CN117660280A - Engineering strain for high yield of NMN and application thereof - Google Patents
Engineering strain for high yield of NMN and application thereof Download PDFInfo
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- DAYLJWODMCOQEW-TURQNECASA-N NMN zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)([O-])=O)O2)O)=C1 DAYLJWODMCOQEW-TURQNECASA-N 0.000 claims abstract description 122
- 241000588724 Escherichia coli Species 0.000 claims abstract description 39
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims abstract description 38
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to engineering strains, and discloses a stable high-yield nicotinamide mononucleotide engineering strain and application thereof. The NMN metabolic pathway taking nicotinamide as a synthesis precursor is modified from three aspects of improving the supply of the synthesis precursor, rationally modifying the NMN degradation pathway and enhancing the energy supply of the synthesis pathway, the endogenous NMN accumulation of escherichia coli is improved, and an engineering strain E.coli-NMN capable of efficiently producing nicotinamide mononucleotide is obtained by optimizing a co-expression strategy and combining a promoter engineering technology, wherein the NMN yield reaches 2.7g/l. The strain has wide application prospect in the fields of foods, medicines, health products and the like.
Description
Technical Field
The invention belongs to genetic engineering bacteria, and in particular relates to an engineering strain for high-yield NMN and application thereof
Background
Beta-nicotinamide mononucleotide (beta-nicotinamide mononucleotide, NMN) is a nucleotide with biological activity, plays a vital role in cell proliferation and function, becomes a research hot spot in the field of physiological medicine in recent years, and becomes a precious raw material in industries such as medicines, functional foods, cosmetics and the like.
NMN exerts its physiological function in humans by converting into NAD+ which is a coenzyme necessary for biological body tissues (Shinchiro S, et al, metab. Eng.,2021; 65:166-177), and can transfer hydrogen during biological oxidation, activate a multienzyme system, promote synthesis and metabolism of nucleic acids, proteins, polysaccharides, enhance substance transport and regulatory control, improve metabolic function, and are vital to regulating cellular senescence, and maintain normal functions of the body. However, NAD+ is too large to be directly absorbed and utilized by cells, and NMN is a natural compound with a smaller molecular weight and can be effectively absorbed by cells. In addition, NMN can penetrate cell membrane fast after entering into organism as the most direct and efficient precursor of NAD+, promote cell to synthesize NAD+ naturally, raise NAD+ level fast and effectively and promote cell energy metabolism and normal operation of various biological processes. In recent years, NMN has been found to have great therapeutic potential, show good therapeutic effects on age-related chronic diseases such as diabetes, cardiovascular problems, cognitive impairment and the like (SomaM, etal.Mol.Biol.Rep.,2022;49: 9737-9748), and as NMN is deeply known, and people are increasingly concerned about aging resistance and health and longevity, NMN is expected to be in great demand in the future.
Because of the complex and costly chemical synthesis of NMN, biosynthesis of NMN has become an alternative approach. Coli is widely applied to the improvement work of high-yield NMN strains due to the advantages of clear genetic background, easy operation, easy regulation, simple culture and requirement, short growth period and the like. There are three main pathways for NMN biosynthesis (HuangZS, et al ACS. Synthetic.biology. Vol., 11:2979-2988): (1) NMN is directly biosynthesized from nicotinamide and PRPP by NAMPT; (2) Biosynthesis of NMN from niacin by niacin phosphoribosyl transferase; (3) synthesis of NMN from nicotinamide riboside phosphate. However, when nicotinic acid is added to the culture medium as a precursor, the nicotinic acid can make the culture medium acidic, thereby affecting the biological metabolism of escherichia coli, and the nicotinic acid is slightly soluble in water, so that the development of large-scale feeding is limited. The nicotinamide riboside is used as a precursor substance, so that the cost is relatively high, and the industrial production is not facilitated. The nicotinamide is used as a precursor substance for NMN synthesis, so that the influence on pH and biological metabolism of a culture medium can be avoided, the price is relatively low, and the requirement of mass production can be better met.
As shown in the above formula, nicotinamide riboside transferase (NAMPT) catalyzes the condensation of nicotinamide with PRPP to beta-Nicotinamide Mononucleotide (NMN), however, the lack of endogenous NAMPT in E.coli limits the high capacity of NMN. In addition, high yields of NMN are often limited by factors such as insufficient supply of PRPP, a precursor required for synthesis, lack of energy supply, inhibition of NMN levels by key enzyme genes of degradation pathways, etc., and if regulation is only maintained at a single factor level, NMN levels are difficult to elevate. Based on the foregoing, it is difficult to achieve stable high yield of NMN in the prior art.
Disclosure of Invention
The present study aims at constructing NMN metabolic pathway using nicotinamide as synthesis precursor in E.coli, and realizing NMN accumulation in E.coli by means of enhancing supply of synthesis precursor, knocking out degradation pathway related gene, enhancing energy supply of synthesis pathway, etc. Meanwhile, the high nicotinamide mononucleotide yield of the engineering strain E.coli-NMN is further optimized by combining a co-expression strategy and a promoter engineering technology. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
1) The nicotinamide riboside phosphate transferase is VsNAMPT (the nucleotide sequence is shown in SEQ ID NO: 1) derived from Variovorax sp.
2) Nicotinamide riboside phosphate transferase constructs plasmids.
3) The co-expression strategy of nicotinamide phosphoribosyl transferase gene and PRPP synthetase gene is optimized, and comprises single plasmid co-expression and fusion expression.
4) Promoter engineering, replacing the T7 promoter with promoters PL1118, PL1744, PL277, PUTRssrA-PUTRinfC-rplT, respectively.
5) Nicotinamide riboside phosphate transferase is expressed in E.coli BL21 (DE 3), a coenzyme NMN metabolic pathway using nicotinamide as a synthesis precursor is constructed, and the heterologous expression host is only an example, and the nicotinamide riboside phosphate transferase can be expressed in lactobacillus, streptomyces, saccharomycetes, bacillus subtilis and the like without limiting the scope of the invention.
6) Enhancing supply of NMN synthesis precursors
The prs gene (the nucleotide sequence is shown as SEQ ID NO: 2) of the coded 5-phosphoribosyl-1-pyrophosphoric acid (PRPP) synthetase is overexpressed, so that the concentration of the intracellular PRPP is increased, and the synthesis of NMN is promoted.
7) Knocking out NMN degradation related genes to realize accumulation of NMN
E.coli BL21 (DE 3) is modified by using CRISPR-Cas9 gene editing technology, deoD, ushA, pncC and nadR are knocked out, and the degradation path of NMN is blocked.
8) Enhancing energy supply of NMN synthesis path to achieve NMN accumulation
E.coli BL21 (DE 3) is modified by using CRISPR-Cas9 gene editing technology, the amb and add are knocked out, the adenine metabolic pathway is regulated, the accumulation of ATP is realized, and energy is provided for NMN synthesis.
The application of the high-yield NMN engineering strain is characterized in that nicotinamide is used as a donor, and NMN is synthesized by the high-yield nicotinamide mononucleotide engineering strain in claim 1.
The beneficial effects are that:
the invention utilizes bioinformatics and data mining technology to obtain a novel nicotinamide phosphoribosyl transferase (NAMPT) and successfully carries out heterologous expression in escherichia coli. Through analysis of NMN synthesis regulation network, the invention adopts various strategies to improve the synthesis level of endogenous NMN of escherichia coli. First, by enhancing the supply of PRPP, a synthesis precursor, more starting material is provided for the synthesis of NMN. Secondly, by knocking out key enzyme genes related to NMN degradation pathways, the NMN degradation pathways are blocked, thereby increasing the accumulation level thereof. Furthermore, by enhancing the energy supply of the NMN synthesis pathway, more energy is provided for the synthesis of NMN. Finally, by optimizing a co-expression strategy and combining a promoter engineering technology, an engineering strain capable of producing nicotinamide mononucleotide at high yield is constructed.
Specifically:
the present invention relates to the heterologous expression of nicotinamide phosphoribosyl transferase from Variovorax sp. In E.coli, and the modification of NMN metabolic pathway in E.coli using Nicotinamide (NAM) as synthesis precursor. Compared with other reported engineering strains for synthesizing nicotinamide mononucleotide, the stable high-yield nicotinamide mononucleotide engineering strain constructed by the invention adopts a multisystem combination to optimize an NMN metabolic system taking nicotinamide as a synthetic substrate, the nicotinamide has low price, the influence of nicotinic acid on pH and biological metabolism of a culture medium is avoided, the restriction of the slightly-soluble nicotinic acid is eliminated, and the requirement of large-scale production is better met.
Based on the combination optimization of a multisystem and the NMN metabolic system taking nicotinamide as a synthetic substrate, the novel consideration of the combination of an optimization co-expression strategy and promoter engineering is added, so that the efficient accumulation of endogenous NMN of escherichia coli is further realized, and finally, the stable and high-yield nicotinamide mononucleotide engineering strain E.coli-NMN is obtained, the production efficiency of NMN is greatly improved, and the potential of industrial production of NMN is provided.
Detailed Description
The specific steps of the present invention are described below by way of examples, but the scope of the present invention is not limited by this example.
The nucleotide sequences referred to in the present invention were delegated Jin Wei intelligent company synthesis.
Example 1 fluorescence detection of NMN content
Principle of detecting NMN content by fluorescence method: the NMN and acetophenone are subjected to condensation reaction under alkaline condition, and then are subjected to acid treatment to obtain the fluorescent 2, 7-naphthyridine derivative, wherein the derivative has the strongest fluorescence intensity under 382nm of excitation light and 445nm of emission light, and the fluorescence is measured by using a Tecan multifunctional enzyme-labeled instrument, so that the NMN content can be detected.
Establishment of a standard curve: the NMN standard substance is utilized to prepare 32mg/LNMN solution, and dilution is carried out on the basis of the NMN standard substance to obtain a series of NMN standard solutions with the concentration of 0.5mg/L, 1mg/L, 2mg/L, 4mg/L, 8mg/L, 16mg/L and 32 mg/L. Adding the standard solutions into the fluorescence reaction system, and measuring fluorescence under 382nm and 445nm of excitation light by using a Tecan multifunctional enzyme-labeled instrument to obtain a standard curve of fluorescence intensity and NMN concentration of y=2539.6x+49.207 (R 2 ＝0.9964)。
TABLE 1 fluorescence reaction System
Extraction of NMN in samples: extracting extracellular NMN, centrifuging 1mL of culture at 4 ℃ and 15700g after bacterial culture is finished, centrifuging for 5min, and sucking the supernatant to be detected; the intracellular NMN was extracted, the pellet was resuspended with 1mL of ultra pure water, sonicated (ice bath, power 120w. Ultrasound 2s, interval 4s. Repeated 30 times), centrifuged at 15700Xg at 4℃for 5min, and the supernatant was aspirated for testing. The fluorescence intensity of the intracellular and extracellular NMN solution is detected by a fluorescence method, and the concentration of NMN is obtained by means of a standard curve.
Example 2CRISPR/Cas9 Gene editing System Gene knockout strategy
Knockout strategy principle: introducing homologous arms at the upstream and downstream of the gene to be knocked out into the escherichia coli BL21 (DE 3), and deleting the gene of the escherichia coli BL21 (DE 3) through homologous recombination by a gene editing technology.
Preparation of E.coli BL21 (DE 3)/pCas electrotransduction competent cells: plasmid pCas into E.coli BL21 (DE 3) competence, spread on solid LB medium (containing 50. Mu.g/mL kanamycin), and culture at 30℃for 8-12h; selecting single colony, inoculating in liquid LB containing kanamycin, culturing at 30deg.C and 220rpm for 12 hr, and collecting seed solution; transferring the seed solution into 50mL culture medium with 2% inoculation amount, culturing at 30deg.C and 220rpm until OD600 is 0.1-0.2, adding L-arabinose to final concentration of 100mM, inducing culture until OD is 0.5-0.6, centrifuging at 5000rpm for 10min, collecting bacteria, making into transformed competent cells, washing with transformed competent cell treatment solution twice, re-suspending thallus with 2mL treatment solution, sub-packaging into 1.5mL EP tube, and storing at-80deg.C.
TABLE 2 preparation of competent cell processing liquid
Preparing homologous arm fragments at the upstream and downstream of the gene to be knocked out: inquiring a genome sequence (GenBank number: NC_ 012971.2) of escherichia coli BL21 (DE 3) in GenBank, selecting 500bp upstream and downstream of a gene to be knocked out as homology arms, respectively designing primers, taking the escherichia coli BL21 (DE 3) genome as a template, obtaining a 500bp homology arm C1 upstream and a 500bp homology arm C2 downstream of the gene to be knocked out by PCR, and connecting the C1 and the C2 by using overlap PCR to obtain a homology arm segment upstream and downstream of the gene to be knocked out.
The sgRNA of the gene to be knocked out was designed using an online website (http:// chopchop. Cbu. Uib. No /), and the primer N20-F/N20-R of the sgRNA was designed using snapgene software. The designed sgRNA sequence was introduced into the primer N20-F/N20-R, and inverse PCR was performed in a 25. Mu.l system using pTargetF as a template to obtain pTargetF containing sgRNA. After preliminary verification by agarose gel electrophoresis, recovery was performed using a gel recovery kit. The gel recovery products were self-ligated using a seamless cloning kit. The ligated plasmid was transferred into E.coli DH 5. Alpha. Competent cells, and the plasmid was extracted using a plasmid extraction kit, and the plasmid with the correct sequence was designated pTargetF-sgRNA by sequencing verification.
The upstream and downstream homology arm fragments of the gene to be knocked out and pTargetF containing sgRNA of the gene to be knocked out were plasmid-transferred into E.coli BL21 (DE 3)/pCas, spread onto solid LB plates (containing 300. Mu.g/mL streptomycin and 50. Mu.g/mL kanamycin) and incubated at 30℃for 16-24h. The obtained monoclonal is picked up and cultured in LB liquid medium at 30 ℃ and 220rpm for 12-14h. Extracting genome according to the instruction of the Norvezan genome extraction kit, performing PCR verification, and if the size of the target band is correct, recovering and sequencing by using a DNA gel recovery kit, and verifying the knockout result.
The other genes need to be continuously knocked out on the basis of the knocked out strain, and pTargetF in bacteria needs to be eliminated: the knocked-out strain was inoculated into liquid LB containing 50. Mu.g/mL kanamycin, IPTG was added at a final concentration of 0.5mM, the Cas9 protein was induced to cleave the pTargetF plasmid, cultured at 30℃and 220rpm for 12-14h, passaged twice, colonies from which pTargetF had been eliminated were screened by the plate photocopying method, and chemocompetent cells were prepared in preparation for the next gene editing.
If no further gene editing is required, further elimination of pCas is required in addition to the elimination of the pTargetF plasmid: the strain with the eliminated pTargetF is inoculated into liquid LB without resistance, cultured for 12-14h at 42 ℃ and 220rpm, passaged twice, and colonies with the eliminated pCas are screened by using a flat-plate photocopying method and preserved for later use.
EXAMPLE 3 construction of Stable high yield NMN engineering Strain E.coli BL21 (DE 3) -DeltadeoD
According to the CRISPR/Cas9 gene editing system gene knockout strategy described in example 2, deoD (the nucleotide sequence is shown as SEQ ID NO: 9) in E.coli BL21 (DE 3) is knocked out, and positive clones are screened to obtain the strain E001 E.coli BL21 (DE 3) delta deoD. E.coli BL21 (DE 3) and E001 were inoculated into LB medium, respectively, and cultured at 37℃and 220rpm for 24 hours, and NMN content was measured as described in example 1. The intracellular NMN content of the strain E001 reaches 3.2mg/L, which is improved by 1.7 times compared with the control group E.coli BL21 (DE 3).
EXAMPLE 4 construction of Stable high yield NMN engineering Strain E.coli BL21 (DE 3) -DeltadeoD DeltaushA
According to the CRISPR/Cas9 gene editing system gene knockout strategy described in example 2, ushA (the nucleotide sequence is shown as SEQ ID NO: 10) is continuously knocked out on the basis of the strain E001, and positive clones are screened to obtain the strain E002 E.coli BL21 (DE 3) delta deoD delta ushA. E.coli BL21 (DE 3) and E002 were inoculated into LB medium, respectively, and cultured at 37℃and 220rpm for 24 hours, and NMN content was measured as described in example 1. The intracellular NMN content of the strain E002 reaches 4.5mg/L, which is improved by 2.4 times compared with the control group.
EXAMPLE 5 construction of Stable high yield NMN engineering Strain E.coli BL21 (DE 3) DeltadeoD DeltaushA DeltanadR
According to the CRISPR/Cas9 gene editing system gene knockout strategy described in example 2, nadR (the nucleotide sequence is shown as SEQ ID NO: 11) is continuously knocked out on the basis of the strain E002, and positive clones are screened to obtain the strain E003 E.coli BL21 (DE 3) delta deoD delta ushA delta nadR. E.coli BL21 (DE 3) and E003 were inoculated into LB medium, respectively, and cultured at 37℃and 220rpm for 24 hours, and the NMN content was measured as described in example 1. The intracellular NMN content of the strain E003 reaches 6.1mg/L, and is improved by 3.3 times compared with a control group.
EXAMPLE 6 construction of stable high yield NMN engineering bacteria E.coli BL21 (DE 3) DeltadeoD DeltaushA DeltanadR DeltapncC
According to the CRISPR/Cas9 gene editing system gene knockout strategy described in example 2, the pncC (the nucleotide sequence is shown as SEQ ID NO: 12) is continuously knocked out on the basis of the strain E003, and positive clones are screened to obtain the strain E004E.coli BL21 (DE 3) delta deoD delta ushA delta nadR delta pncE. E.coli BL21 (DE 3) and E004 were inoculated into LB medium, respectively, and cultured at 37℃and 220rpm for 24 hours, and NMN content was measured as described in example 1. The intracellular NMN content of the strain E004 reaches 7.6mg/L, and is improved by 4.1 times compared with a control group.
EXAMPLE 7 construction of Stable high yield NMN engineering Strain E.coli BL21 (DE 3) DeltajodDeltaushA DeltanadR DeltapncDeltaamb
According to the CRISPR/Cas9 gene editing system gene knockout strategy described in example 2, the amb (the nucleotide sequence is shown as SEQ ID NO: 13) is continuously knocked out on the basis of the strain E004, and positive clones are screened to obtain the strain E005 E.coli BL21 (DE 3) delta deoD delta ushA delta nadR delta pncdelta amb. E.coli BL21 (DE 3) and E005 were inoculated into LB medium, respectively, and cultured at 37℃and 220rpm for 24 hours, and the NMN content was measured as described in example 1. The intracellular NMN content of the strain E005 reaches 9.1mg/L, and is improved by 4.9 times compared with a control group. EXAMPLE 8 construction of stable high yield NMN engineering bacteria E.coli BL21 (DE 3) ΔdeoDΔushA ΔnadR ΔpncΔamnΔadd
According to the CRISPR/Cas9 gene editing system gene knockout strategy described in example 2, the add (the nucleotide sequence is shown as SEQ ID NO: 14) is continuously knocked out on the basis of the strain E005, and positive clones are screened to obtain the strain E006 E.coli BL21 (DE 3) delta deoD delta ushA delta nadR delta pncdelta amp delta add. E.coli BL21 (DE 3) and E006 were inoculated into LB medium, respectively, and cultured at 37℃and 220rpm for 24 hours, and NMN content was measured as described in example 1. The intracellular NMN content of the strain E006 reaches 11.3mg/L, which is improved by 6.1 times compared with the control group.
TABLE 3 sgRNA sequences of different genes
| Gene to be knocked out | sgRNA sequences | Sequence(s) |
| deoD | GACCTGTTCTACTCTCCGGA | SEQ ID NO.15 |
| ushA | GCAAGATCCGGTCTGCATGG | SEQ ID NO.16 |
| nadR | GGTGTGAAAAGACATAATCG | SEQ ID NO.17 |
| pncC | AGTGGCAAAAGCAAACCAGA | SEQ ID NO.18 |
| amn | GTGCGAGGGAAAAATCGACG | SEQ ID NO.19 |
| add | TTGGCAATGACCTGAACGTG | SEQ ID NO.20 |
Example 9 construction of prs overexpression vector (pET 22 b-pPrs)
prs encodes 5-phosphoribosyl-1-pyrophosphate (PRPP) synthetase, which catalyzes the synthesis of NMN from 5-phosphoribosyl-1-pyrophosphate (PRPP) and Nicotinamide (NAM), so that overexpression of prs can realize accumulation of NMN.
TABLE 3 construction of plasmid restriction enzyme System
PCR amplification was performed using the E.coli BL21 (DE 3) genome as a template to obtain prs (nucleotide sequence shown as SEQ ID NO: 2), and NdeI and BamHI were used to cleave the prs fragment and pET22b, respectively, after 2h in a 37℃water bath, agarose gel electrophoresis was performed to verify that the correct DNA product was recovered and ligated overnight at 16 ℃.
TABLE 4 construction of plasmid enzyme ligation System
The overnight enzyme-linked product was transformed into DH 5. Alpha. And the monoclonal was picked up and sent to Jin Weizhi for sequencing, and the monoclonal containing the correct expression plasmid pET22b-PRS was selected. pET22b-PRS was transformed to E006 to give engineering strain E007. E007 was inoculated into LB medium (containing 100. Mu.l)g/mL ampicillin), and the corresponding seed solution was obtained by shaking overnight at 37℃and 220 rpm. The seed solution was transferred to a fresh LB medium (100. Mu.g/mL ampicillin) at 1% inoculum size and incubated at 37℃to OD 600 When =0.8±0.1, the inducer IPTG was added to a final concentration of 0.2mm,37 ℃, and the culture was induced for 24 hours, and the NMN content was measured as described in example 1. The intracellular NMN content of E007 is measured to be 27.2mg/L, which is improved by 2.4 times compared with E006.
EXAMPLE 10 functional expression of nicotinamide riboside phosphate transferase engineering bacteria (E.coli-pVs)
VsNAMPT is a nicotinamide phosphoribosyl transferase (amino acid sequence shown in SEQ ID NO: 1) derived from Variovorax sp. The coding gene thereof is subjected to synthesis, an expression plasmid pET22b-VsNAMPT is constructed according to the plasmid construction strategy described in example 9, and the expression plasmid pET22b-VsNAMPT is transformed into E.coli BL21 (DE 3) to obtain an engineering strain E.coli-pVs.
E.coli-pVs was inoculated into LB medium (containing 100. Mu.g/mL ampicillin) and cultured at 37℃with shaking at 220rpm overnight to obtain a corresponding seed solution. The seed solution was transferred to a fresh LB medium (100. Mu.g/mL ampicillin) at 1% inoculum size and incubated at 37℃to OD 600 When=0.8±0.1, the final concentration was 0.2mm by adding IPTG as an inducer, and the culture was induced for 24 hours at 37 ℃. SDS-PAGE analysis shows that VsNAMPT has soluble expression, and the soluble expression accounts for 42.4%.
After the induction of expression was completed, E.coli-pVs cells were collected, resuspended by dilution with 0.05M Tris-HCl buffer (pH 7.5), and nicotinamide (1.5 g/L) was added to construct a whole cell biosynthesis system. After a reaction time of 12 hours at 37 ℃, the NMN content was measured using the fluorescence method as described in example 1, and the result showed that VsNAMPT could catalyze nicotinamide to NMN with a conversion of 76.3%.
EXAMPLE 11 optimization of Prs and VsNAMPT Co-expression strategy
Fusion co-expression: designing upstream and downstream primers according to constructed plasmids pET22b-PRS and pET22b-VsNAMPT, adding a linker sequence (GGCGGCGGCGGCGGCTCGGGCGGCGGCGGCGGCTCG) between each gene, obtaining pET22b-VsNAMPT-linker-PRS by using an overlap PCR technology, and transforming to a strain E006 to obtain engineering bacteriaStrain E008. E008 was inoculated into LB medium (containing 100. Mu.g/mL ampicillin) and cultured at 37℃with shaking at 220rpm overnight to obtain a corresponding seed solution. The seed solution was transferred to a fresh LB medium (100. Mu.g/mL ampicillin) at 1% inoculum size and incubated at 37℃to OD 600 When =0.8±0.1, the inducer IPTG was added to a final concentration of 0.2mM, nicotinamide (1.5 g/L) was added, and the culture was induced at 37 ℃ for 24 hours, and the NMN content was measured as described in example 1. The intracellular NMN content of E008 is 263.8mg/L, which is improved by 9.7 times compared with E007.
Co-expression of single plasmids: and designing upstream and downstream primers according to constructed plasmids pET22b-PRS and pET22b-VsNAMPT, adding an SD-AS sequence (AGAAGGAGATATACA) between each gene, obtaining pET22b-VsNAMPT-SD-AS-PRS by using an overlap PCR technology, and converting to a strain E006 to obtain an engineering strain E009. E009 was inoculated into LB medium (containing 100. Mu.g/mL ampicillin) and cultured overnight at 37℃with shaking at 220rpm to obtain the corresponding seed solution. The seed solution was transferred to a fresh LB medium (100. Mu.g/mL ampicillin) at 1% inoculum size and incubated at 37℃to OD 600 When =0.8±0.1, the inducer IPTG was added to a final concentration of 0.2mM, nicotinamide (1.5 g/L) was added, and the culture was induced at 37 ℃ for 24 hours, and the NMN content was measured as described in example 1. The content of NMN in E009 cells is 891.6mg/L, which is improved by 33 times compared with E007.
Example 12 promoter screening of Nicotinamide phosphoribosyl transferase engineering bacteria
In order to reduce the cost of raw materials and improve the industrial competitiveness, constitutive promoter replacement was performed. The promoters PL1118, PL1744, PL277, PUTRssrA-PUTRinfC-rplT (nucleotide sequences are shown AS SEQ ID NO:3,SEQ ID NO:4,SEQ ID NO:5,SEQ ID NO:6 respectively) are constructed into an expression plasmid pET22b-VsNAMPT-SD-AS-PRS by utilizing seamless cloning, and the T7 promoter is replaced, so that plasmids pET22b-PL1118-VsNAMPT-SD-AS-PRS, pET22b-PL1744-VsNAMPT-SD-AS-PRS, pET22b-PL277-VsNAMPT-SD-AS-PRS and pET22b-PUTRssrA-PUTRinfC-rplT-VsNAMPT-SD-AS-PRS are obtained by respectively converting the promoters into E006 to obtain engineering strains E.coli-PL1118-NMN, E.coli-PL 4-NMN, E.coli-PL 277-SD-AS-PRS, E.coli-PUTRrA-PUINC-rplNMN.
E.coli-PL1118-NMN, E.coli-PL1744-NMN, E.coli-PL277-NMN and E.coli-PUTRssrA-PUTRinfC-rplT-NMN were inoculated into LB medium (containing 100. Mu.g/mL ampicillin) respectively, and cultured overnight at 37℃with shaking at 220rpm to obtain corresponding seed solutions. The seed solution was transferred to a fresh LB medium (100. Mu.g/mL ampicillin) at 1% inoculum size, incubated at 37℃for 24 hours, and NMN content was measured as described in example 1. The measured intracellular NMN contents of E.coli-PL1118-NMN, E.coli-PL1744-NMN, E.coli-PL277-NMN and E.coli-PUTRssrA-PUTRinfC-rplT-NMN are 1926.6mg/L, 2282.5mg/L, 2736.9mg/L and 1867.4mg/L respectively, which are improved by 2.2, 2.6, 3.1 and 2.1 times compared with E009 respectively.
TABLE 4 comparison of NMN content in different knock-out strains and in obtaining final strains
Note that: e.coli-pVs only verifies the catalytic function of the protein, so specific values are not written.
From the above, the transformation efficiency of engineering bacteria formed by knocking out genes is 7 times or less, but the transformation efficiency of E.coli-NMN is 1471.5 times, which is greatly higher than that of the engineering bacteria, and the transformation efficiency is obviously different, so that the genes form a synergistic effect in the engineering bacteria.
(1) VsNAMPT nucleotide sequence (SEQ ID NO: 1)
ATGACCCGCAACCCGACGAGCCTGGGCGGCAACCTGTTACTGCGCACCGATAGCTATAAAGTGAGCCATTGGATGCAGTATCCGCCGGGCACGCAGACCGTGTTTAGCTATATTGAAAGCCGCGGCGGCGCGTTTAGCCATAGCGTGTTTTTTGGCCTGCAAGCGTATCTGCGCGAATATCTGAGCACCCCGGTGACCGTGGAAGATGTGGATGAAGCGGCCGCGCTGATGGCGCTGCATGGCGAACCGTTTAACCGCGAAGGCTGGCTGCGCCTGATTGAAAAACATGGCGGCCTGATGCCGGTGCGCATTCGCGCGATGCCGGAAGGCAGCGTGGTGCCGGTGCATAACGTGCTGGCGACCATTGAAAACACCGATCCGGAATTTTTTTGGGTGACGAGCTTTCTGGAAACCGAACTGCTGCGCGCGATTTGGTATCCGACCACCGTGGCGACCCTGAGCGGCGCGGCGAAAGCGACCCTGATGCGCTATTTAGACGCGACCTGCGAAGATCCGGCGGCGCAGATTGGCTTTAAACTGCATGATTTTGGCGCGCGCGGCGTGAGCAGCCTGGAAAGCGCGGCGCTGGGCGGCATGGCGCATCTGGTGAACTTTCTGGGCACCGATACCATTGCGGCGCTGGTGGCGGCGCGCCGCTATTATGATTGCGATGTGGCGGGCTTTAGCATTCCGGCGGCGGAACATAGCACCATTACCGGCTGGGGCCGCGATAAAGAAAGCAGCGCGTATCGCAACATGGTGGAACAGTTTGGCAAACCGGGCGCGATTTTTGCGGTGGTGAGCGATAGCTATGATATTTTTAACGCGTGCGATCAGATTTGGGGCACCGAACTGAAAGATCTGGTGGTGCAGAGCGGCGCGACCCTGGTGGTGCGCCCGGATAGCGGCGATCCGGCGGAAACCGTGCTGAAAGTGGTGCGCATTCTGGCGGCGCGCTTTGGCACCACCCGCAACACCAAAGGCTATCTGGTGCTGAACAACGTGCGCGTGATTCAAGGCGATGGCATTAACCTGGATAGCCTGCGCCTGGTGCTGAGCAACCTGTTTCATAACGGCTTTAGCGCGGAAAACGTGGCGTTTGGCATGGGCGGTGGCCTGCTGCAGCAAGTGAACCGCGATACCATGCAGTGGGCGATGAAATGCAGCGCGATGCAAGTGAACGGCGAATGGCGCGATGTGTATAAAGCGCCGGTGGGCGATATGAGCAAAGCGAGCAAAAAAGGCCGCCTGACCCTGACCCGCGATGCGAAAGGCGCGATGCAGACGCAGCGCATTGAAACCCTGGATGCGACGCAGACCGATCTGCTGGAAACCGTGTTTGAAGATGGCCGCATTGTGCGCCAAACGACCTTTGACGCGGTGCGTGAGCGCGCCGCGGCGGGCGCGCGCGCGCTGGTGACGAGCCGCGCGCCGCTGTAAGGATCC
(2) prs nucleotide sequence (SEQ ID NO: 2)
TTAGTGTTCGAACATGGCAGAGATCGATTCTTCGTTGCTGATACGACGAATCGCTTCGGCCAGCATACCTGACAGGGTCAGAGTACGCACGTTCGGCAGTGATTTGATTTCATCGCTCAGCGGAATGGTATCGCAGACAACGACTTCATCAATTACAGAGTTACGCAGGTTGTTCGCCGCGTTGCCAGAGAAGATCGGGTGAGTCGCGTACGCAAATACACGTTTAGCACCACGTTCTTTCAGAGCTTCAGCAGCTTTACACAGCGTACCGCCAGTGTCGATCATATCATCGACCAGTACGCAGTCACGACCTGCAACGTCACCGATGATATGCATCACCTGTGAAACGTTCGCACGCGGACGACGTTTGTCGATGATTGCCATATCGGTATCGTTCAGCAGCTTAGCGATAGCGCGGGCACGCACAACGCCGCCGATGTCCGGAGAAACCACAATTGGGTTATCCAGATTCAGCTGCAGCATGTCTTCCAGCAGGATCGGGCTACCAAATACGTTATCAACCGGAACGTCGAAGAAACCCTGAATCTGTTCAGCGTGCAGATCCACTGTCAGCACACGGTCAACACCGACGCTGGAGAGGAAGTCTGCAACCACTTTCGCAGTGATTGGTACACGAGCGGAACGGACGCGACGGTCCTGGCGCGCATAGCCAAAGTAGGGGATAACAGCGGTGATACGACCTGCGGAAGCACGACGCAGGGCATCAACCATAACGACTAATTCCATCAGGTTGTCGTTAGTAGGGGCACAAGTGGACTGGATGATGAAAATATCACCACCGCGTACATTTTCATTAATTTGTACGCTGACTTCGCCGTCGCTAAAGCGACCTACAGCGGCGTCGCCGAGTGAAGTGTACAGGCGGTTGGCAATACGTTGTGCTAGTTCCGGGGTGGCGTTACCAGCAAAAAGCTTCATATCAGGCAC
(3) Promoter PL1118 nucleotide sequence (SEQ ID NO: 3)
TTGACAATTAATCATCCGGCTCTTATAATGTGTGGAATTGCGAGCGGTTAACAA TTTCACACAGGAAACAGACC
(4) Promoter PL1744 nucleotide sequence (SEQ ID NO: 4)
TTGACAATTAATCATCCGGCTCTTATAATGTGTGGAATTGCGAGCCGATAACAA TTTCACACAGGAAACAGACC
(5) Promoter PL277 nucleotide sequence (SEQ ID NO: 5)
TTGACAATCAATCATCCGGCTCGTATAATGTGTGGAATTGTTATCTGCTCACAA TTCCACACAGGAAACAGACC
(6) Promoter PUTRssrA-PUTRinfC-rplT nucleotide sequence (SEQ ID NO: 6)
ATTGGCTATCACATCCGACACAAATGTTGCCATCCCATTGCTTAATCGAATAAAAATCAGGCTACATGGGTGCTAAATCTTTAACGATAACGCCATTGAGGCTGGTCATGGCGCTCATAAATCTGGTATACTTACCTTTACACATTGCGGGCATTCGTGTTAAAGCAGACTTGAGAAATGAGAAGATTGGCTTTAAAATCCGCGAGCACACTTTGCGTCGCGTCCCATATATGCTGGTCTGTGGTGATAAAGAGGTGGAATCAGGCAAAGTTGCCGTTCGCACCCGCCGTGGTAAAGACCTGGGAAGCATGGACGTAAATGAAGTGATCGAGAAGCTGCAACAAGAGATTCGCAGCCGCAGTCTTAAACAATTGGAGGAATAAGGT
(7) deoD-C1 nucleotide sequence (SEQ ID NO: 7)
TATCACCAAGAAAGTGAAAGCAACTGGCCTGGACGCGCTGTTTGACGCCACCATCAAAGAGATGAAAGAAGCGGGTGATAACACCATCGTCTTCACCAACTTCGTTGACTTCGACTCTTCCTGGGGCCACCGTCGCGACGTCGCCGGTTATGCTGCGGGTCTGGAGCTGTTCGACCGCCGTCTGCCGGAGCTGATGTCTCTGCTGCGCGATGACGACATCCTGATCCTCACTGCTGACCACGGTTGTGATCCGACCTGGACCGGTACTGACCACACGCGTGAACACATTCCGGTACTGGTTTACGGCCCGAAAGTAAAACCGGGCTCACTGGGTCACCGTGAAACCTTCGCGGATATTGGTCAGACTCTGGCAAAATATTTTGGTACTTCTGATATGGAATATGGCAAAGCCATGTTCTGATAATGGGTGCGGCTTGATGCCCTCACCCCGGCCCTCTCCCACAGGGAGAGGGTGAAAACATGTTGATAAGGATAAAACA
(8) deoD-C2 nucleotide sequence (SEQ ID NO: 8)
TTGTGTTTCGCTGAAAGGCGAACGTCGTGTTAAGCCGGAGCAGGAGACTGCTCCGGCTTTTTAGTATCTATTCATCAATAGCAACTGATTTATAGATTTACTATTATTTTTCATATGGATAGAAAATAAAATCCATTCAAGTATCTATTCACGAATCTATTCATTTATGTTCAGCCGTCATCCAGGAAGGGCATATCAGCAACTCTGGTGGACAACCATAAATTTCTGCCATTTTGCTTTTGATGTTTGAGCTAAGCCGTTCCGTCTTTTCGATCTGTTTTAGAGAATTAATCGAGATACCTAACATTTTACTTACTTCAGCAAGCGATAATTTATGCCTGATACGCACGGCGGCTAGCCAGGAAATACGCTGGCTGAACATTAAATTGATAATCTCCCGTTCCTGGTTTTCCAGATCGATCATATTTCCTTCCCTGGATATTTTAATCAGCGAGTTACCTTACCGCCCCCGCTATCCACGTCGACAACTTCCGTAGCTC
(9) deoD nucleotide sequence (SEQ ID NO: 9)
ATGGCTACCCCACACATTAATGCAGAAATGGGCGATTTCGCTGACGTAGTTTTGATGCCAGGCGACCCGCTGCGTGCGAAGTATATTGCTGAAACTTTCCTTGAAGATGCCCGTGAAGTGAACAACGTTCGCGGTATGCTGGGCTTCACCGGTACTTACAAAGGCCGCAAAATTTCCGTAATGGGTCACGGTATGGGTATCCCGTCCTGCTCCATCTACACCAAAGAACTGATCACCGATTTCGGCGTGAAGAAAATTATCCGCGTGGGTTCCTGTGGCGCAGTTCTGCCGCACGTAAAACTGCGCGACGTCGTTATCGGTATGGGTGCCTGCACCGATTCCAAAGTTAACCGTATCCGTTTTAAAGACCATGACTTTGCCGCTATCGCTGACTTTGACATGGTGCGTAACGCGGTAGACGCGGCTAAAGCACTGGGTGTTGATGCTCGCGTGGGTAACCTGTTCTCCGCTGACCTGTTCTACTCTCCGGACGGCGAAATGTTCGACGTGATGGAAAAATACGGCATCCTCGGCGTGGAAATGGAAGCGGCTGGTATCTACGGCGTCGCTGCAGAGTTTGGCGCGAAAGCCCTGACCATCTGCACCGTGTCTGACCACATCCGCACTCACGAGCAGACCACTGCCGCTGAGCGTCAGACCACCTTCAACGACATGATCAAAATCGCACTGGAATCCGTTCTGCTGGGCGATAAAGAGTAA
(10) ushA nucleotide sequence (SEQ ID NO: 10)
ATGAAATTATTGCAGCGGGGCGTGGCGTTAGCGCTGTTAACCACATTTACACTGGCGAGTGAAACTGCTCTGGCGTATGAGCAGGATAAAACCTACAAAATTACAGTTCTGCATACCAATGATCATCATGGGCATTTTTGGCGCAATGAATATGGCGAATATGGTCTGGCGGCGCAAAAAACGCTGGTGGATGGTATCCGCAAAGAGGTTGCGGCTGAAGGCGGTAGCGTGCTGCTACTTTCCGGTGGCGACATTAACACTGGCGTGCCCGAGTCTGACTTACAGGATGCCGAACCTGATTTTCGCGGTATGAATCTGGTGGGCTATGACGCGATGGCGATCGGTAATCATGAATTTGATAATCCGCTCACCGTATTACGCCAGCAGGAAAAGTGGGCCAAGTTCCCGTTGCTTTCCGCGAATATCTACCAGAAAAGTACTGGCGAGCGCCTGTTTAAACCGTGGGCGCTGTTTAAGCGTCAGGATCTGAAAATTGCCGTTATTGGGCTGACAACCGATGACACAGCAAAAATTGGTAACCCGGAATACTTCACTGATATCGAATTTCGTAAGCCCGCCGATGAAGCGAAGCTGGTGATTCAGGAGCTGCAACAGACAGAAAAGCCAGACATTATTATCGCGGCGACCCATATGGGGCATTACGATAATGGTGAGCACGGCTCTAACGCACCGGGCGATGTGGAGATGGCACGCGCGCTGCCTGCCGGATCGCTGGCGATGATCGTCGGTGGTCACTCGCAAGATCCGGTCTGCATGGCGGCAGAAAACAAAAAACAGGTCGATTACGTGCCGGGTACGCCATGCAAACCAGATCAACAAAACGGCATCTGGATTGTGCAGGCGCATGAGTGGGGCAAATACGTGGGACGGGCTGATTTTGAGTTTCGTAATGGCGAAATGAAAATGGTTAACTACCAGCTGATTCCGGTGAACCTGAAGAAGAAAGTGACCTGGGAAGACGGGAAAAGCGAGCGCGTGCTTTACACTCCTGAAATCGCTGAAAACCAGCAAATGATCTCGCTGTTATCACCGTTCCAGAACAAAGGCAAAGCGCAGCTGGAAGTGAAAATAGGCGAAACCAATGGTCGTCTGGAAGGCGATCGTGACAAAGTGCGTTTTGTACAGACCAATATGGGGCGGTTGATTCTGGCAGCCCAAATGGATCGCACTGGTGCCGACTTTGCGGTGATGAGCGGAGGCGGAATTCGTGATTCTATCGAAGCAGGCGATATCAGCTATAAAAACGTGCTGAAAGTGCAGCCATTCGGCAATGTGGTGGTGTATGCCGACATGACCGGTAAAGAGGTGATTGATTACCTGACCGCCGTCGCGCAGATGAAGCCAGATTCAGGTGCCTACCCGCAATTTGCCAACGTTAGCTTTGTGGCGAAAGACGGCAAACTGAACGACCTTAAAATCAAAGGCGAACCGGTCGATCCGGCGAAAACTTACCGTATGGCGACATTAAACTTCAATGCCACCGGCGGTGATGGATATCCGCGCCTTGATAACAAACCGGGCTATGTGAATACCGGCTTTATTGATGCCGAAGTGCTGAAAGCGTATATCCAGAAAAGCTCGCCGCTGGATGTGAGTGTTTATGAACCGAAAGGTGAGGTGAGCTGGCAGTAA
(11) nadR nucleotide sequence (SEQ ID NO: 11)
ATGTCGTCATTTGATTACCTGAAAACTGCCATCAAGCAACAGGGCTGCACGCTACAGCAGGTAGCTGATGCCAGCGGTATGACCAAAGGGTATTTAAGCCAGTTACTGAATGCCAAAATCAAAAGCCCCAGCGCGCAAAAGCTGGAGGCGTTGCACCGTTTTTTGGGGCTTGAGTTTCCCCGGCAGAAGAAAACGATCGGTGTCGTATTCGGTAAGTTCTACCCACTGCATACCGGACATATCTACCTTATCCAGCGCGCCTGTAGCCAGGTTGACGAGCTGCATATCATTATGGGTTTTGACGATACCCGTGACCGCGCGTTGTTCGAAGACAGTGCCATGTCGCAGCAGCCGACCGTGCCGGATCGTCTGCGTTGGTTATTGCAAACTTTTAAATATCAGAAAAATATTCGCATTCATGCTTTCAACGAAGAGGGCATGGAGCCGTATCCGCACGGCTGGGATGTGTGGAGCAACGGCATCAAAAAGTTTATGGCTGAAAAAGGGATCCAGCCGGATCTGATCTACACCTCGGAAGAAGCCGATGCGCCACAGTATATGGAACATCTGGGGATCGAGACGGTGCTGGTCGATCCGAAACGTACCTTTATGAGTATCAGCGGTGCGCAGATCCGCGAAAACCCGTTCCGCTACTGGGAATATATTCCTACCGAAGTGAAGCCGTTTTTTGTGCGTACCGTGGCGATCCTTGGCGGCGAGTCGAGCGGTAAATCCACCCTGGTAAACAAACTTGCCAATATCTTCAACACCACCAGTGCGTGGGAATATGGCCGCGATTATGTCTTTTCACACCTCGGCGGTGATGAGATCGCATTGCAGTATTCTGACTACGATAAAATCGCGCTGGGCCACGCTCAATACATTGATTTTGCGGTGAAATATGCCAATAAAGTGGCATTTATCGATACCGATTTTGTCACCACTCAGGCGTTCTGCAAAAAGTACGAAGGGCGGGAACATCCGTTCGTGCAGGCGCTGATTGATGAATACCGTTTCGATCTGGTGATCCTGCTGGAGAACAACACGCCGTGGGTGGCGGATGGTTTACGCAGCCTCGGCAGTTCGGTGGATCGCAAAGAGTTCCAGAACTTGCTGGTGGAGATGCTCGAAGAGAACAATATCGAATTTGTGCGGGTTGAAGAGGAAGATTACGACAGTCGTTTCCTGCGCTGCGTGGAACTGGTGCGGGAGATGATGGGGGAGCAGAGATAA
(12) pncC nucleotide sequence (SEQ ID NO: 12)
ATGACTGACAGTGAACTGATGCAGTTAAGTGAACAGGTTGGGCAGGCGCTGAAAGCCCGTGGCGCAACCGTAACAACTGCCGAGTCTTGTACCGGTGGTTGGGTAGCGAAAGTGATTACCGATATTGCCGGTAGCTCCGCCTGGTTTGAACGCGGATTTGTCACCTACAGTAACGAAGCCAAAGCGCAGATGATCGGCGTACGCGAAGAGACGCTGGCGCAGCATGGCGCGGTGAGTGAACCCGTCGTGGTGGAAATGGCGATAGGCGCACTGAAAGCGGCTCGTGCTGATTATGCCGTGTCTATTAGTGGTATCGCCGGGCCGGATGGCGGCAGTGAAGAGAAGCCTGTCGGCACCGTCTGGTTTGCTTTTGCCACTGCCCGCGGTGAAGGCATTACCCGGCGGGAATGCTTCAGCGGCGACCGTGATGCGGTGCGTCGTCAGGCTACTGCGTATGCATTGCAGACCTTGTGGCAACAATTTCTACAAAACACTTGA
(13) The amp nucleotide sequence (SEQ ID NO: 13)
ATGAATAATAAGGGCTCCGGTCTGACCCCAGCTCAGGCACTGGATAAACTCGACGCGCTGTATGAGCAATCTGTAGTCGCATTACGCAACGCCATTGGCAACTATATTACAAGTGGCGAATTACCTGATGAAAACGCCCGCAAACAAGGTCTTTTTGTCTATCCATCACTGACCGTAACCTGGGACGGTAGCACAACCAATCCCCCCAAAACGCGCGCATTTGGTCGCTTTACCCACGCAGGCAGCTACACCACCACGATTACTCGCCCTACTCTCTTTCGTTCGTATCTTAATGAACAACTTACGTTGCTGTATCAGGATTATGGTGCGCATATCTCAGTGCAACCCTCGCAGCATGAAATCCCTTATCCTTATGTCATCGATGGCTCTGAATTGACACTTGATCGCTCAATGAGCGCTGGGTTAACTCGCTACTTCCCGACAACAGAACTGGCGCAAATTGGCGATGAAACTGCAGACGGCATTTATCATCCAACTGAATTCTCCCCGCTATCGCATTTTGATGCGCGCCGCGTCGATTTTTCCCTCGCACGGTTGCGCCATTATACCGGTACGCCAGTTGAACATTTTCAGCCGTTCGTCTTGTTTACCAACTACACACGTTATGTGGATGAATTCGTTCGTTGGGGATGCAGCCAGATCCTCGATCCTGATAGTCCCTACATTGCCCTTTCTTGTGCTGGCGGGAACTGGATCACCGCCGAAACCGAAGCGCCAGAAGAAGCCATTTCCGACCTTGCATGGAAAAAACATCAGATGCCAGCATGGCATTTAATTACCGCCGATGGTCAGGGTATTACTCTGGTGAATATTGGCGTGGGACCGTCAAATGCTAAAACCATCTGCGATCATCTGGCAGTGCTACGCCCGGATGTCTGGTTGATGATTGGTCACTGTGGCGGATTACGTGAAAGTCAGGCCATTGGCGATTATGTACTTGCACACGCTTATTTACGCGATGACCACGTTCTTGATGCGGTTCTGCCGCCCGATATTCCTATTCCGAGCATTGCTGAAGTGCAACGTGCGCTTTATGACGCCACCAAGCTGGTGAGCGGCAGGCCCGGTGAGGAAGTCAAACAGCGGCTACGTACTGGTACTGTGGTAACCACAGATGACAGGAACTGGGAATTACGTTACTCAGCTTCTGCACTTCGTTTTAACTTAAGCCGGGCCGTAGCAATTGATATGGAAAGTGCAACCATTGCCGCGCAAGGATATCGTTTCCGCGTGCCATACGGGACACTACTGTGTGTTTCAGATAAACCGTTGCATGGCGAGATTAAACTTCCCGGCCAGGCTAACCGTTTTTATGAAGGCGCTATTTCCGAACATCTGCAAATTGGCATTCGGGCGATCGATTTGCTGCGCGCAGAAGGCGACCGACTGCATTCGCGTAAATTACGAACCTTTAATGAGCCGCCGTTCCGATAA
(14) add nucleotide sequence (SEQ ID NO: 14)
ATGATTGATACCACCCTGCCATTAACTGATATCCATCGCCACCTTGATGGCAACATTCGTCCCCAGACCATTCTTGAACTTGGCCGCCAGTATAATATCTCGCTTCCTGCACAATCCCTGGAAACACTGATTCCCCACGTTCAGGTCATTGCCAACGAACCCGATCTGGTGAGCTTTCTGACCAAACTTGACTGGGGCGTTAAAGTTCTCGCCTCTCTTGATGCCTGTCGCCGCGTGGCATTTGAAAACATTGAAGATGCAGCCCGTCACGGCCTGCACTATGTCGAGCTGCGTTTTTCACCAGGCTACATGGCAATGGCACATCAGCTGCCTGTAGCGGGTGTTGTCGAAGCGGTGATCGATGGCGTACGTGAAGGTTGCCGCACCTTTGGTGTGCAGGCGAAGCTTATCGGCATTATGAGCCGGACCTTCGGCGAAGCCGCCTGTCAGCAAGAGCTGGAGGCCTTTTTAGCCCACCGTGACCAGATTACCGCACTTGATTTAGCCGGTGATGAACTTGGTTTCCCGGGAAGTCTGTTCCTTTCTCACTTCAACCGCGCGCGTGATGCGGGCTGGCATATTACCGTCCATGCAGGCGAAGCTGCCGGGCCGGAAAGCATCTGGCAGGCGATTCGTGAACTGGGTGCGGAGCGTATTGGACATGGCGTAAAAGCCATTGAAGATCGGGCGCTGATGGATTTTCTCGCCGAGCAACAAATTGGTATTGAATCCTGTCTGACCTCCAATATTCAGACCAGCACCGTAGCAGAGCTGGCTGCACATCCGCTGAAAACGTTCCTTGAGCATGGCATTCGTGCCAGCATTAACACTGACGATCCCGGCGTACAGGGAGTGGATATCATTCACGAATATACCGTTGCCGCGCCAGCTGCTGGGTTATCCCGCGAGCAAATCCGCCAGGCACAGATTAATGGTCTGGAAATGGCTTTCCTCAGCGCTGAGGAAAAACGCGCACTGCGAGAAAAAGTCGCCGCGAAGTAA
Claims (9)
1. The high-yield nicotinamide mononucleotide engineering strain is characterized by being constructed according to the following steps: the following genes were expressed in the strain: nicotinamide phosphoribosyl transferase, related genes for enhancing NMN synthesis precursors, knocked-out NMN degradation related genes, knocked-out genes, ATP supply enhancement, optimization of a co-expression mode, and combination of promoters to obtain stable high-yield nicotinamide mononucleotide engineering strains.
2. The high-yield nicotinamide mononucleotide engineering strain of claim 1, wherein the strain is escherichia coli, lactobacillus, streptomyces, saccharomycetes or bacillus subtilis.
3. The high-yield nicotinamide mononucleotide engineering strain according to claim 1, wherein the nicotinamide riboside transferase is VsNAMPT derived from Variovorax sp, and the nucleotide sequence of the nicotinamide mononucleotide engineering strain is shown as SEQ ID NO. 1; and nicotinamide riboside transferase having greater than 30% homology to the VsNAMPT nucleotide sequence.
4. The high-yield nicotinamide mononucleotide engineering strain of claim 1, wherein the enhanced NMN synthesis precursor gene is an overexpressed endogenous 5-phosphoribosyl-1-pyrophosphate synthetase gene prs, and the nucleotide sequence is shown as SEQ ID NO. 2.
5. The high-yield nicotinamide mononucleotide engineering strain according to claim 1, wherein the NMN degradation related genes are deoD, ushA, nadR and pncC; the nucleotide sequence of deoD is shown as SEQ ID NO. 9, the nucleotide sequence of ushA is shown as SEQ ID NO. 10, the nucleotide sequence of nadR is shown as SEQ ID NO. 11, and the nucleotide sequence of pncC is shown as SEQ ID NO. 12.
6. A high nicotinamide mononucleotide engineered strain according to claim 1, characterized in that said enhanced ATP supply is achieved by knocking out an, add; the nucleotide sequence of the amp is shown as SEQ ID NO. 13, and the nucleotide sequence of the add is shown as SEQ ID NO. 14.
7. The high-yield nicotinamide mononucleotide engineering strain according to claim 1, wherein the gene co-expression mode is single plasmid co-expression or fusion expression.
8. The nicotinamide mononucleotide engineering strain with high yield according to claim 1, wherein the promoter comprises PL1118, PL1744, PL277, PUTRssrA-PUTRinffC-rplT, and the nucleotide sequences of the promoters are SEQ ID NO. 3,SEQ ID NO:4,SEQ ID NO:5,SEQ ID NO:6 respectively.
9. The application of the engineering strain in preparing nicotinamide mononucleotide, which is characterized in that nicotinamide is taken as a donor, and NMN is synthesized by the high-yield nicotinamide mononucleotide engineering strain according to any one of claims 1-8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311635722.XA CN117660280A (en) | 2023-12-01 | 2023-12-01 | Engineering strain for high yield of NMN and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311635722.XA CN117660280A (en) | 2023-12-01 | 2023-12-01 | Engineering strain for high yield of NMN and application thereof |
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| Publication Number | Publication Date |
|---|---|
| CN117660280A true CN117660280A (en) | 2024-03-08 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311635722.XA Pending CN117660280A (en) | 2023-12-01 | 2023-12-01 | Engineering strain for high yield of NMN and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117660280A (en) |
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2023
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