to generate NR, and the activity of the UshA enzyme is highest.

TABLE 2 screening of genes encoding NR production by NMN

Gene	NR yield (g/L)
		phoA	1.2
yfbT	0
		yniC	0
appA	0
		aphA	1.96
nadN	0.97
		ushA	8.6

TABLE 3 Trc, P3.1 promoter sequences

2. Plasmid construction: the Escherichia coli ushA gene can encode a bifunctional enzyme having 5' -nucleotidase and UDP-sugar hydrolase activities. It has a relatively broad substrate specificity, UDP sugars and CDP-alcohols. Thus, it can catalyze the removal of a molecular phosphate group from the substrate β -Nicotinamide Mononucleotide (NMN) to produce the product Nicotinamide Ribose (NR). However, if NR production is carried out only by relying on a single copy of ushA on the genome, the expression level of ushA is far from sufficient for continuous catalytic production of NMN, so the ushA gene is respectively constructed on high copy expression vectors pETrc and pET3.1 (wherein pET3.1 vector construction uses pET30a as a template, and a T7 promoter is replaced by a P3.1 promoter, and the sequence is shown in the table 3 above) to be used as an expression plasmid pHQ07 and pHA171 for in vitro enzymatic preparation of NR, and in addition, an expression plasmid pHD03 (plasmid pEZ07-nadV-prs128, wherein pEZ07 is the same as Chinese patent application No. 201510093004.3, and an actinobacter bayli sp.ADP1 derived nadV gene and a large intestine self derived prs gene with a D128A point mutation are sequentially connected on a pEZ07 vector to be used as an engineering bacterium to ferment and produce NR by using the ushA gene as an engineering bacterium pHF 57.

The specific process of plasmid construction is as follows: pHQ07 is taken as an example, Escherichia coli W3110(ATCC27325) is taken as a template, a ushA fragment is amplified by primers pHQ07-F (SEQ ID NO:1)/pHQ07-R (SEQ ID NO:2) respectively, the size of the obtained PCR product is 1700bp, NO impurity band is generated in electrophoresis detection, column recovery and purification (strap-down gel recovery and purification kit) are directly carried out, the obtained purified fragment and a pETrc vector fragment which is recovered by NcoI/XhoI enzyme digestion are seamlessly cloned and constructed (Suzhou Shenzhou gene GBclonart seamless cloning kit) according to the nanomolar ratio of 3:1, a recombinant cloning reaction solution is subjected to warm bath in a water bath kettle at 45 ℃ for 30min, then is transferred to ice and placed for 5min, transferred to TG1 transformation competent cells, hot shock is carried out at 42 ℃ for 2min, 800 muL of recovery culture medium LB is added after ice bath for 2min, after recovery culture is carried out for 1h, an LB plate containing 50mg/L kanamycin resistance is centrifugally coated, the clone culture is picked on the next day and kept overnight, and extracting the plasmid, carrying out enzyme digestion verification, and finally constructing to obtain the plasmid pHQ 07.

SHA04K strain construction: according to the gene knockout method of experiment 1, in order to accumulate NMN in cells using Escherichia coli W3110(ATCC27325) as the starting strain, genes ushA, cdh, pnCC and nadR for degrading and utilizing NMN were sequentially knocked out, while genes pncA and xapA for consuming NAM as substrates were removed, and a gene deoD for degrading NR was removed.

Since NR is produced by continuous catalysis by means of a nucleotidase encoded by ushA, the ushA 04K from which ushA has been knocked out is selected as a starting strain by constructing an overexpression plasmid without relying on the expression of ushA in a single copy of the genome itself.

Example 2: screening for NR-degrading encoding genes

As can be seen from fig. 1, genes related to utilization and degradation of NAM, NMN and NR known in the metabolic pathway have been removed from the genome of the NMN producing strain SHA04K, and theoretically, when SHA04K is used as a host, no significant degradation of NR occurs when an enzyme solution over-expressing pHQ07 (pet-ushA) plasmid is subjected to in vitro catalysis. However, the experimental results show (FIG. 3) that in 3mL reaction system, 200. mu.L of crude pHQ07 enzyme solution expressed by NMN was added at 5g/L (see Experimental method 3 for the preparation method of the enzyme solution), and the reaction was carried out at 35 ℃ for various times in 50mM PBS buffer solution with pH7.0, so that the catalytic product NR was degraded into NAM rapidly with the increase of the reaction time, and the NR was only 0.9g/L after 6h of the reaction, indicating that there still exists an unknown enzyme for degrading NR in the metabolic pathway except the known deoD and XapA for degrading NR.

The main way of degrading NR is realized by hydrolyzing side-chain ribose with ribonucleoside hydrolase, and coding genes with the function of hydrolyzing ribonucleoside in Escherichia coli comprise ribonucleoside hydrolase RihB and RihA with pyrimidine specificity, nonspecific ribonucleoside hydrolase RihC, thymidine phosphorylase DeoA with catalytic action on deoxyribonucleoside, uridine phosphorylase Udp, 5' -monophosphate enzyme PpnN and the like. The invention screens genes capable of catalyzing NR to NAM by constructing an overexpression plasmid and carrying out in-vitro catalysis by taking NR as a reaction substrate.

According to the plasmid construction method in example 1, the genes rihA, rihB, rihC, deoA, udP and ppnN were constructed on the vector pETrc to obtain plasmids pHQ01-pHQ06, the host SHA04K was transformed, and the related enzyme solutions were obtained by overexpressing strains with different plasmids. The catalytic reaction system is as follows: adding substrate NR to 3mL of the reaction solution to a final concentration of 3g/L, adding 100. mu.L of enzyme solution, 10mM of buffer solution, pH6.0 PBS, stirring the mixture in a water bath at 37 ℃ for reaction for 1h, sampling to detect whether NAM is generated, and detecting that only NAM can be detected after the reaction of the enzyme solution expressed by plasmids pHQ01(pETrc-rihA) and pHQ03(pETrc-rihC) (see Table 4 below), which indicates that the enzymes encoded by the genes rihA and rihC can cause the rapid degradation of NR.

TABLE 4 screening of genes encoding NR degradation

Gene	NR(g/L)	Yield of NAM (g/L)
			rihA	1.48	0.67
rihB	2.97	0
			rihC	1.2	0.81
deoA	2.97	0
			udP	2.95	0
ppnN	2.97	0

Example 3: knock-out coding gene for degrading NR to NAM

According to the experimental results of example 2, the genes rihA, rihC are the key causes of degradation of NR, therefore, rihA, rihC were knocked out separately on the basis of the NMN-producing strain SHA04K according to the method of gene knock-out in experimental method 1, strains SHQ01C (SHA 04. DELTA. rihA:: CmFRT), SHQ02C (SHA 04. DELTA. rihC:: CmFRT), expression plasmids pHQ07(pETrc-ushA) were transformed separately, enzyme solutions were prepared according to the method of expression and collection of the enzyme solutions in experimental method 3, the catalytic reaction was carried out in the reaction system in example 2, the results of detection are shown in FIG. 4, after ushA was expressed in the host bacteria SHQ01C, SHQ02C in which rihA, rihC were knocked out, the enzyme solution catalytically produced in a significantly higher yield than SHA04K, wherein the molar conversion rate of the expressed in SHQ02C was high, compared with that the amount of the NR 04, NR was significantly reduced, but the amount of NR 04K was still present.

Example 4: construction of rihA and rihC double knockout strains

According to the results of example 3, since knockout of rhA and rhC, respectively, can significantly improve the production of NR and decrease the amount of the by-product NAM, but the level of not degrading NR at all can not be achieved, the rhC gene was further knocked out on the basis of SHQ01C to construct strain SHQ05C (SHQ 01. DELTA. rhC:: CmFRT), expression plasmids pHF57(pHD03-ushA) and pHQ07(pETrc-ushA) were transferred into host SHQ05C, respectively, and it was verified whether NR could be accumulated by the own metabolic pathway shake flask fermentation and whether there was the by-product NAM by the extra-enzymatic catalysis according to the methods of Experimental methods 2 and 3, respectively.

HPLC detection shows that the UshA enzyme solution is expressed in SHQ05C, 6g/L NMN is used as a substrate, 4.3g/L NR can be obtained through catalysis after 1h of reaction, the molar conversion rate from NMN to NR reaches 93.5%, and the content of a byproduct NAM is maintained at about 20mg/L along with the prolonging of the reaction time, so that the effect is obvious compared with SHA04K (see fig. 5 and 6).

The results of the shake flask fermentation for producing NR show that the cells can ferment to obtain nearly 300mg/L NR (shown in figure 7) by utilizing a substrate NAM through the cell self-metabolism, while only 18mg/L is detected in a control strain SHA04K/pHF57, which further shows that the simultaneous knockout of rihA and rihC can obviously improve the yield of NR and reduce the accumulation of a byproduct NAM.

The recombinant strain W3110 delta ushA delta cdh delta pncC delta nadR delta pncA delta xapA delta deoD delta rihA delta rihC, CF is abbreviated as strain SHQ05C, which is classified and named as Escherichia coli (Escherichia coli), and the strain is deposited at 28 days 5 and 2021 at the general microbiological culture Collection center of China Committee for culture Collection of microorganisms, address: west road No. 1, north west of the republic of kyo, yang, institute of microbiology, academy of sciences of china, zip code: 100101, preservation number is CGMCC NO. 22633.

Example 5: two-plasmid co-expression fermentation in recombinant microorganisms

Although SHQ05C/pHF57 shake flask fermentation can accumulate nearly 300mg/L NR, about 120mg/L NMN is still detected in the fermentation broth and is not transformed into NR, which indicates that the ushA expression amount on the expression plasmid pHF57 is insufficient and can not catalyze the complete transformation of the NMN, therefore, the SHQ05C/pHF57 strain is transferred into a pHA171(pET3.1-ushA) expression plasmid for double-plasmid co-expression, the NMN is transformed into NR completely as much as possible by increasing the ushA expression amount, and the reaction is pushed to the NR so as to utilize the NAM to the maximum extent.

Specifically, the shake flask fermentation was performed according to experiment method 2, and the results are shown in Table 5, after the expression level of UshA was increased, the NR yield in shake flask fermentation could reach 418mg/L, no NAM was detected, and about 40mg/L NMN remained, indicating that the increase of UshA was effective, but the expression level was still insufficient.

TABLE 5 two-plasmid co-expression fermentation results

Bacterial strains	NR(mg/L)	NAM(mg/L)	NMN(mg/L)
				SHA04k/pHF57	18.5	267.1	103.8
SHQ05C/pHF57	299.7	71.7	121.8
				SHQ05C/pHF57&pHA171	418.8	0	41.1

Example 6: construction of higher copy expression plasmids for shake flask fermentation to produce NR

The results of example 5 show that the expression level of UshA needs to be greatly increased to ensure that the fermentation process is completely depleted of NAM and NMN, and the co-expression with the plasmid pHA171 is still insufficient for completely transforming the NMN in the fermentation broth, so that the high expression plasmid pHA172(pUC19-P3.1-ushA) with 200 and 300 copies is further constructed, and pHF57 and pHA172 are co-expressed in SHQ05C, and the NR yield is up to 520mg/L in the shake flask fermentation result, and no NAM and NMN are detected.

Example 7: enzymatic preparation of NR in a fermenter

Carrying out 3L reaction solution catalysis on homogeneous enzyme solution expressed by SHQ05C/pHA171 in a fermentation tank, wherein the catalytic reaction system is as follows: substrate NMN final concentration 120g/L, enzyme solution 36mL, in 136mM pH6.5 sodium acetate buffer (containing 8mM Co²⁺) Stirring and reacting for 1h in a water bath kettle at the temperature of 55 ℃, detecting that NR 89g/L is generated, the NAM by-product only contains 4.13g/L, and the conversion rate is 96.7%.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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