CN110373370A

CN110373370A - A kind of catalyst system of coupling ATP regenerative system and its application during producing glutathione

Info

Publication number: CN110373370A
Application number: CN201910642421.7A
Authority: CN
Inventors: 范超; 洪皓; 齐佳坤; 刘军; 吴文忠
Original assignee: Dalian Promise Biological Ltd By Share Ltd
Current assignee: Dalian Promise Biological Ltd By Share Ltd
Priority date: 2019-07-16
Filing date: 2019-07-16
Publication date: 2019-10-25
Anticipated expiration: 2039-07-16
Also published as: CN110373370B

Abstract

The invention discloses a kind of catalyst system of coupling ATP regenerative system and its applications during producing glutathione.Construct the Escherichia coli of heterogenous expression/genome conformity bifunctional enzyme containing glutathione synthesis and ATP regeneration enzyme respectively first, the optimization of glutathione synthesis reaction condition and catalyst system is carried out again, finally, pass through the optimization of two kinds of Escherichia coli mixed culture and strain ratio and catalysis reaction process, it realizes the production of glutathione, utmostly saves the cost of raw material, equipment and synthetic reaction process.

Description

The catalyst system of a kind of coupling ATP regenerative system and its in production glutathione process In application

Technical field

The invention belongs to biocatalysis technology fields, and in particular to a kind of catalyst system of coupling ATP regenerative system and its Application during producing glutathione.

Background technique

Glutathione (γ-L-glutamyl-cysteinyl-glycine, GSH) is by Pidolidone, L-cysteine A kind of micromolecule polypeptide obtained with three kinds of amino acid condensations of glycine, is widely present in various organisms, be microorganism and The intracellular main small-molecular peptides containing sulfydryl of plant biological.Glutathione is a kind of internal important antioxidant.Reduced form Glutathione have the function of in living tissue many important, in hepatic injury, eye disease, metabolism, adjust cell and wither Die etc. plays an important role.In addition, its effect that no less important is also played in field of food preservation, therefore it is domestic Outer medical market, food, cosmetic field are also promoting the demand of glutathione steadily.

There are many preparation method of glutathione at present, and common includes solvent extraction, chemical synthesis, biological fermentation process And enzyme process.Extraction extracts glutathione from the plant containing a large amount of glutathione such as grain germ, that there are yield is low, at The disadvantages of this height, organic solvent pollution are serious, purity is not high, less use.Chemical synthesis synthesizing glutathion activity Product is not readily separated, and needs chemical resolution, and product purity is not high, it is difficult to promote.Therefore, glutathione is produced both at home and abroad at present It is mostly using fermentation method or enzyme process, fermentation method is to arrive the gene cloning for encoding synthesizing glutathion enzyme system or bifunctional enzyme In bacterium or yeast, fermented and cultured obtains glutathione.Yeast fermentation method technique is more mature in fermentation method, but the production cycle is long, Yield is relatively low, and excessive by-product keeps downstream process processing complicated, and environmental pollution is larger.Patent CN201810844388 and The CN201680013630 heterogenous expression glutathione synthesis bifunctional enzyme gene in yeast and Escherichia coli respectively, reaches fermentation The purpose of glutathione is produced, but yield is very low, is not suitable for mass production.

The advantages that Production by Enzymes glutathione is clear with technique, and the substrate transformation rate is high, and product purity is high, has caused people Extensive concern.Newfound difunctional glutathione synthetase (GshF) gradually replaces original half Guang ammonia of gamma-glutamyl Two kinds of enzymes of acid enzyme (Gsh I, EC 6.3.2.2) and glutathione synthetase (Gsh II, EC 6.3.2.2) are used to enzyme process Catalytic production GSH, feedback inhibition is smaller, is suitably applied large-scale production.But in the prior art, it still remains each Kind problem interferes the industrial applications of the technology, and patent CN201710543648 obtains the different GshF-4 of amino acid sequence Mutant, synthesis of dynamic all increase than living and optimum temperature, relatively low but there are yield；ATP consumption is big, at This high problem is unfavorable for industrialized production.

Enzymatic clarification glutathione needs atriphos (ATP) as energy donor, but the price of ATP can be mentioned substantially High production cost, therefore establishing ATP regenerating system is the key link in glutathione synthesis.Patent CN201310538982 It is overexpressed (external source) glutathione bifunctional enzyme (STH) and acetokinase (ack) in Bacillus coli cells, is weighed Group expression cell, although having reached the regenerated purpose of ATP, Rosetta (DE3) stability is not high, required substrate Acetyl phosphate dilithium salt is at high cost, and Product yields are low, is crushed enzyme solution and improves separation costs, loses in addition, existing there are also plasmid The problem of use not friendly to the environment of possible problem and antibiotic, it is difficult to carry out commercial introduction；Patent CN201710452240 generates gluathione using GshF enzyme, ATP regeneration enzyme (PPK enzyme and PAP enzyme) and AK enzyme in the reactor Peptide substitutes ATP or AMP using adenosine, but ATP therein regeneration enzyme system includes that PPK enzyme, PAP enzyme and a variety of enzymes of AK enzyme are deposited The problem of culture causes cost to increase respectively and immobilization separating step is cumbersome, and energy consumption is high, in addition, there are also plasmids to exist Lose possible problem；The problem of use not friendly to the environment of antibiotic etc., eventually leading to it can not be well in industry Upper popularization.

Summary of the invention

To solve the above-mentioned problems, the present invention has selected to be catalyzed nucleotide phosphodiesterase using AMP/ADP in PPK2 family The source red pseudomonas (Rhodobacter sphaeroides) enzyme of the ppk2 gene as ATP circular regeneration, obtain A kind of catalyst system of coupling ATP regenerative system.ADP and AMP can be catalyzed in the presence of the calgon of low cost by realizing Phosphorylation, the energy-rich phosphate bond of catalysate be fractured into glutathione synthetic reaction energy supply.

An aspect of of the present present invention discloses a kind of catalyst system of coupling ATP regenerative system, respectively by GshF gene and ppk2 On gene integration to genome of E.coli, the Escherichia coli containing GshF gene and the large intestine bar containing ppk2 gene are obtained Bacterium, the two obtain mixed bacterium cell with the ratio mixed culture of 3:2-3:1, and the phosphate group donor of the catalyst system is six inclined Sodium phosphate.

For technique described above scheme, it is preferred that the Escherichia coli containing GshF gene and contain ppk2 base The Escherichia coli of cause are mixed with the ratio of 2:1-5:2.

For technique described above scheme, it is preferred that using genome editing technique by the nucleosides after codon optimization Acid sequence gene GshF as shown in SEQ ID NO.2 is integrated on genome of E.coli.

For technique described above scheme, it is preferred that the preparation step packet of the Escherichia coli containing ppk2 gene It includes: using genome editing technique by the nucleotide sequence after codon optimization as gene ppk2 shown in SEQ ID NO.4 is whole It closes on genome of E.coli.

For technique described above scheme, it is preferred that described to utilize genome editing technique for according to E.coli The method of CRISPRCas9 gene editing kit CR1001 operates.In the embodiment of the present invention, the large intestine bar containing GshF gene The preparation method of bacterium is: by the gene GshF as shown in SEQ ID NO.2 of the nucleotide sequence after codon optimization according to The method of E.coli CRISPRCas9 gene editing kit CR1001 operates.The Escherichia coli containing ppk2 gene Preparation method is: by the gene ppk2 as shown in SEQ ID NO.4 of the nucleotide sequence after codon optimization according to E.coli The method of CRISPRCas9 gene editing kit CR1001 operates.

For technique described above scheme, it is preferred that the method for the mixed culture is: GshF will be contained after activation The Escherichia coli (BL21-GshF) of gene and Escherichia coli (BL21-ppk2) containing ppk2 gene according to 3:2-3:1 ratio It after example mixing, is inoculated with the inoculum concentration of 8%-12% volume ratio, at 37 ± 0.5 DEG C, pH is 6.7 ± 0.1, ventilatory capacity 20- 100L·h^-1, speed of agitator 100-600rpm, make dissolved oxygen control in 10%-40%, it is mixed that thallus is collected in fermentation to late log phase Bacterium cell.

Another aspect of the present invention discloses a kind of catalyst system the answering in production glutathione of coupling ATP regenerative system With at 37-45 DEG C, pH is 6-7.5 condition for the Escherichia coli containing GshF gene and the Escherichia coli containing ppk2 gene Directly catalyze and synthesize reduced glutathione down.

For application described above, it is preferred that the Escherichia coli containing GshF gene and contain ppk2 gene For Escherichia coli at 40-42 DEG C, pH directly catalyzes and synthesizes reduced glutathione under the conditions of being 6.5-7.0.

For application described above, it is preferred that the Escherichia coli containing GshF gene and contain ppk2 gene For the mixed bacterium cell that Escherichia coli mixed culture obtains at 42 DEG C, pH directly catalyzes and synthesizes reduced form gluathione under the conditions of being 7.0 Peptide.

For application described above, it is preferred that described to catalyze and synthesize substrate used in reduced glutathione as L- Glycine 150-170mM, L-sodium 150-170mM, MgSO₄·7H₂O 65-75mM, L-cysteine hydrochloride 135- 145 mM, ATP 1-3mM, calgon 55-65mM.

For application described above, it is preferred that described to catalyze and synthesize substrate used in reduced glutathione as L- Glycine 155-165mM, L-sodium 155-165mM, MgSO₄·7H₂O 65-75mM, L-cysteine hydrochloride 135- 145 mM, ATP 1-3mM, calgon 55-65mM.It is further preferred that substrate used in the application is the sweet ammonia of L- Sour 160mM, L-sodium 160mM, MgSO₄·7H₂O 70mM, 2 mM of L-cysteine hydrochloride 140mM, ATP, six inclined phosphorus Sour sodium 60mM.

Beneficial effect

1. target gene is integrated into genome, plasmid is easy to be lost during solving the problems, such as strain culturing, and saves The use of antibiotic and the subsequent processing containing antibiotic waste water, reduce industrial cost during culture thallus；

2. having saved time cost and equipment cost simultaneously with tank Hybrid NC machine tool, avoid because reaction efficiency is high, remaining The placement issue of cell；

3. having, raw material cheap and easy to get, easily-controllable, route of synthesis green high-efficient easy to operate, biological safety be good, reaction The advantages such as rate is fast, conversion ratio is high obtain glutathione and are up to 40.63g/L, conversion ratio reaches in preferred embodiment To 95.43%.

Detailed description of the invention

Fig. 1 is BL21-pET-30a-GshF and BL21-pET-30a-ppk2 growth curve chart；

Fig. 2 is BL21-GshF and BL21-ppk2 growth curve chart；

It is to urge that Fig. 3, which is with BL21-pET-30a-GshF and BL21-pET-30a-ppk2, BL21-GshF and BL21-ppk2, Change the conversion ratio figure of cell production glutathione；

Fig. 4 is influence diagram of the BL21-ppk2 wet thallus to reaction result of Different adding amount；Wherein (a) is that addition is different The conversion ratio of glutathione when the BL21-ppk2 wet thallus of amount；(b) to add different amounts of BL21-ppk2 wet thallus system when The concentration of middle ATP, ADP, AMP；

Fig. 5 is influence diagram of the BL21-GshF wet thallus to reaction result of Different adding amount；Wherein (a) is that addition is different The conversion ratio of glutathione when the BL21-GshF wet thallus of amount；(b) to add different amounts of BL21-GshF wet thallus system when The concentration of middle ATP, ADP, AMP；

Fig. 6 is the conversion ratio (a) of glutathione and ATP, ADP after BL21-GshF and BL21-ppk2 are inoculated in varing proportions And content (b) figure of AMP；

Fig. 7 is the Yield mapping that BL21-GshF couples the glutathione that different amounts of ATP is reacted.

Specific embodiment

A specific embodiment of the invention is described in detail below, it is to be understood that protection scope of the present invention is simultaneously It is not restricted by specific implementation.Biomaterial used in the embodiment of the present application or reagent, unless otherwise specified, For conventional commercially produced product, it is commercially available.

The experimental method of the catalyst system production glutathione of exploitation design coupling ATP regenerative system.It constructs respectively first The Escherichia coli of exogenous gene expression/genome conformity bifunctional enzyme containing glutathione synthesis and ATP regeneration enzyme, then carry out The optimization of glutathione synthesis reaction condition and catalyst system, finally, passing through two kinds of Escherichia coli mixed culture and strain The production of glutathione is realized in the optimization of ratio and zymotechnique, utmostly saves raw material, equipment and synthetic reaction mistake The cost of journey.

Due to the coupling of glutathione synthesis and ATP regenerative process, need to add two according to a certain percentage in reaction process The cell of kind genetic engineering bacterium, and two kinds of bacterium need secondary cultures in two batches, energy consumption is high, time-consuming long；Growth curve is observed, will be produced The gene of raw two kinds of enzymes is respectively using Escherichia coli as expressive host, according to a certain percentage by two strain gene engineering bacterium of acquisition It is cultivated simultaneously after inoculation, the double enzymes of a kettle shorten the duration of strain culturing and the demand of equipment.Further, by GshF gene Be integrated on genome of E.coli respectively with ppk2 gene, can in the case where not adding antibiotic great expression.Using upper Stating bacterial strain can be glutathione by substrate glutamic acid sodium, cysteine and glycine Efficient Conversion.Solves strain culturing process Middle plasmid problem easy to be lost, and save culture thallus during antibiotic use and containing the subsequent of antibiotic waste water Processing, reduces industrial cost.

Embodiment one, building recombination bacillus coli

1. constructing recombinant expression carrier

It target gene GshF (SEQ ID NO.1) to the source Streptococcus thermophilus and derives from Ppk2 (the Gene ID:3718134, VERSION:CP000143.2 of Rhodobacter sphaeroides；SEQ ID NO.3) Codon optimization is carried out, the GshF gene order (SEQ ID NO.2) after obtaining codon optimization, after codon optimization Ppk2 gene order (SEQ ID NO.4) is drawn with the primers after codon optimization in target gene fragment PCR Object 5 ' hold is introduced into linearized vector end sequence (end sequence of the linearized vector be primer sequence in capital letter It is female) so that the both ends of Insert Fragment 5 ' and 3 ' have the consensus sequence (15-25bp) at carrier both ends, as shown in table 1, primer and Template sequence matches corresponding gel extraction after carrying out High fidelity PCR respectively；The appointing in multiple cloning sites by plasmid pET-30a simultaneously Gel extraction after meaning position digestion, by the plasmid of linearisation respectively with GshF and ppk2 segment after the recovery according to Tiangeng biochemistry " the EasyGeno Quick Casting Cloning Kit " desired ratio and dosage of science and technology establish recombination system, react in 50 DEG C 15min, brief centrifugation after reaction, the cooled on ice that is placed on obtain recombinant products, it is anti-to wait for subsequent transformation It answers.

1 primer sequence of table

Primer	Primer sequence	SEQ ID NO
			GshF-F	CTTTAAGAAGGAGATATACATATGaccctgaaccaactgc	5
GshF-R	GTGGTGGTGGTGGTGGTGCTCGAGttaggtttggcctgccacaatc	6
			ppk2-F	CTTTAAGAAGGAGATATACATATGgccgaagatcgtgctatg	7
ppk2-R	GTGGTGGTGGTGGTGGTGCTCGAGtcaaccttgacgcggtttac	8

It takes E.coli BL21 competent cell to be placed in ice, 5-10 μ L recombinant products is added into competent cell suspension, Mixing is flicked, 30min is stood in ice bath.Centrifuge tube is placed in 42 DEG C of water-baths and places 60-90s, then quickly shifts pipe Into ice bath, make the cooling 2-3min of cell, which not shake centrifuge tube；It is sterile that 350 μ L are added into each centrifuge tube LB culture medium (is free of antibiotic), and mixing is placed on 37 DEG C, 180rpm shaking table shaken cultivation 45min, it is therefore an objective to make phase on plasmid The resistant maker gene of pass is expressed, and thallus is made to recover；Transformation system is mixed, the competent cell that 100 μ L have been converted is drawn and adds To containing Kan^ROn the LB solid agar medium of antibiotic, with spreading rod gently cell is uniformly spreadable.Plate is placed in room Temperature is absorbed up to liquid, is inverted plate, 37 DEG C of culture 12-16h.

By obtained colony inoculation 1-5mL LB (containing Kan^RAntibiotic) culture medium, 37 DEG C of shaking table shaken cultivations are overnight, Plasmid is extracted after saving strain, send sequencing company identification Insert Fragment whether correct.Correct Strain Designation is BL21-pET- 30a-GshF, BL21-pET-30a-ppk2 give over to and test in next step.

2. constructing genomic integrated strains

By after codon optimization gene GshF (SEQ ID NO.2) and ppk2 (SEQ ID NO.4) according to the luxuriant industry of English The operating procedure of kit " E.coli CRISPRCas9 gene editing kit CR1001 " be integrated into e. coli bl21 (DE3) on genome: first the pFN-Cas9 carrier in kit being transformed into BL21 cell, and includes by successful conversion Competent cell BL21-Cas9 is made in the BL21 of pFN-Cas9 carrier.

The sgRNA target sequence of GshF is found in the lacZ gene of BL21, and according to kit " E.coli CRISPRCas9 gene editing kit CR1001 " explanation adds the homologous sequence of upper sgRNA (in sequence with primer at both ends Capitalization), obtain sequence through PCR amplification, SEQ ID NO.9:

TCCTAGGTATAATACTAGTcgtcgtgactgggaaaaccctggGTTTTAGAGCTAGA AATAGC is connected into examination In the pFG-sgRNA carrier that agent box is included, plasmid pFG-sgRNA-GshF is obtained；Ppk2 is found in the ppk gene of BL21 SgRNA target sequence, and the homologous sequence (capitalization in sequence) of upper sgRNA is added at both ends with primer, through PCR Amplification obtains sequence, SEQ ID NO.10:

TCCTAGGTATAATACTAGTgttcaatgaacgcgtgcttcaggGTTTTAGAGCTAGA AATAGC, is connected into In pFG-sgRNA carrier, plasmid pFG-sgRNA-ppk2 is obtained.

PCR amplification homologous recombination template: the upstream and downstream for being inserted into gene GshF or ppk2 is amplified into insertion point respectively 45bp upstream and downstream sequence, after amplification for homologous recombination template HRT-GshF (derive from Streptococcus The recombinant fragment of thermophilus, SEQ ID NO.11) and HRT-ppk2 (derive from Rhodobacter sphaeroides Recombinant fragment, SEQ ID NO.12), by pFG-sgRNA-GshF, homologous recombination template HRT-GshF convert BL21-Cas9 Competent cell；PFG-sgRNA-ppk2, homologous recombination template HRT-ppk2 are converted into BL21-Cas9 competent cell.

The step of after sequencing obtains identifying correct transformant according to kit, removes pFG-sgRNA plasmid and pFN- Cas9 plasmid obtains recombinant bacterium BL21-GshF and BL21-ppk2.

3. growing state compares

By the BL21-pET-30a-GshF of preservation in glycerol tube and BL21-pET-30a-ppk2 respectively according to volume ratio 1% Inoculum concentration accesses the Kan containing 50 μ g/mL^RIn the TB culture medium of antibiotic, according to the inoculation of volume ratio 10% after being activated overnight It in amount access fermentor, is sampled every 2h and surveys biomass, the IPTG that 0.1mM is added after cultivating to logarithmic phase latter stage induces 10h Tank is played afterwards, and thallus is centrifuged, and the cell as reaction is spare.By in glycerol tube BL21-GshF and BL21-ppk2 according to volume Inoculum concentration than 1% is respectively connected in TB culture medium, accesses fermentor according to the inoculum concentration of volume ratio 10% after being activated overnight In, it is sampled every 2h and surveys biomass, fermentation to tank under late log phase, thallus is centrifuged, and the cell as reaction is spare.It grows Curve is as depicted in figs. 1 and 2:

The cell of genome conformity is time-consuming short, at low cost during the cultivation process it can be seen from Fig. 1 and Fig. 2, is not required to resist Raw element and inducer, environmental pollution are small；In contrast, it is depressed in the selection of antibiotic, the necessity of plasmid expression leads to bacterium Body replicative cycle extends, thus in the phase same time, BL21-pET-30a-GshF and BL21-pET-30a-ppk2 are than plasmid-free Engineering bacteria BL21-GshF and BL21-ppk2 cell metabolism burden is big, and biomass is low, and growth cycle is longer.

4. the comparison of reaction effect

Reaction system is as follows: L- glycine 160mM, L-sodium 160mM, L-cysteine 140mM, MgSO₄· 7H₂O 70mM, ATP 2mM, 60mM calgon, 100mL deionized water.By 50mg BL21-pET-30a-GshF and 50mg BL21-pET-30a-ppk2,50mg BL21-GshF and 50mg BL21-ppk2 react 3h under the conditions of 42 DEG C, pH are 7 respectively After sample, after 8000rpm is centrifuged 5min, supernatant is detected to the yield of glutathione with HPLC, reaction result is as shown in Figure 3. Target gene is integrated into plasmid vector inducing expression (BL21-pET-30a-GshF and BL21-pET-30a-ppk2) and integration Effect to enzyme coupling and catalyzing caused by genome (BL21-GshF and BL21-ppk2) is slightly lower.Now in view of environmental protection, enzyme Purify into, although and plasmid vector copy number it is high, express the enzyme generated in intracellular or cell surface, with substrate knot The probability of conjunction depends on the number and distribution consistency degree of cell, without being decided by total enzyme amount.Therefore, genome conformity is selected Recombinant bacterium (BL21-GshF and BL21-ppk2) carry out ATP reproducible glutathione synthesis reaction.

The optimization of embodiment two, glutathione synthesis reaction bacterium additive amount

In order to reduce cost, the additive amount of BL21-GshF and BL21-ppk2 are optimized.Reaction system is as follows: L- is sweet Propylhomoserin 160mM, L-sodium 160mM, L-cysteine 140mM, MgSO₄·7H₂O 70mM, ATP 2mM, 60mM six are inclined Sodium phosphate, 100mL deionized water.Wet thallus is added in reaction system, at 42 DEG C, pH is sampled after reacting 3h under the conditions of being 7, After 8000rpm is centrifuged 5min, supernatant is detected to the yield of glutathione and the content of ATP, ADP and AMP with HPLC.

When the additive amount of BL21-GshF wet thallus is 50mg, compare the BL21-ppk2 wet thallus of Different adding amount to anti- Answer the influence of result.As shown in figure 4, the BL21-ppk2 wet thallus of 20-50mg does not constitute a limitation the synthesis of glutathione Factor, the system GSH-PX activity conversion ratio of the BL21-ppk2 wet thallus of 10mg only have 84.67%, and adenosine A TP and ADP disappear Consumption becomes AMP, and energy smooth can not recycle and provide energy for glutathione synthesis；And the body of the BL21-ppk2 wet thallus of 20mg The conversion ratio for being GSH-PX activity is 95.17%, almost the same with the BL21-ppk2 wet thallus effect of 30-40mg.Therefore it selects The BL21-ppk2 wet thallus for selecting 20mg carries out the circulation of ATP regenerating system.

When the additive amount of BL21-ppk2 wet thallus is 20mg, compare the BL21-GshF wet thallus of Different adding amount to anti- Answer the influence of result.As shown in figure 5, the BL21-GshF wet thallus of 30mg can reach 95.11% conversion ratio, with 50mg item Glutathione conversion ratio under part is similar with energy proportion, illustrates that the BL21-GshF wet thallus of 30mg or more is excessive；And Wet thallus less than 30mg can then become the limiting factor of glutathione production.

To sum up, the wet thallus 20mg of BL21-GshF wet thallus 30mg and BL21-ppk2 can carry out ATP circulation to economy Glutathione synthesis reaction is coupled, i.e. BL21-GshF and BL21-ppk2 take 50mg after mixing with the ratio of 3:2.

Example IV, the optimization for being mixed inoculum concentration

BL21-GshF and BL21-ppk2 to be obtained must control the inoculation ratio of the two with the mixed mixed bacterium of the ratio of 3:2 Example.After being mixed after BL21-GshF and BL21-ppk2 are activated overnight respectively according to different ratios (3:2,2:1,5:2,3:1) Mixed bacterium is accessed in fermentor (TB culture medium) with the inoculum concentration of volume ratio 10%, maintains temperature at 37 DEG C ± 0.5 DEG C, pH is 6.7 ± 0.1, by adjusting ventilatory capacity (20-100Lh^-1) and speed of agitator (100-600rpm) by dissolved oxygen control System is centrifuged in 10%-40%, culture to tank under late log phase, thallus, and the cell as reaction is spare.

By BL21-GshF and BL21-ppk2 with the wet thallus 50mg of different vaccination ratio mixed fermentation at 42 DEG C, pH 7 Under the conditions of with L- glycine 160mM, L-sodium 160mM, MgSO₄·7H₂O 70mM, L-cysteine hydrochloride 140mM, It is reacted in ATP 2mM, calgon 60mM, 100mL deionized water, establishes ATP regenerative coupling glutathione synthesis system, It is sampled after 3h, after 8000rpm is centrifuged 5min, supernatant is detected to the yield and ATP, ADP and AMP of glutathione with HPLC Content.As shown in fig. 6, inoculative proportion can reach within the scope of 2:1-5:2 95% or more conversion ratio and ATP effective use Effect.Inoculative proportion range can efficiently accomplish technique between 3:1 and 3:2.

The optimization of ATP consumption in embodiment five, non ATP coupled system

By the wet thallus 50mg of BL21-GshF fermentation at 42 DEG C, pH be 7 under the conditions of with L- glycine 160mM, L- paddy ammonia Sour sodium 160mM, MgSO₄·7H₂O 70mM, L-cysteine hydrochloride 140mM, 100mL deionized water, with different amounts of ATP (100mM, 120mM, 130mM, 140mM, 150mM) is reacted, and is sampled after 3h, after 8000 rpm are centrifuged 5min, by supernatant With the yield of HPLC detection glutathione.

As shown in fig. 7, the ATP of 140mM can reach maximum yield, and lower ATP shows energy insufficient supply. Therefore, if not coupling with ATP regenerating system, 70 times of ATP is needed to can be only achieved same effect, industrialization cost is huge Greatly.

Ack the and ppk2 comparative analysis of embodiment six, ATP regenerating system

By BL21-GshF and BL21-ppk2 with the wet thallus 50mg of 2:1 mixed fermentation at 42 DEG C, pH be 7 under the conditions of with L- glycine 160mM, L-sodium 160mM；MgSO₄·7H₂O 70mM, L-cysteine hydrochloride 140mM and ATP 2mM, 60mM calgon, 100mL deionized water are established ATP regenerative coupling glutathione synthesis system, are sampled after 3h, After 8000rpm is centrifuged 5min, supernatant is detected to the yield of glutathione with HPLC.Glutathione 40.63g/L can be obtained, turn Rate reaches 95.43%.

By BL21-GshF and BL21-ack (the genome conformity mistake of BL21-ppk2 in construction method reference implementation example three Journey) with the wet thallus 50mg of 2:1 mixed fermentation at 42 DEG C, pH be 7 under the conditions of with L- glycine 160mM, L-sodium 160 mM、MgSO₄·7H₂O 70mM, L-cysteine hydrochloride 140mM, acetyl phosphate dilithium salt 210mM and ATP 10 mM, 100mL Deionized water is established ATP regenerative coupling glutathione synthesis system, is sampled after 3h, after 8000rpm is centrifuged 5min, by supernatant With the yield of HPLC detection glutathione.Glutathione 35.75g/L, conversion ratio 85.35% can be obtained.

It can be seen that although ack enzyme can be used for the regeneration of ATP, but it is at high cost, conversion capability is weak, glutathione yield It is low.

The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto, Within the technical scope of the present disclosure, any changes or substitutions that can be easily thought of by those skilled in the art, should all cover at this Within the protection scope of invention.Therefore, the scope of protection of the invention shall be subject to the scope of protection specified in the patent claim.

Sequence table

<110>Dalian Yi Nuo Biological Co., Ltd.

<120>a kind of catalyst system of coupling ATP regenerative system and its application during producing glutathione

<160> 12

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caaattgttg tcaaacccaa gtcaactaac tttggtctgg gaatttccat tttccaagaa 1620

cctgccagtc ttgacaacta tcaaaaagcc cttgaaattg ctttcgcaga agatacctct 1680

gtccttgttg aagaatttat tccaggaacc gaataccgtt tcttcatctt ggatgggcgt 1740

tgtgaggctg ttcttctgcg tgtcgctgcc aatgttattg gtgatggcaa acacaccatt 1800

cgtgaactag tcgctcagaa aaatgctaat ccattgcgtg gccgtgatca ccggtcacct 1860

ctggaaatca ttgagctagg agacatcgaa caactaatgt tagctcaaca gggttacaca 1920

cctgatgata ttctcccaga aggaaaaaag gtcaatctgc gtcgtaattc caacatctct 1980

acaggtggtg actctattga tatcactgag accatggatt cctcttacca agaattagcc 2040

gcagccatgg caactagcat gggcgcctgg gcttgcgggg ttgatctgat aattccagat 2100

gaaactcaaa ttgccaccaa ggaaaatcct cattgcacct gcattgagct caactttaac 2160

ccttcgatgt atatgcacac ctactgtgct gagggtcctg gccaagctat cactactaaa 2220

atcctagata aactttttcc agaaatagtg gctggtcaaa cttaa 2265

<210> 2

<211> 2271

<212> DNA

<213>the GshF gene order after codon optimization

<400> 2

atggagctca ccctgaacca actgctgcag aaactggagg caacaagccc gatcctgcag 60

gccaacttcg gcatcgagcg cgagagcctg cgtgtggacc gtcagggcca gctggtgcat 120

acacctcatc cgagttgcct gggcgcacgt agctttcacc cgtatatcca aaccgacttc 180

tgcgagttcc aaatggaact gattacaccg gttgccaaga gtaccaccga ggcacgccgt 240

ttcttaggcg ccattaccga cgttgccggc cgtagcattg ccaccgatga ggtgttatgg 300

ccgctgagca tgccgccgcg tttaaaagcc gaggaaattc aggtggccca actggaaaac 360

gacttcgagc gccactaccg caattacctg gccgagaaat atggcaccaa actgcaggcc 420

atcagtggca ttcactacaa catggaactg ggcaaagatt tagtggaggc actgtttcag 480

gaaagcggcc agaccgacat gatcgccttt aagaacgccc tgtatctgaa gctggcccag 540

aactatctgc gctaccgctg ggttatcacc tacctgttcg gtgccagtcc gattgcagaa 600

cagggctttt tcgaccagga agttccggag ccggttcgta gtttccgtaa cagcgaccac 660

ggctacgtga acaaggaaga gattcaagtg agttttgtga gtctggagga ttacgttagc 720

gccattgaaa cctacatcga gcagggtgat ctgaacgccg aaaaagagtt ttatagtgca 780

gtgcgctttc gtggccagaa agtgaatcgc agcttcctgg acaaaggcat cacctatctg 840

gaatttcgta attttgatct gaacccgttc gagcgcattg gcattagcca gaccaccatg 900

gacaccgttc atctgctgat cctggcattc ctgtggttag atagcccgga aaacgtggat 960

caggcactgg cccagggtca cgcactgaat gagaagatcg ccctgagcca tcctctggag 1020

ccgctgccga gcgaggcaaa gacccaggac attgtgaccg ccttagacca actggtgcaa 1080

cactttggcc tgggcgacta ccaccaggac ctggtgaagc aagtgaaagc agcctttgca 1140

gatccgaacc aaacactgag tgcccagctg ttaccgtaca tcaaggacaa gagcctggcc 1200

gagttcgcac tgaataaagc cctggcctac catgactatg attggaccgc ccactatgcc 1260

ctgaagggtt atgaggagat ggagctgagc acccaaatgc tgttatttga cgccattcaa 1320

aaaggcattc acttcgaaat cttagatgag caggatcagt ttctgaagct gtggcaccag 1380

gaccatgtgg aatatgtgaa aaacggtaac atgaccagta aggataatta cgtggttccg 1440

ctggccatgg caaacaaaac cgtgaccaag aaaatcctgg ccgacgccgg ctttccggtg 1500

ccgagtggcg atgaattcac cagcctggaa gaaggtctgg catactatcc gctgatcaaa 1560

gacaaacaaa ttgttgttaa accgaaaagc accaactttg gtctgggcat cagcatcttt 1620

caggaaccgg ccagcctgga taattaccaa aaagcattag aaatcgcctt cgcagaagat 1680

acaagcgttc tggtggagga gtttatcccg ggcaccgaat atcgcttttt tatcctggat 1740

ggtcgttgtg aagccgttct gttacgcgtt gcagcaaacg tgattggtga cggcaaacac 1800

acaattcgcg aactggtggc ccagaagaac gcaaatccgc tgcgcggccg cgatcatcgt 1860

agccctctgg aaatcatcga gctgggtgat atcgagcagc tgatgctggc ccagcagggc 1920

tacacccctg atgatatctt accggaaggc aagaaagtga acctgcgccg taacagcaac 1980

atcagcacag gtggcgacag cattgacatc acagaaacca tggacagcag ctatcaggaa 2040

ctggcagcag ccatggccac cagtatgggc gcctgggcat gtggcgttga tctgatcatc 2100

ccggatgaaa cccagatcgc caccaaagaa aatccgcact gcacctgcat cgaactgaac 2160

ttcaacccga gcatgtacat gcatacctac tgcgcagagg gtccgggtca ggcaattacc 2220

acaaaaatct tagataaact gtttccggag attgtggcag gccaaaccta a 2271

<210> 3

<211> 1011

<212> DNA

<213>the ppk2 gene order in the source Rhodobacter sphaeroides

<400> 3

atggccgaag accgcgcgat gcccgtgatg ccgcctgccg cagacgcggc cgaggcggtg 60

cctgccgccc ccacggccct gccggaagag gggcccgcag ggcccgaggc ccctcttcag 120

acgctgcacg gaccgcgcca tttcccggcc gtggatgcga atgcgatacg gcaggccttc 180

gagggcgggc attatcccta tccgcgccgg ttgggccgcg tggtctacga ggccgagaaa 240

gccagattgc aggccgaact gctgaaggtg cagatctggg cgcaggagac cgggcagaag 300

ttcgtcatcc tgatggaagg gcgcgacgcc gccggcaagg gtggcacgat caagcgcttc 360

atggagcatc tcaatccgcg ctatgcccgc gtcgtggccc tgaccaagcc cggcgagcgc 420

gagcgcggcc aatggttctt tcagcgctat atcgaacatc tgccgaccgc gggcgagatc 480

gtctttttcg accgcagctg gtataaccgc gcgggcgtcg agcgggtgat ggggttctgc 540

accccctcgg aatatctcga attcatgcgc caggcgcccg agctcgagcg gatgctggtc 600

cgctcgggga tccggctcta caaatactgg ttctcggtga cgcgcgacga acagcgcgcc 660

cgcttcctcg cccgcgagac cgatcccctg aaacgctgga agctctcgcc catcgacaag 720

gcgagcctcg acaagtggga cgattatacc gaggcgaagg aggcgatgtt cttctacacc 780

gacacggccg atgcgccctg gaccatcgtc aagtccaacg acaagaagcg cgcgcggctg 840

aactgcatgc ggcacttcct gtcgagcctc gactatccgg gcaaggaccc ggaggtggtg 900

ggcgtgcccg atccgctgat cgtgggacgt gcggcgcagg tgatcggcac ggcggccgac 960

atcctcgaca gcgccacgcc gcccgcgctg cgcaagccgc gtcagggatg a 1011

<210> 4

<211> 1011

<212> DNA

<213>the ppk2 gene order after codon optimization

<400> 4

atggccgaag atcgtgctat gccggttatg ccgccggctg ctgacgctgc tgaagccgtc 60

ccggccgctc cgaccgccct gccggaagaa ggtccggcag gtccggaagc accgctgcaa 120

accctgcatg gtccgcgtca ctttccggca gttgatgcga acgccattcg ccaggctttc 180

gaaggcggtc attatccgta cccgcgtcgc ctgggccgtg tggtttatga agcggaaaaa 240

gcccgcctgc aggcagaact gctgaaggtc cagatttggg cgcaagaaac cggtcagaaa 300

tttgtgatcc tgatggaagg ccgtgatgcg gccggtaaag gcggtacgat caagcgcttc 360

atggaacatc tgaacccgcg ttatgcacgc gtcgtggctc tgaccaaacc gggcgaacgt 420

gaacgcggtc aatggttttt ccagcgttac attgaacacc tgccgacggc cggcgaaatc 480

gtgtttttcg atcgcagctg gtataatcgt gcaggcgtgg aacgcgttat gggtttttgc 540

accccgtctg aatacctgga atttatgcgt caagcgccgg aactggaacg tatgctggtt 600

cgctcaggta ttcgtctgta taaatactgg ttttcggtca cccgcgatga acagcgtgca 660

cgcttcctgg cccgtgaaac ggacccgctg aaacgctgga agctgagtcc gattgataaa 720

gcgtccctgg acaagtggga tgactatacc gaagcaaaag aagctatgtt tttctacacc 780

gatacggcag acgctccgtg gacgatcgtg aagtccaacg ataaaaagcg tgcccgcctg 840

aattgtatgc gtcactttct gagctctctg gattatccgg gcaaagaccc ggaagttgtc 900

ggtgtcccgg acccgctgat tgtgggtcgt gcagctcagg ttatcggtac cgctgccgac 960

attctggact ccgccacccc gccggccctg cgtaaaccgc gtcaaggttg a 1011

<210> 5

<211> 40

<212> DNA

<213> GshF-F

<400> 5

ctttaagaag gagatataca tatgaccctg aaccaactgc 40

<210> 6

<211> 46

<212> DNA

<213> GshF-R

<400> 6

gtggtggtgg tggtggtgct cgagttaggt ttggcctgcc acaatc 46

<210> 7

<211> 42

<212> DNA

<213> ppk2-F

<400> 7

ctttaagaag gagatataca tatggccgaa gatcgtgcta tg 42

<210> 8

<211> 44

<212> DNA

<213> ppk2-R

<400> 8

gtggtggtgg tggtggtgct cgagtcaacc ttgacgcggt ttac 44

<210> 9

<211> 62

<212> DNA

<213>gene order

<400> 9

tcctaggtat aatactagtc gtcgtgactg ggaaaaccct gggttttaga gctagaaata 60

gc 62

<210> 10

<211> 62

<212> DNA

<213>gene order

<400> 10

tcctaggtat aatactagtg ttcaatgaac gcgtgcttca gggttttaga gctagaaata 60

gc 62

<210> 11

<211> 2361

<212> DNA

<213>homologous recombination template HRT-GshF

<400> 11

tgtgtgaaat tgtgagcgga taacaatttc acacaggaaa cagctatgga gctcaccctg 60

aaccaactgc tgcagaaact ggaggcaaca agcccgatcc tgcaggccaa cttcggcatc 120

gagcgcgaga gcctgcgtgt ggaccgtcag ggccagctgg tgcatacacc tcatccgagt 180

tgcctgggcg cacgtagctt tcacccgtat atccaaaccg acttctgcga gttccaaatg 240

gaactgatta caccggttgc caagagtacc accgaggcac gccgtttctt aggcgccatt 300

accgacgttg ccggccgtag cattgccacc gatgaggtgt tatggccgct gagcatgccg 360

ccgcgtttaa aagccgagga aattcaggtg gcccaactgg aaaacgactt cgagcgccac 420

taccgcaatt acctggccga gaaatatggc accaaactgc aggccatcag tggcattcac 480

tacaacatgg aactgggcaa agatttagtg gaggcactgt ttcaggaaag cggccagacc 540

gacatgatcg cctttaagaa cgccctgtat ctgaagctgg cccagaacta tctgcgctac 600

cgctgggtta tcacctacct gttcggtgcc agtccgattg cagaacaggg ctttttcgac 660

caggaagttc cggagccggt tcgtagtttc cgtaacagcg accacggcta cgtgaacaag 720

gaagagattc aagtgagttt tgtgagtctg gaggattacg ttagcgccat tgaaacctac 780

atcgagcagg gtgatctgaa cgccgaaaaa gagttttata gtgcagtgcg ctttcgtggc 840

cagaaagtga atcgcagctt cctggacaaa ggcatcacct atctggaatt tcgtaatttt 900

gatctgaacc cgttcgagcg cattggcatt agccagacca ccatggacac cgttcatctg 960

ctgatcctgg cattcctgtg gttagatagc ccggaaaacg tggatcaggc actggcccag 1020

ggtcacgcac tgaatgagaa gatcgccctg agccatcctc tggagccgct gccgagcgag 1080

gcaaagaccc aggacattgt gaccgcctta gaccaactgg tgcaacactt tggcctgggc 1140

gactaccacc aggacctggt gaagcaagtg aaagcagcct ttgcagatcc gaaccaaaca 1200

ctgagtgccc agctgttacc gtacatcaag gacaagagcc tggccgagtt cgcactgaat 1260

aaagccctgg cctaccatga ctatgattgg accgcccact atgccctgaa gggttatgag 1320

gagatggagc tgagcaccca aatgctgtta tttgacgcca ttcaaaaagg cattcacttc 1380

gaaatcttag atgagcagga tcagtttctg aagctgtggc accaggacca tgtggaatat 1440

gtgaaaaacg gtaacatgac cagtaaggat aattacgtgg ttccgctggc catggcaaac 1500

aaaaccgtga ccaagaaaat cctggccgac gccggctttc cggtgccgag tggcgatgaa 1560

ttcaccagcc tggaagaagg tctggcatac tatccgctga tcaaagacaa acaaattgtt 1620

gttaaaccga aaagcaccaa ctttggtctg ggcatcagca tctttcagga accggccagc 1680

ctggataatt accaaaaagc attagaaatc gccttcgcag aagatacaag cgttctggtg 1740

gaggagttta tcccgggcac cgaatatcgc ttttttatcc tggatggtcg ttgtgaagcc 1800

gttctgttac gcgttgcagc aaacgtgatt ggtgacggca aacacacaat tcgcgaactg 1860

gtggcccaga agaacgcaaa tccgctgcgc ggccgcgatc atcgtagccc tctggaaatc 1920

atcgagctgg gtgatatcga gcagctgatg ctggcccagc agggctacac ccctgatgat 1980

atcttaccgg aaggcaagaa agtgaacctg cgccgtaaca gcaacatcag cacaggtggc 2040

gacagcattg acatcacaga aaccatggac agcagctatc aggaactggc agcagccatg 2100

gccaccagta tgggcgcctg ggcatgtggc gttgatctga tcatcccgga tgaaacccag 2160

atcgccacca aagaaaatcc gcactgcacc tgcatcgaac tgaacttcaa cccgagcatg 2220

tacatgcata cctactgcgc agagggtccg ggtcaggcaa ttaccacaaa aatcttagat 2280

aaactgtttc cggagattgt ggcaggccaa acctaataat aaccgggcag gccatgtctg 2340

cccgtatttc gcgtaaggaa a 2361

<210> 12

<211> 1101

<212> DNA

<213>homologous recombination template HRT-ppk2

<400> 12

aatatccagg cagtgtcccg tgaataaaac ggagtaaaag tggtaatggc cgaagatcgt 60

gctatgccgg ttatgccgcc ggctgctgac gctgctgaag ccgtcccggc cgctccgacc 120

gccctgccgg aagaaggtcc ggcaggtccg gaagcaccgc tgcaaaccct gcatggtccg 180

cgtcactttc cggcagttga tgcgaacgcc attcgccagg ctttcgaagg cggtcattat 240

ccgtacccgc gtcgcctggg ccgtgtggtt tatgaagcgg aaaaagcccg cctgcaggca 300

gaactgctga aggtccagat ttgggcgcaa gaaaccggtc agaaatttgt gatcctgatg 360

gaaggccgtg atgcggccgg taaaggcggt acgatcaagc gcttcatgga acatctgaac 420

ccgcgttatg cacgcgtcgt ggctctgacc aaaccgggcg aacgtgaacg cggtcaatgg 480

tttttccagc gttacattga acacctgccg acggccggcg aaatcgtgtt tttcgatcgc 540

agctggtata atcgtgcagg cgtggaacgc gttatgggtt tttgcacccc gtctgaatac 600

ctggaattta tgcgtcaagc gccggaactg gaacgtatgc tggttcgctc aggtattcgt 660

ctgtataaat actggttttc ggtcacccgc gatgaacagc gtgcacgctt cctggcccgt 720

gaaacggacc cgctgaaacg ctggaagctg agtccgattg ataaagcgtc cctggacaag 780

tgggatgact ataccgaagc aaaagaagct atgtttttct acaccgatac ggcagacgct 840

ccgtggacga tcgtgaagtc caacgataaa aagcgtgccc gcctgaattg tatgcgtcac 900

tttctgagct ctctggatta tccgggcaaa gacccggaag ttgtcggtgt cccggacccg 960

ctgattgtgg gtcgtgcagc tcaggttatc ggtaccgctg ccgacattct ggactccgcc 1020

accccgccgg ccctgcgtaa accgcgtcaa ggttgaccct atgccaatac acgataaatc 1080

ccctcgtccg caggagtttg c 1101

Claims

1. a kind of catalyst system of coupling ATP regenerative system, which is characterized in that respectively arrive GshF gene and ppk2 gene integration On genome of E.coli, the Escherichia coli containing GshF gene and the Escherichia coli containing ppk2 gene are obtained, the two is with 3: The ratio mixed culture of 2-3:1 obtains mixed bacterium cell.

2. the catalyst system of coupling ATP regenerative system according to claim 1, which is characterized in that described to contain GshF base The Escherichia coli of cause and Escherichia coli containing ppk2 gene are mixed with the ratio of 2:1-5:2.

3. the catalyst system of coupling ATP regenerative system according to claim 1, which is characterized in that described to contain GshF base The preparation step of the Escherichia coli of cause includes: using genome editing technique by such as SEQ of the nucleotide sequence after codon optimization Gene GshF shown in ID NO.2 is integrated on genome of E.coli.

4. the catalyst system of coupling ATP regenerative system according to claim 1, which is characterized in that described to contain ppk2 base The preparation step of the Escherichia coli of cause includes: using genome editing technique by such as SEQ of the nucleotide sequence after codon optimization Gene ppk2 shown in ID NO.4 is integrated on genome of E.coli.

5. according to the catalyst system of the described in any item coupling ATP regenerative systems of claim 3 or 4, which is characterized in that the benefit It is to be operated according to the method for E.coli CRISPRCas9 gene editing kit CR1001 with genome editing technique.

6. the catalyst system of coupling ATP regenerative system according to claim 1, which is characterized in that the mixed culture Method is: by the Escherichia coli containing GshF gene after activation and the Escherichia coli containing ppk2 gene according to 3:2-3:1's It after ratio mixing, is inoculated with the inoculum concentration of 8%-12% volume ratio, at 37 ± 0.5 DEG C, pH is 6.7 ± 0.1, ventilatory capacity 20- 100L·h^-1, speed of agitator 100-600rpm, make dissolved oxygen control in 10%-40%, fermentation to late log phase is collected thallus and must be mixed Bacterium cell.

7. application of the catalyst system of coupling ATP regenerative system as described in claim 1 in production glutathione, feature It is, the mixed bacterium cell of the Escherichia coli containing GshF gene and the mixed culture acquisition of the Escherichia coli containing ppk2 gene At 37-45 DEG C, pH directly catalyzes and synthesizes reduced glutathione under the conditions of being 6-7.5.

8. application according to claim 7, which is characterized in that the Escherichia coli containing GshF gene and contain ppk2 For the Escherichia coli of gene at 40-42 DEG C, pH directly catalyzes and synthesizes reduced glutathione under the conditions of being 6.5-7.0.

9. application according to claim 7, which is characterized in that described to catalyze and synthesize bottom used in reduced glutathione Object is L- glycine 150-170mM, L-sodium 150-170mM, MgSO₄·7H₂O 65-75mM, L-cysteine hydrochloride 135-145mM, ATP 1-3mM, calgon 55-65mM.

10. application according to claim 9, which is characterized in that described to catalyze and synthesize used in reduced glutathione Substrate is L- glycine 155-165mM, L-sodium 155-165mM, MgSO₄·7H₂O 65-75mM, L-cysteine hydrochloric acid Salt 135-145mM, ATP 1-3mM, calgon 55-65mM.