CN114107160A

CN114107160A - Nicotinamide ribokinase gene engineering bacterium and application thereof

Info

Publication number: CN114107160A
Application number: CN202111610085.1A
Authority: CN
Inventors: 袁围; 刘梦珠; 徐诗艺; 王松茂; 杨源源; 俞科臣
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang Yingwodi Biotechnology Co ltd
Priority date: 2021-12-27
Filing date: 2021-12-27
Publication date: 2022-03-01

Abstract

The invention discloses a nicotinamide ribokinase gene engineering bacterium and an application thereof in synthesizing beta-nicotinamide mononucleotide, wherein the engineering bacterium is obtained by transferring a nicotinamide ribokinase gene into a host bacterium; the nucleotide sequence of the nicotinamide ribokinase gene is shown in SEQ ID NO. 1. Under the condition that the input amount of nicotinamide ribokinase and polyphosphate kinase is kept unchanged, the total reaction time is 2 hours, the maximum concentration of synthesized beta-nicotinamide mononucleotide reaches 86.9g/L through HPLC determination, and the conversion rate of substrate nicotinamide ribochloride reaches more than 94 percent, which is the currently known highest production level.

Description

Nicotinamide ribokinase gene engineering bacterium and application thereof

(I) technical field

The invention relates to construction of nicotinamide ribokinase (NR kinase, NRK) genetic engineering bacteria derived from Trichoderma asperellum and application of the nicotinamide ribokinase (NR kinase, NRK) genetic engineering bacteria in synthesis of beta-nicotinamide mononucleotide.

(II) background of the invention

beta-Nicotinamide Mononucleotide (NMN) is an important intermediate in a synthetic pathway of Nicotinamide adenine dinucleotide (NAD +) in mammals. In recent years, research proves that NMN has obvious anti-aging function, so that functional health-care food taking NMN as an active ingredient has great development potential and market prospect, and related health-care products are sold on the market in Japan and America.

The traditional NMN is produced by chemical synthesis, taking Nicotinamide Ribose (NR) as a raw material and carrying out phosphorylation by phosphorus oxychloride. The overall yield is very low; meanwhile, the dosage of the organic solvent is large, and the environmental pollution is serious, so that the NMN is mainly prepared by a biological enzyme method at present.

The biological enzyme method preparation of NMN mainly has two ways: the first one is that D-ribose and nicotinamide are used as initial raw materials, and NMN is obtained through three steps of catalytic reaction under the action of ribokinase, phosphoribosyl pyrophosphate synthetase, nicotinamide phosphoribosyl transferase and the like; the conversion rate of the substrate in the route is not high (calculated by nicotinamide, the highest conversion rate is not more than 50 percent), and the intermediate products are more, and the subsequent separation and purification are more difficult, so the overall yield is low, and the production cost is high. In addition, D-ribose and nicotinamide in the raw materials used in the preparation method are unstable in properties and expensive in price; nicotinamide is not a common industrial raw material, is not easy to purchase, needs to be chemically synthesized additionally, and has low catalytic efficiency of single enzyme of Nicotinamide Ribokinase (NRK), which is not favorable for efficient and environment-friendly large-scale production of NMN.

The second route is to obtain the NMN by one-step reaction with Nicotinamide Riboside (NR) as the starting material under the action of nicotinamide riboside kinase (NR kinase, NRK) and ATP, and has high yield and high product purity. However, the reaction time is too long, and the amount of by-products is still a limiting factor. Most importantly, nicotinamide ribokinase activity is lower and needs to be further improved. In view of this, it is of great significance to find new NRK enzymes to further improve the production capacity of β -nicotinamide mononucleotide.

Disclosure of the invention

The invention aims to provide a nicotinamide ribokinase genetic engineering bacterium for efficiently synthesizing beta-nicotinamide mononucleotide and application thereof in synthesizing the beta-nicotinamide mononucleotide, and effectively improves the yield of the beta-nicotinamide mononucleotide.

The technical scheme adopted by the invention is as follows:

in a first aspect, the invention provides a nicotinamide ribokinase gene engineering bacterium, which is obtained by transferring a nicotinamide ribokinase gene derived from Trichoderma asperellum (bio-40993, purchased from China center for culture collection and management of industrial microorganisms) into a host bacterium to construct the nicotinamide ribokinase gene; the nucleotide sequence of the nicotinamide ribokinase gene is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.

Further, the host bacterium may be Escherichia coli, preferably Escherichia coli BL21(DE 3).

Further, the engineering bacteria are constructed according to the following steps: the nicotinamide ribokinase gene is cut by restriction enzymes BamH I and Hind III, then connected with pET-28a (+) plasmid cut by restriction enzymes, and then transferred into escherichia coli BL21(DE3) competent cells, and screened to obtain the nicotinamide ribokinase gene engineering bacteria.

In a second aspect, the invention also provides an application of the genetically engineered bacterium of nicotinamide ribokinase in the synthesis of beta-nicotinamide mononucleotide, wherein the application comprises the following steps: taking a crushed mixed solution obtained by carrying out ultrasonic crushing on wet thalli obtained by fermenting and culturing nicotinamide ribokinase genetic engineering bacteria as a catalyst, taking polyphosphate kinase (PPK) as an ATP regeneration coenzyme, taking nicotinamide ribochloride (NR-Cl) and ATP (adenosine triphosphate) as substrates, taking sodium hexametaphosphate as an ATP regenerant, taking magnesium chloride hexahydrate as a kinase activator, taking purified water as a reaction medium to form a conversion system, adjusting the pH to 6.5-6.8, carrying out catalytic reaction at the temperature of 20-30 ℃, and separating and purifying a reaction liquid after the reaction is finished to obtain the beta-nicotinamide mononucleotide.

Further, in the conversion system, the adding amount of the catalyst is 1-5g/L, preferably 1-3g/L, based on the weight of wet bacteria before crushing; the adding amount of the nicotinamide riboside chloride is 20-100g/L, preferably 40-100 g/L; the adding amount of ATP is 4.5-5.5g/L, preferably 5 g/L; the adding amount of the sodium hexametaphosphate is 28-35g/L, preferably 30 g/L; the addition amount of the magnesium chloride hexahydrate is 5-5.8g/L, and preferably 5.5 g/L.

Further, the polyphosphate kinase is added in the form of a crushed mixed solution obtained by carrying out ultrasonic crushing on wet thalli obtained by fermenting and culturing a polyphosphate kinase (PPK) gene engineering bacterium, wherein the nucleotide sequence of the polyphosphate kinase gene is shown as SEQ ID NO.3, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 4; the addition amount of the polyphosphate kinase is 1-5g/L, preferably 1-3g/L based on the weight of wet bacteria before crushing.

Further, the catalyst and the ATP regeneration coenzyme are cultured in a shake flask and prepared according to the following method: inoculating nicotinamide ribokinase gene engineering bacteria in LB culture medium, culturing overnight at 37 deg.C, diluting with fresh LB culture medium to OD₆₀₀Is 0.1; further incubation at 37 ℃ for 2 hours to OD₆₀₀After 0.8, adding IPTG with the final concentration of 0.3mM, performing induction culture at 25 ℃ for 10 hours, centrifuging (preferably, centrifuging at 4000rpm for 15min), collecting wet bacteria, resuspending the wet bacteria with purified water, ultrasonically crushing cells, and collecting a cell crushing mixed solution; the procedure of ultrasonic cell disruption was: working for 4s, stopping for 3s, performing 250w, and performing ultrasonic treatment for 99 cycles; the preparation method of the ATP regenerated coenzyme is the same as the preparation of the catalyst, namely, the nicotinamide ribokinase gene engineering bacteria are replaced by polyphosphate kinase (PPK) gene engineering bacteria.

Further, the catalyst and the ATP regeneration coenzyme are prepared by adopting a fermentation tank: inoculating nicotinamide ribokinase gene engineering bacteria into LB culture solution, and culturing overnight at 37 ℃ to obtain seed solution; transferring the seed liquid to a fermentation medium containing a 5% inoculum sizeIn a fermentation tank, setting the temperature at 37 ℃, stirring speed at 800rpm, dissolved oxygen at more than or equal to 30%, air inflow at 1.2vvm and tank pressure at 0.03-0.04 MPa, starting fermentation, and controlling the pH value of ammonia water to 6.8-6.9 in the process; when the OD600 reaches 25 ℃, beginning to reduce the temperature to 25-26 ℃, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.3mM, inducing expression for 14-16 h, centrifuging fermentation liquor (preferably at 4000rpm for 15min), collecting wet bacteria, carrying out resuspension on the wet bacteria by using purified water, carrying out ultrasonic cell disruption, and collecting cell disruption mixed liquor; the ultrasonic cell disruption procedure is as follows: working for 4s, stopping for 3s, performing 250w, and performing ultrasonic treatment for 99 cycles; the fermentation medium adopts an inorganic salt medium and comprises the following components: 12g/L peptone, 14g/L yeast extract, 5g/L anhydrous glycerol and 2g/L KH₂PO₄，16g/L K₂HPO₄·3H₂O, water as solvent, and pH 6.5-6.8. The preparation method of the fermentation tank of the ATP regenerated coenzyme is the same as that of the catalyst.

The catalyst and the coenzyme are respectively added in the form of cell disruption mixed liquor, and the volume ratio of the catalyst to the coenzyme is 1:1.2-1:2, and preferably 1: 1.5.

Further, the reaction solution separation and purification method can be a method commonly used in the field, for example, the reaction solution is subjected to ultrafiltration, nanofiltration, electrodialysis, nanofiltration concentration, alcohol precipitation crystallization and vacuum drying to obtain the high-purity beta-nicotinamide mononucleotide.

Further, the nicotinamide ribochloride is supplemented in the reaction process, and the supplementing time is determined by the residual quantity of the nicotinamide ribochloride in the reaction solution: the concentration and pH value of nicotinamide riboside chloride can inhibit enzyme irreversibly, and along with the reaction, the pH value in the reaction solution fluctuates, the enzyme activity of the enzyme gradually decreases, and the accumulation of byproduct nicotinamide increases. Therefore, after the reaction is carried out for 30min, the residual quantity of nicotinamide riboside chloride is lower than 6g/L, the material can be fed, and the pH value is readjusted to 6.5-6.8. The supplementing method comprises the following steps: when the residual amount of nicotinamide riboside is lower than 6g/L, adding 30-50g/L nicotinamide riboside, regulating pH to 6.5-6.8, continuing the reaction, and repeating the operation until the product beta-nicotinamide mononucleotide reaches 80-90 g/L.

Compared with the prior art, the invention has the following beneficial effects: under the condition that the input amount of nicotinamide ribokinase and polyphosphate kinase is kept unchanged, the total reaction time is 2 hours, the maximum concentration of synthesized beta-nicotinamide mononucleotide reaches 86.9/L through HPLC determination, the substrate conversion rate reaches more than 94.7 percent, and the nicotinamide ribokinase gene engineering bacteria are the currently known highest production level.

(IV) description of the drawings

FIG. 1 shows the recombinant expression plasmid pET28a-NRK constructed according to the present invention.

FIG. 2 is the relationship between yield and productivity of beta-nicotinamide mononucleotide synthesized by engineering bacteria LMZ001 and reaction time.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

the purified water in the embodiment of the invention is water with the resistivity of more than 0.1x10^6 omega cm when the temperature is more than 25 ℃.

The nicotinamide riboside chloride (3-carbamyl-1-beta-D-ribofuranosyl pyridine chloride) has a molecular formula C₁₁H₁₅N₂O₅·Cl。

Example 1 cloning of nicotinamide ribokinase Gene (TA-NRK)

1. Engineering bacterium LMZ001

Referring to fig. 1, through designing primers, using Trichoderma asperellum (bio-40993, purchased from china industrial microbial strain collection management center) genome as a template, successfully cloning nicotinamide ribokinase gene (TA-NRK), and through performing codon optimization on gene sequence, obtaining codon-optimized nicotinamide ribokinase gene, wherein a nucleotide sequence is shown as SEQ ID No.1, and an amino acid sequence of encoded protein is shown as SEQ ID No. 2.

The primer sequence is as follows:

KL-F (5 'gccgacaagaaggcgctgat 3') and KL-R (5 'tcaggcgtacttcttcatctcctcc 3')

The codon-optimized nicotinamide ribokinase gene was digested simultaneously with restriction enzymes BamH I and Hind III (NEB from New England Biolabs) at 37 ℃ for 4h, separated by electrophoresis on a 1% agarose gel and recovered by gel cutting (gel recovery kit from Hangzhou Haofeng Biotechnology, Inc.). Subsequently, the cells were ligated with the expression vector pET28a (+) (Novagen) which had been similarly double-digested, overnight at 16 ℃ under the action of T4 DNA ligase (purchased from Takara). The ligation solution was transformed into competent cells of E.coli BL21(DE3) (purchased from Biotech, Oncorhynchs, Ltd.), and colony PCR screening and sequencing verification were performed to obtain the positive recombinant plasmid pET28 a-NRK. The positive transformant containing NRK gene is marked as engineering bacteria LMZ001 and stored at-80 ℃.

SEQ ID NO.1

gaattcatggcagacaaaaaggcgttaatcgtggcgttaagcgggtgtagcagcagcgggaaaacgacgctggcgcggttactgcgggacattttcccgagcaccttcattctgcacgaagacgacttttatcgtccggaaaccgaactgccgaaaaaggatgatctgttagattgggattgcgccgaaagtattgatatccctgcaatggccgaaagcctgtcctatattcgccagcatgcagcatttcctcctaccctggatagctatcaggacaagaattccgttggtgaatgtccggttccgccgagcaccatttctgcactgaaatctaaagttagctccctgttacccagcacccatccgcttgcaacctctgcactgcacttatgtattctggatggttttctgctgtatccgccttcaatgagcgcaattcagccgcatctggatattaaaatttttctgcgtgcatcatacgcacaggcaaaggcccgtcgtgaagcccgtgacggttatgttacccttgaaggtttttgggccgacccgccgggttatgttgataaaattgtgtggccgaattatgttgcagagcatagttggatgtttcaggatggtgatgttgaaggggaatataaagcagatgttctggaacaggaagcaatacgtgttccgtcgggtagcggcgttgatggtgagatggataaaatactggaatggatggttgatctgattctggaggaaatgaaaaagtatgcataaaagctt.

SEQ ID NO.2

MADKKALIVALSGCSSSGKTTLARLLRDIFPSTFILHEDDFYRPETELPKKDDLLDWDCAESIDIPAMAESLSYIRQHAAFPPTLDSYQDKNSVGECPVPPSTISALKSKVSSLLPSTHPLATSALHLCILDGFLLYPPSMSAIQPHLDIKIFLRASYAQAKARREARDGYVTLEGFWADPPGYVDKIVWPNYVAEHSWMFQDGDVEGEYKADVLEQEAIRVPSGSGVDGEMDKILEWMVDLILEEMKKYA.

2. PPK engineering bacteria

According to NCBI (https:// www.ncbi.nlm.nih.gov/protein/CNW32538.1report ═

The polyphosphate kinase (PPK) for ATP regeneration, which is derived from Mycobacterium tuberculosis ATCC 25618/H37Rv in genbank & log & blast _ rank & RID & VBW3MVBJ016, is synthesized by Protecobacterium tuberculosis Inc. (Hangzhou), the nucleotide sequence is shown as SEQ ID NO.3, the amino acid sequence of the encoded protein is shown as SEQ ID NO.4, the subsequent construction of the recombinant expression plasmid is the same as the construction of pET28a-NRK plasmid, the expression strain is obtained after the plasmid is transferred into a competent cell of Escherichia coli BL21(DE3), and the transformant is positive as PPK engineering bacteria.

SEQ ID NO.3

atgaaccgcgaactgagctggctggattttaacgcgcgcgtgctggcgctggcggcggataaaagcatgccgctgctggaacgcgcgaaatttctggcgatttttgcgagcaacctggatgaattttatatggtgcgcgtggcgggcctgaaacgccgcgatgaaatgggcctgagcgtgcgcagcgcggatggcctgaccccgcgcgaacagctgggccgcattggcgaacagacccagcagctggcgagccgccatgcgcgcgtgtttctggatagcgtgctgccggcgctgggcgaagaaggcatttatattgtgacctgggcggatctggatcaggcggaacgcgatcgcctgagcacctattttaacgaacaggtgtttccggtgctgaccccgctggcggtggatccggcgcatccgtttccgtttgtgagcggcctgagcctgaacctggcggtgaccgtgcgccagccggaagatggcacccagcattttgcgcgcgtgaaagtgccggataacgtggatcgctttgtggaactggcggcgcgcgaagcgagcgaagaagcggcgggcaccgaaggccgcaccgcgctgcgctttctgccgatggaagaactgattgcggcgtttctgccggtgctgtttccgggcatggaaattgtggaacatcatgcgtttcgcattacccgcaacgcggattttgaagtggaagaagatcgcgatgaagatctgctgcaggcgctggaacgcgaactggcgcgccgccgctttggcagcccggtgcgcctggaaattgcggatgatatgaccgaaagcatgctggaactgctgctgcgcgaactggatgtgcatccgggcgatgtgattgaagtgccgggcctgctggatctgagcagcctgtggcagatttatgcggtggatcgcccgaccctgaaagatcgcacctttgtgccggcgacccatccggcgtttgcggaacgcgaaaccccgaaaagcatttttgcgaccctgcgcgaaggcgatgtgctggtgcatcatccgtatgatagctttagcaccagcgtgcagcgctttattgaacaggcggcggcggatccgaacgtgctggcgattaaacagaccctgtatcgcaccagcggcgatagcccgattgtgcgcgcgctgattgatgcggcggaagcgggcaaacaggtggtggcgctggtggaaattaaagcgcgctttgatgaacaggcgaacattgcgtgggcgcgcgcgctggaacaggcgggcgtgcatgtggcgtatggcctggtgggcctgaaaacccattgcaaaaccgcgctggtggtgcgccgcgaaggcccgaccattcgccgctattgccatgtgggcaccggcaactataacagcaaaaccgcgcgcctgtatgaagatgtgggcctgctgaccgcggcgccggatattggcgcggatctgaccgatctgtttaacagcctgaccggctatagccgcaaactgagctatcgcaacctgctggtggcgccgcatggcattcgcgcgggcattattgatcgcgtggaacgcgaagtggcggcgcatcgcgcggaaggcgcgcataacggcaaaggccgcattcgcctgaaaatgaacgcgctggtggatgaacaggtgattgatgcgctgtatcgcgcgagccgcgcgggcgtgcgcattgaagtggtggtgcgcggcatttgcgcgctgcgcccgggcgcgcagggcattagcgaaaacattattgtgcgcagcattctgggccgctttctggaacatagccgcattctgcattttcgcgcgattgatgaattttggattggcagcgcggatatgatgcatcgcaacctggatcgccgcgtggaagtgatggcgcaggtgaaaaacccgcgcctgaccgcgcagctggatgaactgtttgaaagcgcgctggatccgtgcacccgctgctgggaactgggcccggatggccagtggaccgcgagcccgcaggaaggccatagcgtgcgcgatcatcaggaaagcctgatggaacgccatcgcagcccg.

SEQ ID NO.4

MNRELSW LDFNARVLAL AADKSMPLLE RAKFLAIFAS NLDEFYMVRV AGLKRRDEMG LSVRSADGLT PREQLGRIGE QTQQLASRHA RVFLDSVLPA LGEEGIYIVT WADLDQAERD RLSTYFNEQV FPVLTPLAVD PAHPFPFVSG LSLNLAVTVR QPEDGTQHFA RVKVPDNVDR FVELAAREAS EEAAGTEGRT ALRFLPMEEL IAAFLPVLFP GMEIVEHHAF RITRNADFEV EEDRDEDLLQ ALERELARRR FGSPVRLEIA DDMTESMLEL LLRELDVHPG DVIEVPGLLD LSSLWQIYAV DRPTLKDRTF VPATHPAFAE RETPKSIFAT LREGDVLVHH PYDSFSTSVQ RFIEQAAADP NVLAIKQTLY RTSGDSPIVR ALIDAAEAGK QVVALVEIKA RFDEQANIAW ARALEQAGVH VAYGLVGLKT HCKTALVVRR EGPTIRRYCH VGTGNYNSKT ARLYEDVGLL TAAPDIGADL TDLFNSLTGY SRKLSYRNLL VAPHGIRAGI IDRVEREVAA HRAEGAHNGK GRIRLKMNAL VDEQVIDALY RASRAGVRIE VVVRGICALR PGAQGISENI IVRSILGRFL EHSRILHFRA IDEFWIGSAD MMHRNLDRRV EVMAQVKNPR LTAQLDELFE SALDPCTRCW ELGPDGQWTA SPQEGHSVRD HQESLMERHR SP.

Example 2 verification of beta-Nicotinamide mononucleotide Synthesis by engineering bacteria LMZ001 by Shake flask fermentation

(1) The engineering bacteria LMZ001 and PPK engineering bacteria constructed in example 1 were respectively inoculated into a 250mL triangular flask containing 50mL LB medium, cultured overnight at 37 ℃, and measured for OD₆₀₀Then diluting the bacterial liquid into a fresh 50mL LB culture medium to OD₆₀₀Is 0.1. Further incubation at 37 ℃ for 2 hours to OD₆₀₀After 0.8, 0.3mM IPTG was added, induction culture was carried out at 25 ℃ for 10 hours, and then centrifugation was carried out at 4000rpm for 15 minutes, and the wet cells were collected and resuspended in purified water to prepare a cell suspension having a cell content of 100 g/L. Carrying out ultrasonic cell disruption on the bacterial suspension of the engineering bacteria LMZ001 (the ultrasonic cell disruption procedure is that the work is 4s, the stop is 3s, 250W and the ultrasonic is carried out for 99 times of circulation), obtaining cell disruption mixed liquor which is NRK free enzyme liquor, and obtaining 15mL of cell disruption mixed liquor every 15.7mL of bacterial suspensionAnd (4) mixing the liquid.

Under the same condition, 100g/L bacterial suspension of the PPK engineering bacteria is subjected to ultrasonic crushing to obtain cell crushing mixed liquor, namely PPK free enzyme liquor, and 10mL of cell crushing mixed liquor can be obtained every 10.7mL of bacterial suspension.

(2) Mixing 15mL (equivalent to the using amount of wet thalli before crushing 2.24g/L) of NRK free enzyme liquid prepared in the step (1) and 10mL (equivalent to the using amount of wet thalli before crushing 1.52g/L) of PPK free enzyme liquid prepared in the step (1) to serve as a catalyst, adding nicotinamide ribochloride with the final concentration of 80g/L, sodium hexametaphosphate with the final concentration of 30g/L, ATP with the final concentration of 5g/L and magnesium chloride hexahydrate with the final concentration of 5.5g/L into the mixture to form a 700mL reaction system by using purified water as a reaction medium, carrying out catalytic reaction for 2 hours under the condition of water bath at 25 ℃, after the reaction is finished, putting 1mL of reaction liquid into a 1.5mL centrifuge tube, centrifuging the mixture at 12000rpm for 1min, taking supernatant, measuring the contents of beta-nicotinamide mononucleotide and nicotinamide ribotide by HPLC, wherein the content of the beta-nicotinamide mononucleotide is 75.1g/L, the conversion rate of nicotinamide riboside chloride (NR) reaches 81.8%.

HPLC detection method of beta-nicotinamide mononucleotide:

a chromatographic column: agilent SB-C185 μm 4.6 mm 150mm

Elution procedure: gradient (table 1); initial flow rate: 0.8 mL/min; analysis time: 12 min; detection wavelength: 260 nm;

sample introduction volume: 5 mu L of the solution; column temperature: 25 ℃; retention time: 3.2 min; mobile phase A: trifluoroacetic acid (TFA) in 0.1% strength by volume aqueous solution; mobile phase B: chromatographic grade methanol.

TABLE 1 gradient program

Time (min)	Phase A (% v/v)	Phase B (% v/v)	Flow rate (mL/min)
				0	100	0	0.8
3.0	100	0	0.8
				5.3	80	20	0.8
6.6	80	20	1.0
				8.6	80	20	1.2
9	100	0	1.2
				12	100	0	1.2

Example 3, 20L fermenter amplification culture of engineering bacteria LMZ001 Synthesis of beta-Nicotinamide mononucleotide

(1) Seed liquid: respectively inoculating the engineering bacteria LMZ001 and PPK engineering bacteria constructed in the example 1 into a triangular flask containing 500mL of LB culture solution, and culturing overnight at 37 ℃ to respectively obtain LMZ001 seed solution and PPK seed solution;

(2) fermentation liquor: transferring the LMZ001 seed solution prepared in the step (1) into a 20L fermentation tank containing 13L fermentation medium by an inoculation amount with the volume concentration of 5%, setting the temperature at 37 ℃, stirring speed at 800rpm, dissolved oxygen at least equal to 30%, air inflow at 1.2vvm and tank pressure at 0.03-0.04 MPa, starting fermentation, and controlling the pH value of ammonia water to be 6.8-6.9 in the process. And when the OD600 reaches 25 ℃, beginning to reduce the temperature to 25-26 ℃, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.3mM, carrying out induced expression for 14-16 h, and ending fermentation. The fermentation liquor is centrifuged for 30min at 4000rpm, wet bacteria are collected and resuspended by purified water to bacterial suspension with the bacterial content of 200g/L, cells are ultrasonically crushed by adopting the same conditions of the embodiment 2, and the obtained cell crushing mixed liquor is NRK free enzyme liquor. 11.5ml of cell disruption mixture can be obtained for every 11.7ml of bacterial suspension.

Under the same condition, PPK seed liquid is prepared into 200g/L bacterial suspension of PPK engineering bacteria according to the method in the step (2), and cell crushing mixed liquid obtained by crushing the bacterial suspension is PPK free enzyme liquid. 7.5ml of cell disruption mixture can be obtained for every 8.2ml of bacterial suspension.

The fermentation medium is composed of an inorganic salt medium: 12g/L peptone, 14g/L yeast extract, 5g/L anhydrous glycerol and 2g/L KH₂PO₄，16g/L K₂HPO₄·3H₂O, water as solvent, and pH 6.5-6.8.

(3) And (3) catalytic reaction: taking 11.5mL (equivalent to the wet thallus content before crushing) of the NRK free enzyme liquid in the step (2) and 7.5mL (equivalent to the wet thallus content before crushing of 1.5g/L) of the PPK free enzyme liquid in the step (2), adding 47g/L of nicotinamide ribochloride, 30g/L of sodium hexametaphosphate, 5g/L of ATP and 5.5g/L of magnesium chloride hexahydrate into the obtained solution, forming a 1000mL reaction system by using purified water as a reaction medium, adjusting the pH to 6.5-6.8 by using an aqueous solution of sodium hydroxide, sampling every 30min after reacting for 30min at 25 ℃, detecting the content of beta-nicotinamide mononucleotide and the residual quantity of nicotinamide ribochloride by adopting the method in example 2, when the substrate is lower than 6g/L, continuously adding 33g/L of the substrate to ensure that the total adding amount of the substrate is 80g/L and adjusting the pH to 6.5-6.8, the reaction was continued. Sampling every 30min to detect the content of the beta-nicotinamide mononucleotide, and detecting that the final concentration of the beta-nicotinamide mononucleotide is 86.9g/L after reacting for 2h, wherein the substrate conversion rate reaches 94.7 percent.

Sequence listing

<110> Zhejiang industrial university

<120> nicotinamide ribokinase gene engineering bacteria and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 768

<212> DNA

<213> Trichoderma asperellum (Trichoderma asperellum)

<400> 1

gaattcatgg cagacaaaaa ggcgttaatc gtggcgttaa gcgggtgtag cagcagcggg 60

aaaacgacgc tggcgcggtt actgcgggac attttcccga gcaccttcat tctgcacgaa 120

gacgactttt atcgtccgga aaccgaactg ccgaaaaagg atgatctgtt agattgggat 180

tgcgccgaaa gtattgatat ccctgcaatg gccgaaagcc tgtcctatat tcgccagcat 240

gcagcatttc ctcctaccct ggatagctat caggacaaga attccgttgg tgaatgtccg 300

gttccgccga gcaccatttc tgcactgaaa tctaaagtta gctccctgtt acccagcacc 360

catccgcttg caacctctgc actgcactta tgtattctgg atggttttct gctgtatccg 420

ccttcaatga gcgcaattca gccgcatctg gatattaaaa tttttctgcg tgcatcatac 480

gcacaggcaa aggcccgtcg tgaagcccgt gacggttatg ttacccttga aggtttttgg 540

gccgacccgc cgggttatgt tgataaaatt gtgtggccga attatgttgc agagcatagt 600

tggatgtttc aggatggtga tgttgaaggg gaatataaag cagatgttct ggaacaggaa 660

gcaatacgtg ttccgtcggg tagcggcgtt gatggtgaga tggataaaat actggaatgg 720

atggttgatc tgattctgga ggaaatgaaa aagtatgcat aaaagctt 768

<210> 2

<211> 251

<212> PRT

<213> Trichoderma asperellum (Trichoderma asperellum)

<400> 2

Met Ala Asp Lys Lys Ala Leu Ile Val Ala Leu Ser Gly Cys Ser Ser

1 5 10 15

Ser Gly Lys Thr Thr Leu Ala Arg Leu Leu Arg Asp Ile Phe Pro Ser

20 25 30

Thr Phe Ile Leu His Glu Asp Asp Phe Tyr Arg Pro Glu Thr Glu Leu

35 40 45

Pro Lys Lys Asp Asp Leu Leu Asp Trp Asp Cys Ala Glu Ser Ile Asp

50 55 60

Ile Pro Ala Met Ala Glu Ser Leu Ser Tyr Ile Arg Gln His Ala Ala

65 70 75 80

Phe Pro Pro Thr Leu Asp Ser Tyr Gln Asp Lys Asn Ser Val Gly Glu

85 90 95

Cys Pro Val Pro Pro Ser Thr Ile Ser Ala Leu Lys Ser Lys Val Ser

100 105 110

Ser Leu Leu Pro Ser Thr His Pro Leu Ala Thr Ser Ala Leu His Leu

115 120 125

Cys Ile Leu Asp Gly Phe Leu Leu Tyr Pro Pro Ser Met Ser Ala Ile

130 135 140

Gln Pro His Leu Asp Ile Lys Ile Phe Leu Arg Ala Ser Tyr Ala Gln

145 150 155 160

Ala Lys Ala Arg Arg Glu Ala Arg Asp Gly Tyr Val Thr Leu Glu Gly

165 170 175

Phe Trp Ala Asp Pro Pro Gly Tyr Val Asp Lys Ile Val Trp Pro Asn

180 185 190

Tyr Val Ala Glu His Ser Trp Met Phe Gln Asp Gly Asp Val Glu Gly

195 200 205

Glu Tyr Lys Ala Asp Val Leu Glu Gln Glu Ala Ile Arg Val Pro Ser

210 215 220

Gly Ser Gly Val Asp Gly Glu Met Asp Lys Ile Leu Glu Trp Met Val

225 230 235 240

Asp Leu Ile Leu Glu Glu Met Lys Lys Tyr Ala

245 250

<210> 3

<211> 2067

<212> DNA

<213> Unknown (Unknown)

<400> 3

atgaaccgcg aactgagctg gctggatttt aacgcgcgcg tgctggcgct ggcggcggat 60

aaaagcatgc cgctgctgga acgcgcgaaa tttctggcga tttttgcgag caacctggat 120

gaattttata tggtgcgcgt ggcgggcctg aaacgccgcg atgaaatggg cctgagcgtg 180

cgcagcgcgg atggcctgac cccgcgcgaa cagctgggcc gcattggcga acagacccag 240

cagctggcga gccgccatgc gcgcgtgttt ctggatagcg tgctgccggc gctgggcgaa 300

gaaggcattt atattgtgac ctgggcggat ctggatcagg cggaacgcga tcgcctgagc 360

acctatttta acgaacaggt gtttccggtg ctgaccccgc tggcggtgga tccggcgcat 420

ccgtttccgt ttgtgagcgg cctgagcctg aacctggcgg tgaccgtgcg ccagccggaa 480

gatggcaccc agcattttgc gcgcgtgaaa gtgccggata acgtggatcg ctttgtggaa 540

ctggcggcgc gcgaagcgag cgaagaagcg gcgggcaccg aaggccgcac cgcgctgcgc 600

tttctgccga tggaagaact gattgcggcg tttctgccgg tgctgtttcc gggcatggaa 660

attgtggaac atcatgcgtt tcgcattacc cgcaacgcgg attttgaagt ggaagaagat 720

cgcgatgaag atctgctgca ggcgctggaa cgcgaactgg cgcgccgccg ctttggcagc 780

ccggtgcgcc tggaaattgc ggatgatatg accgaaagca tgctggaact gctgctgcgc 840

gaactggatg tgcatccggg cgatgtgatt gaagtgccgg gcctgctgga tctgagcagc 900

ctgtggcaga tttatgcggt ggatcgcccg accctgaaag atcgcacctt tgtgccggcg 960

acccatccgg cgtttgcgga acgcgaaacc ccgaaaagca tttttgcgac cctgcgcgaa 1020

ggcgatgtgc tggtgcatca tccgtatgat agctttagca ccagcgtgca gcgctttatt 1080

gaacaggcgg cggcggatcc gaacgtgctg gcgattaaac agaccctgta tcgcaccagc 1140

ggcgatagcc cgattgtgcg cgcgctgatt gatgcggcgg aagcgggcaa acaggtggtg 1200

gcgctggtgg aaattaaagc gcgctttgat gaacaggcga acattgcgtg ggcgcgcgcg 1260

ctggaacagg cgggcgtgca tgtggcgtat ggcctggtgg gcctgaaaac ccattgcaaa 1320

accgcgctgg tggtgcgccg cgaaggcccg accattcgcc gctattgcca tgtgggcacc 1380

ggcaactata acagcaaaac cgcgcgcctg tatgaagatg tgggcctgct gaccgcggcg 1440

ccggatattg gcgcggatct gaccgatctg tttaacagcc tgaccggcta tagccgcaaa 1500

ctgagctatc gcaacctgct ggtggcgccg catggcattc gcgcgggcat tattgatcgc 1560

gtggaacgcg aagtggcggc gcatcgcgcg gaaggcgcgc ataacggcaa aggccgcatt 1620

cgcctgaaaa tgaacgcgct ggtggatgaa caggtgattg atgcgctgta tcgcgcgagc 1680

cgcgcgggcg tgcgcattga agtggtggtg cgcggcattt gcgcgctgcg cccgggcgcg 1740

cagggcatta gcgaaaacat tattgtgcgc agcattctgg gccgctttct ggaacatagc 1800

cgcattctgc attttcgcgc gattgatgaa ttttggattg gcagcgcgga tatgatgcat 1860

cgcaacctgg atcgccgcgt ggaagtgatg gcgcaggtga aaaacccgcg cctgaccgcg 1920

cagctggatg aactgtttga aagcgcgctg gatccgtgca cccgctgctg ggaactgggc 1980

ccggatggcc agtggaccgc gagcccgcag gaaggccata gcgtgcgcga tcatcaggaa 2040

agcctgatgg aacgccatcg cagcccg 2067

<210> 4

<211> 1033

<212> PRT

<213> Unknown (Unknown)

<400> 4

Met Gly Ser Ser His His His His His His Ser Gln Asp Pro Met Ala

1 5 10 15

Phe Ser Ala Asp Thr Pro Glu Ile Val Tyr Thr His Asp Thr Gly Leu

20 25 30

Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn Pro Leu

35 40 45

Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro Ser Glu

50 55 60

Ala Arg Ile Pro Ile Phe Asp Gln Gly Phe Tyr Thr Ser Asp Ala Thr

65 70 75 80

Tyr Thr Thr Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu Gly Asp

85 90 95

His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Ile Arg Leu Ile Pro

100 105 110

Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu Val Ala

115 120 125

Lys Thr Glu Leu Arg Glu Ala Gln Val Thr Val Thr Ile Thr Arg Gly

130 135 140

Tyr Ser Ser Thr Pro Phe Glu Arg Asp Ile Thr Lys His Arg Pro Gln

145 150 155 160

Val Tyr Met Ser Ala Cys Pro Tyr Gln Trp Ile Val Pro Phe Asp Arg

165 170 175

Ile Arg Asp Gly Val His Leu Met Ile Ala Gln Ser Val Arg Arg Thr

180 185 190

Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp Gly Asp

195 200 205

Leu Ile Arg Ala Ile Gln Glu Thr His Asp Arg Gly Phe Glu Leu Pro

210 215 220

Leu Leu Leu Asp Cys Asp Asn Leu Leu Ala Glu Gly Thr Gly Phe Asn

225 230 235 240

Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg Arg Ala

245 250 255

Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu Ser Leu

260 265 270

Gly His Glu Ala Ile Leu Ala Asp Ile Thr Pro Ala Glu Leu Tyr Asp

275 280 285

Ala Asp Glu Val Leu Gly Cys Ser Thr Gly Gly Gly Val Trp Pro Phe

290 295 300

Val Ser Val Asp Gly Asn Ser Ile Ser Asp Gly Val Pro Gly Pro Val

305 310 315 320

Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu Pro Ser

325 330 335

Ser Leu Leu Thr Pro Val Gln Tyr Met Asn Arg Glu Leu Ser Trp Leu

340 345 350

Asp Phe Asn Ala Arg Val Leu Ala Leu Ala Ala Asp Lys Ser Met Pro

355 360 365

Leu Leu Glu Arg Ala Lys Phe Leu Ala Ile Phe Ala Ser Asn Leu Asp

370 375 380

Glu Phe Tyr Met Val Arg Val Ala Gly Leu Lys Arg Arg Asp Glu Met

385 390 395 400

Gly Leu Ser Val Arg Ser Ala Asp Gly Leu Thr Pro Arg Glu Gln Leu

405 410 415

Gly Arg Ile Gly Glu Gln Thr Gln Gln Leu Ala Ser Arg His Ala Arg

420 425 430

Val Phe Leu Asp Ser Val Leu Pro Ala Leu Gly Glu Glu Gly Ile Tyr

435 440 445

Ile Val Thr Trp Ala Asp Leu Asp Gln Ala Glu Arg Asp Arg Leu Ser

450 455 460

Thr Tyr Phe Asn Glu Gln Val Phe Pro Val Leu Thr Pro Leu Ala Val

465 470 475 480

Asp Pro Ala His Pro Phe Pro Phe Val Ser Gly Leu Ser Leu Asn Leu

485 490 495

Ala Val Thr Val Arg Gln Pro Glu Asp Gly Thr Gln His Phe Ala Arg

500 505 510

Val Lys Val Pro Asp Asn Val Asp Arg Phe Val Glu Leu Ala Ala Arg

515 520 525

Glu Ala Ser Glu Glu Ala Ala Gly Thr Glu Gly Arg Thr Ala Leu Arg

530 535 540

Phe Leu Pro Met Glu Glu Leu Ile Ala Ala Phe Leu Pro Val Leu Phe

545 550 555 560

Pro Gly Met Glu Ile Val Glu His His Ala Phe Arg Ile Thr Arg Asn

565 570 575

Ala Asp Phe Glu Val Glu Glu Asp Arg Asp Glu Asp Leu Leu Gln Ala

580 585 590

Leu Glu Arg Glu Leu Ala Arg Arg Arg Phe Gly Ser Pro Val Arg Leu

595 600 605

Glu Ile Ala Asp Asp Met Thr Glu Ser Met Leu Glu Leu Leu Leu Arg

610 615 620

Glu Leu Asp Val His Pro Gly Asp Val Ile Glu Val Pro Gly Leu Leu

625 630 635 640

Asp Leu Ser Ser Leu Trp Gln Ile Tyr Ala Val Asp Arg Pro Thr Leu

645 650 655

Lys Asp Arg Thr Phe Val Pro Ala Thr His Pro Ala Phe Ala Glu Arg

660 665 670

Glu Thr Pro Lys Ser Ile Phe Ala Thr Leu Arg Glu Gly Asp Val Leu

675 680 685

Val His His Pro Tyr Asp Ser Phe Ser Thr Ser Val Gln Arg Phe Ile

690 695 700

Glu Gln Ala Ala Ala Asp Pro Asn Val Leu Ala Ile Lys Gln Thr Leu

705 710 715 720

Tyr Arg Thr Ser Gly Asp Ser Pro Ile Val Arg Ala Leu Ile Asp Ala

725 730 735

Ala Glu Ala Gly Lys Gln Val Val Ala Leu Val Glu Ile Lys Ala Arg

740 745 750

Phe Asp Glu Gln Ala Asn Ile Ala Trp Ala Arg Ala Leu Glu Gln Ala

755 760 765

Gly Val His Val Ala Tyr Gly Leu Val Gly Leu Lys Thr His Cys Lys

770 775 780

Thr Ala Leu Val Val Arg Arg Glu Gly Pro Thr Ile Arg Arg Tyr Cys

785 790 795 800

His Val Gly Thr Gly Asn Tyr Asn Ser Lys Thr Ala Arg Leu Tyr Glu

805 810 815

Asp Val Gly Leu Leu Thr Ala Ala Pro Asp Ile Gly Ala Asp Leu Thr

820 825 830

Asp Leu Phe Asn Ser Leu Thr Gly Tyr Ser Arg Lys Leu Ser Tyr Arg

835 840 845

Asn Leu Leu Val Ala Pro His Gly Ile Arg Ala Gly Ile Ile Asp Arg

850 855 860

Val Glu Arg Glu Val Ala Ala His Arg Ala Glu Gly Ala His Asn Gly

865 870 875 880

Lys Gly Arg Ile Arg Leu Lys Met Asn Ala Leu Val Asp Glu Gln Val

885 890 895

Ile Asp Ala Leu Tyr Arg Ala Ser Arg Ala Gly Val Arg Ile Glu Val

900 905 910

Val Val Arg Gly Ile Cys Ala Leu Arg Pro Gly Ala Gln Gly Ile Ser

915 920 925

Glu Asn Ile Ile Val Arg Ser Ile Leu Gly Arg Phe Leu Glu His Ser

930 935 940

Arg Ile Leu His Phe Arg Ala Ile Asp Glu Phe Trp Ile Gly Ser Ala

945 950 955 960

Asp Met Met His Arg Asn Leu Asp Arg Arg Val Glu Val Met Ala Gln

965 970 975

Val Lys Asn Pro Arg Leu Thr Ala Gln Leu Asp Glu Leu Phe Glu Ser

980 985 990

Ala Leu Asp Pro Cys Thr Arg Cys Trp Glu Leu Gly Pro Asp Gly Gln

995 1000 1005

Trp Thr Ala Ser Pro Gln Glu Gly His Ser Val Arg Asp His Gln Glu

1010 1015 1020

Ser Leu Met Glu Arg His Arg Ser Pro

1025 1030

Claims

1. A nicotinamide ribokinase gene engineering bacterium is characterized in that the engineering bacterium is obtained by transferring a nicotinamide ribokinase gene derived from Trichoderma asperellum into a host bacterium to construct; the nucleotide sequence of the nicotinamide ribokinase gene is shown in SEQ ID NO. 1.

2. The genetically engineered nicotinamide ribokinase of claim 1, wherein the host bacterium is Escherichia coli BL21(DE 3).

3. The genetically engineered nicotinamide ribokinase of claim 1, wherein the engineered bacterium is constructed by the following steps: the nicotinamide ribokinase gene is cut by restriction enzymes BamH I and Hind III, then connected with pET-28a (+) plasmid cut by restriction enzymes, and then transferred into escherichia coli BL21(DE3) competent cells, and screened to obtain the nicotinamide ribokinase gene engineering bacteria.

4. An application of the genetically engineered bacterium of nicotinamide ribokinase of claim 1 in synthesizing beta-nicotinamide mononucleotide.

5. The use according to claim 4, characterized in that said use is: taking a crushed mixed solution obtained by carrying out ultrasonic crushing on wet thalli obtained by fermenting and culturing nicotinamide ribokinase genetic engineering bacteria as a catalyst, taking polyphosphate kinase as ATP regenerated coenzyme, taking nicotinamide riboside chloride and adenosine triphosphate as substrates, taking sodium hexametaphosphate as an ATP regenerated body, taking magnesium chloride hexahydrate as a kinase activator and purified water as a reaction medium to form a conversion system, adjusting the pH to 6.5-6.8, carrying out catalytic reaction at the temperature of 20-30 ℃, and separating and purifying the reaction solution after the reaction is finished to obtain the beta-nicotinamide mononucleotide.

6. The use according to claim 5, wherein in the conversion system, the catalyst is added in an amount of 1-5g/L based on the weight of wet cells before disruption; the adding amount of the nicotinamide riboside chloride is 20-100 g/L; the adding amount of the adenosine triphosphate is 4.5-5.5 g/L; the adding amount of the sodium hexametaphosphate is 28-35 g/L; the addition amount of the magnesium chloride hexahydrate is 5-5.8 g/L.

7. The use of claim 5, wherein the polyphosphate kinase is added in the form of a crushed mixed solution obtained by ultrasonic crushing of wet bacteria obtained by fermentation culture of polyphosphate kinase gene engineering bacteria, and the nucleotide sequence of the polyphosphate kinase gene is shown as SEQ ID No. 3; the adding amount of the polyphosphate kinase is 1-5g/L calculated by the weight of wet thalli before crushing.

8. The use according to claim 5, wherein the catalyst is prepared by the following process: inoculating nicotinamide ribokinase gene engineering bacteria in LB culture medium, culturing overnight at 37 deg.C, diluting with fresh LB culture medium to OD₆₀₀Is 0.1; further incubation at 37 ℃ for 2 hours to OD₆₀₀After 0.8, adding IPTG with the final concentration of 0.3mM, carrying out induction culture at 25 ℃ for 10 hours, centrifuging, collecting wet bacteria, carrying out resuspension on the wet bacteria by using purified water, carrying out ultrasonic cell disruption, and collecting a disrupted mixed solution; the procedure of cell disruption by sonication was: working for 4s, stopping for 3s, 250W, and performing ultrasonic treatment for 99 cycles.

9. The use according to claim 8, characterized in that the wet biomass is prepared using a fermenter: nicotinamide ribokinase baseInoculating the engineering bacteria into LB culture solution, and culturing overnight at 37 ℃ to obtain seed solution; transferring the seed solution into a fermentation tank filled with a fermentation culture medium according to the inoculation amount with the volume concentration of 5%, setting the temperature at 37 ℃, the stirring speed at 800rpm, the dissolved oxygen at least equal to 30%, the air inflow at 1.2vvm and the tank pressure at 0.03-0.04 MPa, starting fermentation, and controlling the pH value of ammonia water to be 6.8-6.9 in the process; when the OD600 reaches 25 ℃, beginning to reduce the temperature to 25-26 ℃, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.3mM, carrying out induced expression for 14-16 h, centrifuging fermentation liquor, and collecting wet thalli; the fermentation medium comprises the following components: 12g/L peptone, 14g/L yeast extract, 5g/L anhydrous glycerol and 2g/L KH₂PO₄，16g/L K₂HPO₄·3H₂O, water as solvent, and pH 6.5-6.8.

10. The application of claim 5, wherein in the reaction process, when the residual amount of nicotinamide riboside chloride is less than 6g/L, 20-40g/L of nicotinamide riboside chloride is added, the pH is regulated to 6.5-6.8, the reaction is continued, and the operation is repeated until the product beta-nicotinamide mononucleotide reaches 80-90 g/L.