CN106635943A - Method for improving content of intracellular oxidation type coenzymes I - Google Patents

Abstract

The invention discloses a method for improving the content of intracellular oxidation type coenzymes I, and belongs to the technical field of cofactor engineering. The method mainly aims at the NAD<+> in escherichia coli for synthesizing a plurality of key genes of the NAD<+> through co-expression; meanwhile, different precursor substances are added in the aerobic fermentation process of the recombination strains; the content of the intracellular oxidation type coenzymes I of the escherichia coli is improved. Therefore the content of the intracellular NAD<+> reaches the maximum value. The ideal and the scheme are provided for solving the problems to efficiently utilize substrates, increase the target products, improve the NAD (H) relay type biological catalysis reaction efficiency and the like.

Description

A kind of method for improving intracellular NAD content

Technical field

The present invention relates to a kind of method for improving intracellular NAD content, belongs to co-factor field of engineering technology.

Background technology

DPN, also known as NADH (nicotinamide adenine dinucleotide), be The coenzyme of one kind transmission electronics (hydrogen ion), KEGG database displaying NADH/NAD⁺Participate in the overall process as important co-factor micro- More than 740 metabolic response in biological cell, including the expression of redox reaction, energy metabolism, controlling gene, maintenance Ca⁺Stable state and regulation cell ageing etc., especially playing in glycolysis, gluconeogenesis, tricarboxylic acid cycle and respiratory chain to replace The effect in generation.NAD⁺Energy and redox power can be provided for various reactions in metabolism network, its concentration can regulate and control metabolism net Network approach, increases metabolism circulation, improves yield of living things catalysis efficiency and target product etc. in redox reaction.

NAD⁺Biosynthesis mainly has two approach：De novo synthesis and salvage route (Fig. 1).De novo formation way Footpath generates quinolinic acid (QA) with aspartic acid or tryptophan as precursor by series of steps, and then Jing genes nadC is encoded Quinolate phosphoribosyl transferase catalysis generates NAMN (NaMN), then the adenylase encoded by gene nadD is urged Change polyadenylation generation nicotinate adenine dinucleotide (NaAD), finally in the NAD of gene nadE codings⁺Amino under synthesis enzymatic Chemical conversion NAD⁺.Remedial pathway is that the nicotinamidase hydrolysis niacinamide of gene pncA codings generates nicotinic acid, then Jing genes pncB codings Nicotinic acid phosphoribosyltransferase catalysis nicotinic acid generate NAMN (NaMN), ultimately produce NAD⁺.When intracellular a large amount of There is NAD⁺When precursor niacinamide (NAM), nicotinic acid (NA), remedial pathway is to intracellular NAD⁺Content play an important role.

At present, intracellular NAD is improved⁺Content mainly considers from metabolic engineering and Biochemical Engineering both direction, wherein wrapping Include：Overexpression NADH metabolizing enzymes, disappearance NADH competition approach, introducing NADH exogenous metabolism approach, addition exogenous electron Acceptor, different redox state substrates and NAD synthesis precursors, regulation culture environment and redox potential etc..

It is excellent that Escherichia coli are understood due to genetic background, easy to operate, easy-regulating, culture medium requirement are simple and growth cycle is short etc. Point, in being widely used in research work.However, its intracellular coenzyme content is relatively low, it is impossible to play enhancing associated catalytic anti- Answer the effect of efficiency.Therefore, intracellular NAD is strengthened by engineered means⁺Content is for raising intracellular NAD Content there is important function.

The content of the invention

First purpose of the present invention is to provide at least two in a kind of coexpression pncA, pncB, nadC, nadD, nadE The recombination bacillus coli of gene.

In one embodiment of the invention, the recombination bacillus coli is with E.coli BL21 as host.

In one embodiment of the invention, the recombination bacillus coli be coexpression pncA, pncB, nadC, nadD, Any three kinds of genes in nadE.

In one embodiment of the invention, recombination bacillus coli coexpression pncA, nadD, nadE gene.

In one embodiment of the invention, recombination bacillus coli coexpression pncB, nadE, nadD gene.

In one embodiment of the invention, recombination bacillus coli coexpression nadC, nadD, nadE gene.

In one embodiment of the invention, recombination bacillus coli coexpression pncB, nadE, pncA gene.

Second object of the present invention is to provide the construction method of the recombination bacillus coli, be with pET-21a as carrier, At least 2 genes in pncA, pncB, nadC, nadD, nadE are expressed in E. coli BL21.

Third object of the present invention is to provide a kind of side of raising recombination bacillus coli intracellular NAD content Method, methods described is that the recombination bacillus coli is seeded in fermentation medium, 30～37 DEG C of 8～72h of culture.

In one embodiment of the invention, the inoculation is that by volume 1% inoculum concentration is inoculated with.

In one embodiment of the invention, the color ammonia in the fermentation medium also containing 1～100mg/L of final concentration At least one in acid, aspartic acid, quinolinic acid, nicotinic acid, niacinamide.

In one embodiment of the invention, methods described is thalline OD after inoculation₆₀₀When reaching 0.6-2.0, add The IPTG of final concentration of 0.1-10mM, 16-37 DEG C of induction 8-48h.

The present invention also provides application of the recombination bacillus coli in terms of food, field of medicaments production NAD.

The present invention also provides application of the methods described in product of the production containing NAD.

Beneficial effect：The present invention is added in the fermentation medium different by adopting the polygenic combination of coexpression Precursor substance, polygenes is combined with the precursor substance of multiple types, expand network-based Metabolic Flux, the coenzyme of Escherichia coli intracellular Content can reach 20-30 μm of ol/g DCW after the regulation and control of two means of metabolic engineering and Biochemical Engineering.Compare the bacterium that sets out Strain BL21/pET-21a improves nearly 10 times, NAD⁺The efficiency of generation is significantly improved, Escherichia coli intracellular NAD⁺Content also enter one Step increases.

Description of the drawings

Fig. 1 is coenzyme NAD⁺Route of synthesis schematic diagram, wherein NAD⁺Chemical constitution and its related intermediate (R：Ribosomes, P： Phosphoric acid, Ad：Adenine) compound is abbreviated as：L-Trp：L-Trp；Asp：Aspartic acid；QA：Quinolinic acid；NA：Nicotinic acid； NaMN：NAMN；NaAD：Nicotinate adenine dinucleotide；NAD⁺：NADH；NAM：Nicotinoyl Amine；NR：Nicotinamide riboside；NMN：Nicotinamide mononucleotide.

Specific embodiment

Fermentation medium：NaCl 10g/L peptone 10g/L, yeast extract 5g/L, pH7.2, with deionized water preparation, Using the front addition μ g/mL of ampicillin 100.

Fermentation condition：Initial temperature is 37 DEG C, and shaking speed is 200r/min, thalline OD₆₀₀When reaching 0.6-2.0, add The IPTG of final concentration of 0.1-10mM, 16-37 DEG C of induction 8-48h, the concentration for adding precursor substance is 1-100mg/L.

Embodiment 1

(1) expression plasmid of overexpression gene pncA, pncB, nadC, nadD, nadE is built：

According to sequence (the Gene ID of gene pncA in the Escherichia coli reported on NCBI:946276), the sequence of gene pncB Row (Gene ID:8182321), sequence (the Gene ID of gene nadC:948869), sequence (the Gene ID of gene nadD: 8180157), sequence (the Gene ID of gene nadE:8179982) upstream and downstream primer is designed, and is synthesized with BamH I and Xho The primer of I restriction enzyme sites：

pncA-F:GGATCCATGCCCCCTCGCGCCCTGTT

pncA-R：CCGCTCGAGTTACCCCTGTGTCTCTTCCCAGTCTG

pncB-F:CGCGGATCCATGACACAATTCGCTTCT

pncB-R:CCGCTCGAGTTAACTGGCTTTTTTAATATGCGG

nadC-F:GGATCCATGCCGCCTCGCCGCTATAACC

nadC-R:CTCGAGTTAGCGAAAACGCATTGAAAGGTCGAGTG

nadD-F:CGCGGATCCATGAAATCTTTACAGGCTC

nadD-R:CCGCTCGAGTCAGCGATACAAGCCTTGTT

nadE-F:CGCGGATCCATGACATTGCAACAACAAAT

nadE-R:CCGCTCGAGTTACTTTTTCCAGAAATCATCG

Extract E.coli BL21 (DE3) genome, with it as template, PCR clonal expansions obtain genes of interest pncA, PncB, nadC, nadD and nadE fragment, reaction condition is：98 DEG C of denaturations 30s；98 DEG C of denaturation 10s；55 DEG C of annealing 30s；72 DEG C each extend over 42s, 80s, 58s, 42s, 55s, 30 circulations；72 DEG C of extension 10min.

After by genes of interest product purification, pMD-19T carriers are connected to, transformation and selection positive colony is simultaneously sequenced.To carry again Genes of interest in body pET-21a and T load restriction endonuclease BamH I and Xho I digestion 90min, Jing agaroses coagulate Gel electrophoresis recovery purifying genes of interest fragment and plasmid backbone, 16 DEG C connect overnight under T4 connection enzyme effects.Then will connection Product converts JM109 cloning host competent cells, the screening positive clone transformant on amicillin resistance flat board.

(2) by plasmid pET-21a-pncA, pET-21a-pncB, pET-21a-nadC, pET-21a-nadD, pET-21a- NadE imports E.coli BL21 (DE3) competence, obtains recombinant bacterial strain.

(3) fermented and cultured is carried out to recombinant bacterial strain, it by 1%, the IPTG induced concentrations of volume is 0.1-10mM that inoculum concentration is, Determine the NAD of its intracellular⁺Content (table 1).Wherein effect is obvious has：E.coli BL21/pET-21a-pncB and E.coli BL21/pET-21a-nadE.The coenzyme content of its intracellular respectively reaches 12.475 μm of ol/g DCW and 13.398 μ Mol/g DCW, improve compared with the control 3 times and 3.3 times.

The overexpression individual gene recombinant bacterial strain growing state of table 1 and NAD⁺Content

Embodiment 2

(1) the polygenic E. coli recombinant stain of coexpression is built：

According to having been built up successful plasmid pET-21a-pncA, pET-21a-pncB, pET-21a-nadC, pET-21a- NadD, pET-21a-nadE design upstream and downstream primer, and SD-AS sequences are added between two multiple genes (AGAAGGAGATATACA), using overlap extension pcr, expressing in series of multiple genes on same plasmid is realized.

Synthesis with BamH I and Xho I restriction enzyme sites primer, with plasmid pET-21a-pncA, pET-21a-pncB, PET-21a-nadC, pET-21a-nadD, pET-21a-nadE are template, and Overlap extension PCR clonal expansion obtains genes of interest pncA-pncB、pncA-nadD、pncA-nadE、pncB-nadD、pncB-nadE、nadD-nadE、nadC-nadD、nadC- NadE and pncA-pncB-nadD, pncA-nadD-nadE, pncB-nadD-nadE, nadC-nadD-nadE, pncA- PncB-nadE fragments, reaction condition is：98 DEG C of denaturations 30s；98 DEG C of denaturation 10s；60 DEG C of annealing 15s；72 DEG C each extend over 112s, 80s, 90s, 112s, 125s, 90s, 95s, 105s, and 155s, 130s, 165s, 145s, 165s, 15 circulations；72 DEG C extend 10min, add each 1 μ L of upstream and downstream primer, continue above-mentioned condition, 15 circulations, 72 DEG C of extension 10min, 12 DEG C 30min。

After by genes of interest product purification, pMD-19T carriers are connected to, transformation and selection positive colony is simultaneously sequenced.To carry again Genes of interest in body pET-21a and T load restriction endonuclease BamH I and Xho I digestion 90min, Jing agaroses coagulate Gel electrophoresis recovery purifying genes of interest fragment and plasmid backbone, 16 DEG C connect overnight under T4 connection enzyme effects.Then will connection Product converts JM109 cloning host competent cells, the screening positive clone transformant on amicillin resistance flat board.

(2) plasmid for arriving obtained as above is imported into E.coli BL21 (DE3) competence, obtains the weight of polygenes coexpression Group bacterial strain

(3) fermented and cultured is carried out to polygenes coexpression recombinant bacterial strain, first activates recombinant bacterium with LB ampicillin plates Strain, picking single bacterium colony accesses 5mL and contains in the LB test tubes of the μ g/mL ampicillins of final concentration 100, cultivates after 12h again by 1% Inoculum concentration is transferred in the LB fermentation mediums of 50mL, 37 DEG C, 200r/min cultivated to OD₆₀₀For 0.6-2.0 when add final concentration For the IPTG of 0.1-10mmol/L, 16-37 DEG C, 200r/min inductions carry out intracellular NAD after protein expression is normal⁺The survey of content Fixed (table 2).

The polygenes of table 2 coexpression recombinant bacterial strain intracellular NAD⁺Content

In polygenes coexpression recombinant bacterial strain, E.coli BL21/pET-21a-nadE-pncB, E.coli BL21/pET- 21a-pncB-nadE-nadD and E.coli BL21/pET-21a-pncB-nadE-pncA intracellulars NAD⁺Content can reach 18 μ mol/g DCW.Compare starting strain E.coli BL21/pET-21a and improve nearly 5 times.

Embodiment 3

(1) function equipment factor accelerator：By tryptophan, aspartic acid, quinolinic acid, nicotinic acid, niacinamide respectively with sterilizing Ultrapure water dissolves afterwards, are formulated as the solution of final concentration 100mg/L.

(2) fermented and cultured is carried out to initial strains E.coli BL21/pET-21a, using 50ml shaking flask aerobic fermentations.Press 1% inoculum concentration is accessed in the LB test tubes of 5mL from cryopreservation tube, while the ampicillin of final concentration of 100 μ g/mL is accessed, training Transfer in the LB fermentation mediums of 50mL by 1% inoculum concentration again after foster 12h, the function of final concentration of 40mg/L is added respectively Factor accelerator, 37 DEG C, 200r/min cultivated to OD₆₀₀For 0.6-2.0 when add the IPTG of final concentration of 0.1-10mmol/L, 16-37 DEG C, 200r/min inductions.

3rd, initial strains E.coli BL21/pET-21a intracellulars NAD after the different precursor substance cultures of addition are determined⁺Contain Amount.As a result as shown in table 3, wherein with the addition of after quinolinic acid, nicotinic acid and niacinamide to the growing amount facilitation of intracellular coenzyme compared with Greatly, blank control group is compared, has been respectively increased nearly 64%, 105% and 67%.

Table 3 adds E.coli BL21/pET-21a intracellulars NAD after different precursor substances⁺Content

Embodiment 4

(1) according to the method function equipment factor accelerator of embodiment 3.

(2) recombinant bacterial strain (recombinant bacterium prepared by embodiment 2) that is co-expressed to polygenes carries out fermented and cultured, first with LB ammonia benzyls Penicillin flat board activates recombinant bacterial strain, and picking single bacterium colony accesses the LB test tubes that 5mL contains the μ g/mL ampicillins of final concentration 100 In, transfer in the LB fermentation mediums of 50mL by 1% inoculum concentration again after culture 12h, add final concentration of 1-100mg/L's Precursor substance, 37 DEG C, 200r/min cultivated to OD₆₀₀For 0.6-2.0 when add the IPTG, 16- of final concentration of 0.1-10mmol/L 37 DEG C, 200r/min inductions.

(3) collecting the thalline after fermented and cultured carries out SDS-PAGE analyses, and the crucial rate-limiting enzyme enzyme for combining coexpression is normal Intracellular NAD is carried out after expression⁺The measure of content, as a result as shown in table 4, with the addition of the coexpression recombinant bacterial strain intracellular of precursor substance Coenzyme content increase.Wherein effect the most it is apparent that E.coli BL21/pET-21a-nadE-pncB and E.coliBL21/pET-21a-pncB-nadE-pncA addition nicotinic acid after, NAD⁺Content can reach nearly 32 μm of ol/g DCW.

The recombinant bacterial strain of the polygenes of table 4 coexpression combines different precursor substances to intracellular NAD⁺The impact of content

The present invention prepares the function factor accelerator of 0～100mg/L, and uses it for the fermentation training of recombination bacillus coli Support, as a result show, can effectively facilitate intracellular NAD⁺Content, double gene coexpression combination in, quinolinic acid is to E.coli The facilitation of BL21/pET-21a-nadC-nadD and E.coli BL21/pET-21a-nadC-nadE is maximum so as to intracellular Oxidized coenzyme content respectively reaches 15.7 μm of ol/g DCW and 19.9 μm of ol/g DCW.Nicotinic acid is to E.coli BL21/pET- 21a-pncB-nadE and E.coli BL21/pET-21a-nadE-pncB intracellular coenzyme contents increase rate is substantially, reachable respectively To 30.6 μm of ol/g DCW and 32.5 μm of ol/g DCW.Niacinamide promotes E.coli BL21/pET-21a-pncA-nadE to make With larger, intracellular coenzyme content is up to 24.8 μm of ol/g DCW.

In three gene co-expressing combinations, raising of the niacinamide to E.coli BL21/pET-21a-nadC-nadD-nadE Amplitude is larger, and other bacterial strains are that nicotinic acid serves the effect for more obviously improving coenzyme content.

The effective obvious recombinant bacterial strain of institute improves more than 56% compared with precursor substance is not added with,.

Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention Enclosing should be by being defined that claims are defined.

Claims (9)

1. the recombination bacillus coli that a kind of intracellular NAD content is improved, it is characterised in that coexpression pncA, pncB, The recombination bacillus coli of at least two genes in nadC, nadD, nadE.

2. recombination bacillus coli according to claim 1, it is characterised in that be co-expressed by host of E.coli BL21 At least two gene in pncA, pncB, nadC, nadD, nadE.

3. the construction method of recombination bacillus coli described in claim 3, it is characterised in that with pET-21a as carrier, in large intestine bar At least 2 genes in pncA, pncB, nadC, nadD, nadE are expressed in bacterium E.coli BL21.

4. it is a kind of improve recombination bacillus coli intracellular NAD content method, it is characterised in that methods described is to weigh Profit require 1 or 2 described in recombination bacillus coli be seeded in fermentation medium, 30～37 DEG C culture 8～72h.

5. method according to claim 4, it is characterised in that the inoculation is inoculated with by the inoculum concentration of volume 1%.

6. the method according to claim 4 or 5, it is characterised in that also contain 1～100mg/L in the fermentation medium Tryptophan, aspartic acid, quinolinic acid, nicotinic acid, niacinamide at least one.

7. method according to claim 6, it is characterised in that methods described is thalline OD after inoculation₆₀₀Reach 0.6- When 2.0, the IPTG of final concentration of 0.1-10mM, 16-37 DEG C of induction 8-48h are added.

8. application of the recombination bacillus coli described in claim 1 or 2 in terms of food, field of medicaments production NAD.

9. application of the arbitrary methods described of claim 4-5,7 in product of the production containing NAD.