CN107779423A - Cofactor regeneration mycobacteria and its application in the fermentation of profit Two Liquid Phases - Google Patents
Cofactor regeneration mycobacteria and its application in the fermentation of profit Two Liquid Phases Download PDFInfo
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Abstract
The invention belongs to biocatalysis technology field, and in particular to a kind of Mycobacterium tuberculosis genes engineering bacteria with cofactor regeneration function and its application in Two Liquid Phases fermentation system.The present invention transforms the genetic engineering bacterium constructed for phytosterol bio-conversion by being carried out to intracellular NADH oxidative systems, solve the problems, such as that androstenedione transformation efficiency is low in profit Two Liquid Phases fermentation system, and the extensive use for profit Two Liquid Phases fermentation system in industrial production androstenedione provides possibility.The genetic engineering bacterium MNR M3N2 that the present invention is built can make 1.4 2.9 times of AD (D) output increased during profit Bi-liquid phase system fermentation method degrading plant sterol prepares androstenedione.
Description
Technical field：
The invention belongs to biocatalysis technology field, and in particular to a kind of mycobacteria base with cofactor regeneration function
Application because of engineering bacteria and its in Two Liquid Phases fermentation system.
Background technology：
Androstenedione, 4-AD (AD) and Androsta-1,4-diene-3,17-dione (ADD), it is production
The weight of other steroid hormone class medicines such as cortisone, mineral-corticoids thing, conventional oral contraceptives medicine and protein anabolic hormone
Intermediate is wanted, can be obtained by microbial degradation phytosterol side chain.Phytosterol, it is hydrophobic compound, solubility is very
It is low, be easily gathered in aqueous phase surface, and Steroid Transformation enzyme belongs to endocellular enzyme, substrate only diffuse into cell could be contacted with enzyme and
Conversion reaction is carried out, this results in phytosterol and can not contacted well with invertase, causes conversion ratio relatively low, fermentation time length
The problems such as.The method for improving androstenedione yield at present for problem above focuses primarily upon：1st, in phytosterol metabolic pathway
The transformation of enzyme；2nd, the dissolubility and utilization rate of substrate phytosterol are improved.
On the transformation of enzyme in phytosterol metabolic pathway, focus primarily upon and aoxidized by being overexpressed or knocking out phytosterol
Enzyme in approach improves AD (D) production efficiencys and yield.Such as it is overexpressed cholesterol oxidase and 3 beta-hydroxy steroid dehydrogenase types
Deng, or -9 α of inactivation 3- ketone steroidals-hydroxylase (Ksh) and 3- ketone steroidal -1- dehydrogenases (KstD).Result of study display knocks out
After certain gene, the conversion ratio and yield of phytosterol can be reduced to varying degrees, that is, causes thalline integrally to produce intensity
Decline, limit the application of industrialized production.So further investigation, system in terms of thalline entirety metabolic regulation are needed at present
Research, to seek the method for improving androstenedione production efficiency.
The research of Two Liquid Phases fermentation refers to add in zymotic fluid a kind of with the immiscible organic phase of water, promotion phytosterol
Dissolving.Due to environmental constraints and advocating for Green Chemistry is adapted to, the utilization reduction of organic solvent in the industrial production turns into
Gesture, natural oil are organic solvent good replacements as " green solvent ".But converted under the pressure of the phytosterol of oil-based system
It is inefficient, it have impact on its extensive use in androstenedione production.
General metabolic engineering research focuses primarily upon the genetic modification of enzyme coding gene in specific metabolic pathway, including gene
Overexpression or gene knockout.However, the direct transformation to enzyme coding gene in metabolic pathway be able to might not bring it is expected
Effect.Not only enzyme is controlled the metabolic pathway that co-factor relies in by metabolic pathway, but also by co-factor concentration and co-factor
The control of redox state ratio.Therefore, for metabolic engineering, co-factor transformation is a kind of potential strong transformation bacterium
The method of strain.Co-factor engineering uses molecular biology method, transforms intracellular cofactor regeneration approach, closely regulating and controlling microbial
The form and concentration of intracellular co-factor, directed change and optimization cell metabolism function, realize metabolic fluxes maximization, rapid guiding
The engineering of target metabolic product.It is considered as a Ge Xin branches of metabolic engineering, has caused increasing concern.
The content of the invention：
The technical problems to be solved by the invention are that bacterial strain is metabolized slow and bottom during overcoming phytosterol bio-conversion
The problem of thing solubility is low, cofactor regeneration system is incorporated into Steroid Transformation microorganism, to improve recombinant microorganism in oil
The transformation efficiency of androstenedione in water Two Liquid Phases fermentation system, new method slowly is provided to solve the metabolism of thalline phytosterol,
And the extensive use for profit Two Liquid Phases fermentation system in industrial production androstenedione provides possibility.
The present invention realizes that the technical scheme of above-mentioned purpose is as follows：The genetic engineering bacterium of one plant of production androstenedione, it is with new gold
Mycobacteria is host cell, what heterogenous expression nadh oxidase gene nox-2 was obtained；
The nucleotide sequence of the nox-2 is as shown in sequence table SEQ ID NO.1
Preferably, the host cell is new golden mycobacteria (Mycobacterium sp.) MNR M3, can be from Chinese work
Industry Microbiological Culture Collection administrative center (abbreviation CICC) purchase obtains, and numbering CICC 21097, is host with CICC 21097
Cell, the genetic engineering bacterium that heterogenous expression nadh oxidase gene nox-2 is obtained are named as MNR M3N2.
The construction method of the genetic engineering bacterium MNR M3N2, it is specific as follows：
(1) nox-2 genes (SEQ ID NO.1) and expression plasmid pMV261 connection, are built into pMV261- by digestion
Nox-2 recombinant plasmids；
(2) recombinant plasmid pMV261-nox-2 is imported in MNR M3 competent cell, the positive transformant of acquisition is
For new structure bacterial strain MNR M3N2 of the invention.
The two of technical scheme provided by the invention are the MNR M3N2 bacterial strains using above-mentioned structure in profit biliquid phase system
The method of middle fermenting and producing androstenedione, it is specific as follows：
MNR M3N2 are inoculated with profit Two Liquid Phases fermentation medium by 5-10% (v/v) inoculum concentration, 28-32 DEG C,
Under the conditions of 130-250r/min, pH6.5-7.8, ferment 4-8 days；
The profit Two Liquid Phases fermentation medium composition is as follows：K2HPO40.5g/L, MgSO4·7H2O 0.5g/L, lemon
Sour iron ammonium 0.05g/L, citric acid 2g/L, diammonium hydrogen phosphate 3.5g/L, glucose 10g/L, oil phase 10-20% (v/v), remaining is
Water, pH6.5-7.8；
The oil phase is vegetable oil, in soybean oil, sunflower oil, rapeseed oil, peanut oil, rice bran oil or tall oil extremely
Few one kind；
Further, 0.5-8g/L emulsifying agent is also added in the profit Two Liquid Phases fermentation medium；
Described emulsifying agent is at least one of lecithin, Tween 80, Span 80, sucrose ester and monoglyceride.
Beneficial effect：
(1) present invention transforms the base constructed for phytosterol bio-conversion by being carried out to intracellular NADH oxidative systems
Because of engineering bacteria, solve the problems, such as that androstenedione transformation efficiency is low in profit Two Liquid Phases fermentation system, and sent out for profit Two Liquid Phases
Extensive use of the ferment system in industrial production androstenedione provides may.
(2) the genetic engineering bacterium MNR M3N2 that the present invention is built are in profit Bi-liquid phase system fermentation method degrading plant sterol system
During standby androstenedione, 1.4-2.9 times of AD (D) output increased can be made.
Brief description of the drawings：
Fig. 1 nox-2 gene magnification electrophoretograms；
Fig. 2 positive transformant pMV261-nox-2 plasmid double digestion proof diagrams.
Embodiment：
In order that the object, technical solution and advantage of this patent are more clearly understood, below in conjunction with specific embodiment, to this
Patent is further elaborated.It should be appreciated that specific embodiment described herein is only to explain this patent, not
For limiting the present invention.
The recombinant bacterial strain MNR M3N2 of embodiment 1 structure
1st, pMV261-nox-2 plasmids are built, its process includes：
(1) target gene is synthesized, according to lactococcus lactis subsp (Lactococcus lactis
Subsp.cremoris) NZ9000 nadh oxidases gene nox-2 nucleotide sequence (Gene ID:13019046,SEQ ID
NO.1), restriction enzyme site (BamH I and Sal I) is made an addition to the both ends of nox-2 sequences, designed sequence is transferred into Jin Weizhi
Company carries out gene chemical synthesis；
(2) PCR amplifying target genes fragment nox-2 genes (proof diagram Fig. 1), purpose fragment nox-2 and expression plasmid
PMV261 uses BamHI and SalI double digestions respectively, and purifying, connection obtains recombinant plasmid pMV261-nox-2.
2nd, bacterial strain MNR M3N2 are built：
(1) prepared by mycobacteria MNR M3 competent cells：First order seed culture is to OD600For 1.0 or so, connect by 10%
Kind amount is transferred to progress secondary seed culture in seed culture medium；2% glycine is added after 24h to continue to cultivate 24h.It is collected by centrifugation
Thalline, rinse suspension thalline with 10% pre- cold glycerol in four times and centrifuge, be eventually adding glycerine suspension thalline, and dispense preservation；
Seed culture medium forms:K2HPO40.5g/L, MgSO4·7H2O 0.5g/L, ferric citrate 0.05g/L, lemon
Sour 2g/L, ammonium nitrate 2g/L, glycerine 20g/L, glucose 5g/L, CaCO310g/L, remaining is water, pH7.2；
(2) electricity conversion：10 μ L step 1 gained recombinant plasmids, which are added in 100 μ L competence thalline, places 10min, and shock by electricity bar
Part is as follows：Electricity turns twice under the conditions of 1.5KV, each 4-5ms；
(3) recombinant screen and checking：Electricity is changed the line of production after thing addition seed culture medium recovery culture 3-5h, is coated on containing card
On that mycin (50mg/L) seed culture medium flat board, 30 DEG C of quiescent culture 7d, picking single bacterium drops down onto liquid seed culture medium culture,
To extract plasmid and carry out double digestion checking (proof diagram Fig. 2), there are 4500bp and 1300bp sizes two bands in double digestion theory,
The correct positive transformant of digestion verification is recombinant bacterium MNR M3N2.
NAD (H) contents and NAD in the recombinant bacterium MNR M3N2 of embodiment 2+The comparison of/NADH ratio.
1st, strain activation and culture：
Recombinant bacterium MNR M3N2 and original bacteria MNR M3 are transferred on fresh slant medium, 29 DEG C of culture 2-4d, used
The strain that 20mL 0.5% aseptic aqueous solutions of Tween 80 are washed on lower inclined plane culture medium obtains eluent, draws 1mL eluents and adds
Enter into 30mL seed culture mediums, at 29 DEG C, shaking table culture 36h obtains seed liquor under the conditions of 200r/min；
Slant medium forms:K2HPO40.5g/L, MgSO4·7H2O 0.5g/L, ferric citrate 0.05g/L, lemon
Sour 2g/L, ammonium nitrate 2g/L, glycerine 20g/L, glucose 5g/L, CaCO310g/L, agar 20g/L, remaining is water, pH7.2；
Seed culture medium forms:K2HPO40.5g/L, MgSO4·7H2O 0.5g/L, ferric citrate 0.05g/L, lemon
Sour 2g/L, ammonium nitrate 2g/L, glycerine 20g/L, glucose 5g/L, CaCO310g/L, remaining is water, pH7.2.
2nd, strain enzyme activity and coenzyme measure
By 7% inoculum concentration, seed liquor is seeded in the 250mL baffle plate bottles containing fermentation medium, at 30 DEG C,
Culture is carried out under the conditions of 150r/min and obtains zymotic fluid within 96 hours.
Fermentation medium forms:K2HPO40.5g/L, MgSO4·7H2O 0.5g/L, ferric citrate 0.05g/L, lemon
Sour 2g/L, diammonium hydrogen phosphate 3.5g/L, glucose 10g/L, remaining is water, pH7.2.
(1) nadh oxidase enzyme activity determination：
Collect zymotic fluid somatic cells and carry out ultrasonication, 4 DEG C of 12000r/min centrifuge 30min, take supernatant as thick
Enzyme liquid.
Enzymatic reaction system：1mL reaction systems are by 100 microlitres of crude enzyme liquids, 800 microlitres of 0.1M phosphate buffers (pH
7.0), 100 microlitres of 0.1mM NADH compositions；Start reaction after adding above-mentioned each component, light absorption value is detected under 340nm absorbances
Change (NADH has special absworption peak between wavelength 340nm, and under the oxidation of nadh oxidase, its product NAD+
But there is no absworption peak.Therefore, the light absorption value of the sample at 340nm wavelength is determined, is inhaled by calculating reactant solution at 340nm
The decrement of luminosity, the thick enzyme enzyme activity of nadh oxidase can be obtained.)；
1 unit nadh oxidase work (U) property is defined as the enzyme amount needed for 1 micromole NADH of 1min oxidations.
(2) intracellular NAD (H) measurement：
1. the extraction of co-factor：
NADH extraction：The 0.3mol/L KOH of 400 μ L precoolings are added in 800 μ L zymotic fluids, stay in 10min on ice, 30
Put back on ice after DEG C water-bath 10min, be added dropwise in 0.3mol/L HCl and pH is between 7.5-8.0, place 10min on ice, 4
DEG C, 12000r/min centrifuges 10min, and supernatant is to be measured.
NAD+Extraction：The 0.3mol/L HCl of 400 μ L precoolings are added in 800 μ L zymotic fluids, stay in 10min on ice, 50
Put back on ice after DEG C water-bath 10min, be added dropwise in 0.3mol/L KOH and pH is between 7.2-7.4, place 10min on ice, 4
DEG C, 12000r/min centrifuges 10min, and supernatant is to be measured.
2. measure and analysis method：
Using enzyme parameters, 270 μ L co-factors circulation fluids are added into 96 orifice plates, and (composition is：16.3g/L Bicine
Buffer, 1.52g/L EDTA, 11.2g/L PES, 0.17g/L MTT, 10% ethanol), the 30 μ L steps 1. supernatant
(NADH extracts supernatant or NAD+Extract supernatant), 10 μ L alcohol dehydrogenases (250U/mL) initiation reactions, determined with ELIASA
Reaction solution tries to achieve enzyme's reaction speeding in 570nm absorbance versus time curves；Enzyme reaction speed is in co-factor concentration
Linear relationship, accordingly, contrasted by the reaction rate with standard items, can accurately measure co-factor concentration in sample.
3rd, results contrast
As shown in table 1, recombinant mycobacterium MNR M3N2 nadh oxidase enzyme activity compared with original strain increases.Born of the same parents
Interior NADH contents reduce, NAD+/ NADH ratio improves.Compared with the control, NAD in MNR M3N2+/ NADH ratio improves 192%.
The intracellular nadh oxidase enzyme activity of table 1 and NAD (H) assay
The MNR M3N2 bacterial strains of embodiment 3 are catalyzed phytosterol production androstenedione in aqueous phase system
1st, strain activation and culture:
Recombinant bacterium MNR M3N2 and original bacteria MNR M3 are transferred on fresh slant medium respectively, 29 DEG C are cultivated 3d,
The strain washed with 20mL 0.5% aseptic aqueous solutions of Tween 80 on lower inclined plane culture medium, eluent is well mixed to obtain, drawn
1mL eluents are added in 30mL seed culture mediums, and at 29 DEG C, shaking table culture 36h obtains seed culture fluid under the conditions of 200r/min；
Slant medium forms:K2HPO40.5g/L, MgSO4·7H2O 0.5g/L, ferric citrate 0.05g/L, lemon
Sour 2g/L, ammonium nitrate 2g/L, glycerine 20g/L, glucose 5g/L, CaCO310g/L, agar 20g/L, remaining is water, pH7.2；
Seed culture medium forms:K2HPO40.5g/L, MgSO4·7H2O 0.5g/L, ferric citrate 0.05g/L, lemon
Sour 2g/L, ammonium nitrate 2g/L, glycerine 20g/L, glucose 5g/L, CaCO310g/L, remaining is water, pH7.2.
2nd, phytosterol microorganism conversion：
The seed culture fluid activated in step 1 is transferred in equipped with containing 5g/L phytosterols with 7% inoculum concentration and
In the 250mL baffle plate bottles of the 50mL fermentation mediums of 25mM hydroxypropylβ-cyclodextrins, at 29 DEG C, shaking table is trained under the conditions of 150r/min
Support 120h；
Fermentation medium forms:K2HPO40.5g/L, MgSO4·7H2O 0.5g/L, ferric citrate 0.05g/L, lemon
Sour 2g/L, diammonium hydrogen phosphate 3.5g/L, glucose 10g/L, remaining is water, pH 7.2.
3rd, the detection of androstenedione mole production rate：
With ethyl acetate ultrasonic extraction zymotic fluid, centrifugation, ethyl acetate phase 0.1mL is taken in 1.5mL pipes, after natural air drying
Add 1mL mobile phase, dissolve, HPLC analyses are carried out after centrifugation.Chromatographic condition：C18 posts, mobile phase are methanol：Water (4：1), flow
Speed is 1mL/min, and column temperature is 30 DEG C, Detection wavelength 254nm.
4th, results contrast：
As shown in table 2, after bioconversion 48h, engineered strain MNR M3N2AD (D) growing amount is 0.68g/L, is original bacteria
1.13 times of strain；After 72h, engineered strain MNR M3N2AD (D) growing amount is 1.85g/L, is 0.94 times of original strain；96h
Afterwards, engineered strain MNR M3N2AD (D) growing amount is 2.50g/L, is 1.02 times of original strain；After 120h, engineered strain MNR
M3N2AD (D) growing amount is 2.81g/L, is 1.01 times of original strain.Show, in aqueous phase system, engineering bacteria NADH oxidations
The expression of enzyme has little to no effect to the ability of mycobacteria transformation phytosterin.
AD (D) yield (g/L) in the hydroxypropylβ-cyclodextrin system of table 2
The MNR M3N2 bacterial strains of embodiment 4 are catalyzed phytosterol production androstenedione in water oil biliquid phase system
1st, strain activation and culture is the same as embodiment 3
2nd, phytosterol microorganism conversion：
The seed culture fluid of activation is transferred with 7% inoculum concentration and trained in being fermented equipped with the 50mL containing 5g/L phytosterols
In the 250mL baffle plate bottles for supporting base, at 29 DEG C, shaking table culture 192h under the conditions of 150r/min.
Fermentation medium forms:K2HPO40.5g/L, MgSO4·7H2O 0.5g/L, ferric citrate 0.05g/L, lemon
Sour 2g/L, diammonium hydrogen phosphate 3.5g/L, glucose 10g/L, soybean oil 16% (v/v), remaining is water, pH 7.2.
3rd, the detection of androstenedione mole production rate：
Oil phase, aqueous phase are separately sampled, and oil phase is pressed after centrifugation：Aqueous phase=16：84 (v/v) are mixed, then add ethyl acetate ultrasound
Extractive fermentation liquid, centrifugation, takes ethyl acetate phase 0.1mL in 1.5mL pipes, and 1mL mobile phase is added after natural air drying, dissolves, from
HPLC analyses are carried out after the heart.Chromatographic condition：C18 posts, mobile phase are methanol：Water (4：1), flow velocity 1mL/min, column temperature 30
DEG C, Detection wavelength 254nm.
4th, results contrast：
As shown in table 3, after 48h, engineered strain MNR M3N2AD (D) growing amount is 0.08g/L, is the 0.26 of original strain
Times；After 96h, engineered strain MNR M3N2AD (D) growing amount is 0.87g/L, is 1.14 times of original strain；After 144h, engineering
Bacterial strain MNR M3N2AD (D) growing amount is 1.46g/L, is 1.51 times of original strain；After 192h, engineered strain MNR M3N2AD
(D) growing amount is 1.61g/L, is 1.63 times of original strain.Show in water oil biliquid phase system, the expression of nadh oxidase
The ability of mycobacteria transformation phytosterin can be significantly improved.
AD (D) yield (g/L) in the oil-based system of table 3
It is male that the MNR M3N2 bacterial strains of embodiment 5 are catalyzed phytosterol production in the water oil biliquid phase system containing emulsifying agent
Alkene diketone
1st, strain activation and culture is the same as embodiment 3；
2nd, phytosterol microorganism conversion：
The seed culture fluid of activation is transferred with 7% inoculum concentration and trained in being fermented equipped with the 50mL containing 5g/L phytosterols
In the 250mL baffle plate bottles for supporting base, at 29 DEG C, shaking table culture 192h under the conditions of 150r/min.
Fermentation medium forms:K2HPO40.5g/L, MgSO4·7H2O 0.5g/L, ferric citrate 0.05g/L, lemon
Sour 2g/L, diammonium hydrogen phosphate 3.5g/L, glucose 10g/L, soybean oil (16%, v/v), Tween 80 3g/L, remaining is water, pH
7.2。
3rd, the detection of androstenedione mole production rate：
Oil phase, aqueous phase are separately sampled, and oil phase is pressed after centrifugation：Aqueous phase=16：84 (v/v) are mixed, then add ethyl acetate ultrasound
Extractive fermentation liquid, centrifugation, takes ethyl acetate phase 0.1mL in 1.5mL pipes, and 1mL mobile phase is added after natural air drying, dissolves, from
HPLC analyses are carried out after the heart.Chromatographic condition：C18 posts, mobile phase are methanol：Water (4：1), flow velocity 1mL/min, column temperature 30
DEG C, Detection wavelength 254nm.
4th, results contrast：
As shown in table 4, after 48h, engineered strain MNR M3N2AD (D) growing amount is 0.38g/L, is the 1.41 of original strain
Times；After 96h, engineered strain MNR M3N2AD (D) growing amount is 1.51g/L, is 1.99 times of original strain；After 144h, engineering
Bacterial strain MNR M3N2AD (D) growing amount is 2.88g/L, is 2.44 times of original strain；After 192h, engineered strain MNR M3N2AD
(D) growing amount is 2.90g/L, is 2.38 times of original strain.Show to add emulsifying agent (tween in water oil biliquid phase system
80) after, the ability of original bacteria and engineering bacteria transformation phytosterin all improves, and from the point of view of production ratio, is not added with compared with embodiment 4
During emulsifying agent, the expression of nadh oxidase becomes apparent to the effect for improving the ability of mycobacteria transformation phytosterin.
AD (D) yield (g/L) in the soya-bean oil of table 4 and Tween 80 combination system
It is male that the MNR M3N2 bacterial strains of embodiment 6 are catalyzed phytosterol production in the water oil biliquid phase system containing emulsifying agent
Alkene diketone
1st, strain activation and culture is the same as embodiment 3；
2nd, phytosterol microorganism conversion：
The seed culture fluid of activation is transferred with 5% inoculum concentration and trained in being fermented equipped with the 50mL containing 5g/L phytosterols
In the 250mL baffle plate bottles for supporting base, at 29 DEG C, shaking table culture 192h under the conditions of 150r/min.
Fermentation medium forms:K2HPO40.5g/L, MgSO4·7H2O 0.5g/L, ferric citrate 0.05g/L, lemon
Sour 2g/L, diammonium hydrogen phosphate 3.5g/L, glucose 10g/L, sunflower oil (10%, v/v), lecithin 0.5g/L, remaining is water, pH
7.2。
3rd, the detection of androstenedione mole production rate：
Oil phase, aqueous phase are separately sampled, and oil phase is pressed after centrifugation：Aqueous phase=10：90 (v/v) are mixed, then add ethyl acetate ultrasound
Extractive fermentation liquid, centrifugation, takes ethyl acetate phase 0.1mL in 1.5mL pipes, and 1mL mobile phase is added after natural air drying, dissolves, from
HPLC analyses are carried out after the heart.Chromatographic condition：C18 posts, mobile phase are methanol：Water (4：1), flow velocity 1mL/min, column temperature 30
DEG C, Detection wavelength 254nm.
4th, results contrast：
As shown in table 5, after 48h, engineered strain MNR M3N2AD (D) growing amount is 0.35g/L, is the 1.52 of original strain
Times；After 96h, engineered strain MNR M3N2AD (D) growing amount is 1.58g/L, is 2.25 times of original strain；After 144h, engineering
Bacterial strain MNR M3N2AD (D) growing amount is 2.67g/L, is 2.42 times of original strain；After 192h, engineered strain MNR M3N2AD
(D) growing amount is 2.80g/L, is the 2.15 of original strain.Show to add emulsifying agent (lecithin) in water oil biliquid phase system
Afterwards, original bacteria and the ability of engineering bacteria transformation phytosterin are all improved, and from the point of view of production ratio, breast is not added with compared with embodiment 4
During agent, the expression of nadh oxidase becomes apparent to the effect for improving the ability of mycobacteria transformation phytosterin.
AD (D) yield (g/L) in the sunflower oil of table 5 and lecithin combination system
It is male that the MNR M3N2 bacterial strains of embodiment 7 are catalyzed phytosterol production in the water oil biliquid phase system containing emulsifying agent
Alkene diketone
1st, strain activation and culture is the same as embodiment 3；
2nd, phytosterol microorganism conversion：
The seed culture fluid of activation is transferred with 7% inoculum concentration and trained in being fermented equipped with the 50mL containing 5g/L phytosterols
In the 250mL baffle plate bottles for supporting base, at 28 DEG C, shaking table culture 192h under the conditions of 250r/min.
Fermentation medium forms:K2HPO40.5g/L, MgSO4·7H2O 0.5g/L, ferric citrate 0.05g/L, lemon
Sour 2g/L, diammonium hydrogen phosphate 3.5g/L, glucose 10g/L, rapeseed oil (18%, v/v), Span80 3g/L, remaining is water, pH
7.2。
3rd, the detection of androstenedione mole production rate：
Oil phase, aqueous phase are separately sampled, and oil phase is pressed after centrifugation：Aqueous phase=18：82 (v/v) are mixed, then add ethyl acetate ultrasound
Extractive fermentation liquid, centrifugation, takes ethyl acetate phase 0.1mL in 1.5mL pipes, and 1mL mobile phase is added after natural air drying, dissolves, from
HPLC analyses are carried out after the heart.Chromatographic condition：C18 posts, mobile phase are methanol：Water (4：1), flow velocity 1mL/min, column temperature 30
DEG C, Detection wavelength 254nm.
4th, results contrast：
As shown in table 6, after 48h, engineered strain MNR M3N2AD (D) growing amount is 0.4g/L, is 2 times of original strain；
After 96h, engineered strain MNR M3N2AD (D) growing amount is 1.78g/L, is 2.23 times of original strain；After 144h, engineered strain
MNR M3N2AD (D) growing amount is 2.5g/L, is 2.1 times of original strain；After 192h, engineered strain MNR M3N2AD (D) are raw
It is 2.80g/L into amount, is 2.3 times of original strain.Show after adding emulsifying agent (Span80) in water oil biliquid phase system, it is former
The ability of beginning bacterium and engineering bacteria transformation phytosterin is all improved, and from the point of view of production ratio, emulsifying agent is not added with compared with embodiment 4
When, the expression of nadh oxidase becomes apparent to the effect for improving the ability of mycobacteria transformation phytosterin.
AD (D) yield (g/L) in the rapeseed oil of table 6 and Span80 combination systems
It is male that the MNR M3N2 bacterial strains of embodiment 8 are catalyzed phytosterol production in the water oil biliquid phase system containing emulsifying agent
Alkene diketone
1st, strain activation and culture is the same as embodiment 3；
2nd, phytosterol microorganism conversion：
The seed culture fluid of activation is transferred with 5% inoculum concentration and trained in being fermented equipped with the 50mL containing 5g/L phytosterols
In the 250mL baffle plate bottles for supporting base, at 32 DEG C, shaking table culture 192h under the conditions of 130r/min.
Fermentation medium forms:K2HPO40.5g/L, MgSO4·7H2O 0.5g/L, ferric citrate 0.05g/L, lemon
Sour 2g/L, diammonium hydrogen phosphate 3.5g/L, glucose 10g/L, sunflower oil (10%, v/v), (emulsifying agent forms emulsifying agent 3g/L
Tween80:Span80=1:1), remaining is water, pH 7.2.
3rd, the detection of androstenedione mole production rate：
Oil phase, aqueous phase are separately sampled, and oil phase is pressed after centrifugation：Aqueous phase=10：90 (v/v) are mixed, then add ethyl acetate ultrasound
Extractive fermentation liquid, centrifugation, takes ethyl acetate phase 0.1mL in 1.5mL pipes, and 1mL mobile phase is added after natural air drying, dissolves, from
HPLC analyses are carried out after the heart.Chromatographic condition：C18 posts, mobile phase are methanol：Water (4：1), flow velocity 1mL/min, column temperature 30
DEG C, Detection wavelength 254nm.
4th, results contrast：
As shown in table 7, after 48h, engineered strain MNR M3N2AD (D) growing amount is 0.45g/L, is the 1.84 of original strain
Times；After 96h, engineered strain MNR M3N2AD (D) growing amount is 1.60g/L, is 2.0 times of original strain；After 144h, engineering bacteria
Strain MNR M3N2AD (D) growing amount is 2.90g/L, is 2.42 times of original strain；After 192h, engineered strain MNR M3N2AD
(D) growing amount is 2.88g/L, is 2.30 times of original strain.Show to add emulsifying agent in water oil biliquid phase system
After (Tween80 and Span80), the ability of original bacteria and engineering bacteria transformation phytosterin all improves, and is not added with compared with embodiment 4
During emulsifying agent, the expression of nadh oxidase becomes apparent to the effect for improving the ability of mycobacteria transformation phytosterin.
AD (D) yield (g/L) in the sunflower oil of table 7 and Tween80/Span80 combination systems
Embodiment 9MNR M3N2 bacterial strains are catalyzed phytosterol production androstene in the water oil biliquid phase system containing emulsifying agent
Diketone
1st, strain activation and culture is the same as embodiment 3；
2nd, phytosterol microorganism conversion：
The seed culture fluid of activation is transferred with 10% inoculum concentration and fermented in equipped with the 50mL containing 5g/L phytosterols
In the 250mL baffle plate bottles of culture medium, at 29 DEG C, shaking table culture 192h under the conditions of 200r/min.
Fermentation medium forms:K2HPO40.5g/L, MgSO4·7H2O 0.5g/L, ferric citrate 0.05g/L, lemon
Sour 2g/L, diammonium hydrogen phosphate 3.5g/L, glucose 10g/L, peanut oil (20%, v/v), sucrose ester 3g/L, remaining is water, pH
7.2。
3rd, the detection of androstenedione mole production rate：
Oil phase, aqueous phase are separately sampled, and oil phase is pressed after centrifugation：Aqueous phase=20：80 (v/v) are mixed, then add ethyl acetate ultrasound
Extractive fermentation liquid, centrifugation, takes ethyl acetate phase 0.1mL in 1.5mL pipes, and 1mL mobile phase is added after natural air drying, dissolves, from
HPLC analyses are carried out after the heart.Chromatographic condition：C18 posts, mobile phase are methanol：Water (4：1), flow velocity 1mL/min, column temperature 30
DEG C, Detection wavelength 254nm.
4th, results contrast：
As shown in table 8, after 48h, engineered strain MNR M3N2AD (D) growing amount is 0.45g/L, is the 1.54 of original strain
Times；After 96h, engineered strain MNR M3N2AD (D) growing amount is 1.70g/L, is 1.80 times of original strain；After 144h, engineering
Bacterial strain MNR M3N2AD (D) growing amount is 2.5g/L, is 2.08 times of original strain；After 192h, engineered strain MNR M3N2AD
(D) growing amount is 2.70g/L, is 1.93 times of original strain.Show to add emulsifying agent (sucrose in water oil biliquid phase system
Ester) after, the ability of original bacteria and engineering bacteria transformation phytosterin all improves, and from the point of view of production ratio, is not added with compared with embodiment 4
During emulsifying agent, the expression of nadh oxidase becomes apparent to the effect for improving the ability of mycobacteria transformation phytosterin.
AD (D) yield (g/L) in the peanut oil of table 8 and sucrose ester combination system
It is male that the MNR M3N2 bacterial strains of embodiment 10 are catalyzed phytosterol production in the water oil biliquid phase system containing emulsifying agent
Alkene diketone
1st, strain activation and culture is the same as embodiment 3；
2nd, phytosterol microorganism conversion：
The seed culture fluid of activation is transferred with 8% inoculum concentration and trained in being fermented equipped with the 50mL containing 5g/L phytosterols
In the 250mL baffle plate bottles for supporting base, at 29 DEG C, shaking table culture 192h under the conditions of 150r/min.
Fermentation medium forms:K2HPO40.5g/L, MgSO4·7H2O 0.5g/L, ferric citrate 0.05g/L, lemon
Sour 2g/L, diammonium hydrogen phosphate 3.5g/L, glucose 10g/L, rice bran oil (20%, v/v), (emulsifying agent forms emulsifying agent 5g/L：Sugarcane
Sugar ester：Monoglyceride：Span=7:2:1), remaining is water, pH 7.2.
3rd, the detection of androstenedione mole production rate：
Oil phase, aqueous phase are separately sampled, and oil phase is pressed after centrifugation：Aqueous phase=20：80 (v/v) are compared to mixing, then add ethyl acetate
Ultrasonic extraction zymotic fluid, centrifugation, takes ethyl acetate phase 0.1mL in 1.5mL pipes, and 1mL mobile phase is added after natural air drying, molten
Solution, HPLC analyses are carried out after centrifugation.Chromatographic condition：C18 posts, mobile phase are methanol：Water (4：1), flow velocity 1mL/min, column temperature
For 30 DEG C, Detection wavelength 254nm.
4th, results contrast：
As shown in table 9, after 48h, engineered strain MNR M3N2AD (D) growing amount is 0.38g/L, is the 2.53 of original strain
Times；After 96h, engineered strain MNR M3N2AD (D) growing amount is 1.46g/L, is 2.65 times of original strain；After 144h, engineering
Bacterial strain MNR M3N2AD (D) growing amount is 2.53g/L, is 2.53 times of original strain；After 192h, engineered strain MNR M3N2AD
(D) growing amount is 2.65g/L, is 2.28 times of original strain.Show to add emulsifying agent (sucrose in water oil biliquid phase system
Ester：Monoglyceride：Span after), the ability of original bacteria and engineering bacteria transformation phytosterin all improves, and is not added with breast compared with embodiment 4
During agent, the expression of nadh oxidase becomes apparent to the effect for improving the ability of mycobacteria transformation phytosterin.
AD (D) yield (g/L) in the rice bran oil of table 9 and sucrose ester/monoglyceride/Span combination systems
It is male that the MNR M3N2 bacterial strains of embodiment 11 are catalyzed phytosterol production in the water oil biliquid phase system containing emulsifying agent
Alkene diketone
1st, strain activation and culture is the same as embodiment 3；
2nd, phytosterol microorganism conversion：
The seed culture fluid of activation is transferred with 5% inoculum concentration and trained in being fermented equipped with the 50mL containing 5g/L phytosterols
In the 250mL baffle plate bottles for supporting base, at 29 DEG C, shaking table culture 192h under the conditions of 150r/min.
Fermentation medium forms:K2HPO40.5g/L, MgSO4·7H2O 0.5g/L, ferric citrate 0.05g/L, lemon
Sour 2g/L, diammonium hydrogen phosphate 3.5g/L, glucose 10g/L, tall oil (20%, v/v), monoglyceride 8g/L, remaining is water, pH
7.2。
3rd, the detection of androstenedione mole production rate：
Oil phase, aqueous phase are separately sampled, oil phase: aqueous phase=20: 80 (v/v) mixing are pressed after centrifugation, then add ethyl acetate ultrasound
Extractive fermentation liquid, centrifugation, takes ethyl acetate phase 0.1mL in 1.5mL pipes, and 1mL mobile phase is added after natural air drying, dissolves, from
HPLC analyses are carried out after the heart.Chromatographic condition：C18 posts, mobile phase are methanol：Water (4：1), flow velocity 1mL/min, column temperature 30
DEG C, Detection wavelength 254nm.
4th, results contrast：
As shown in table 10, after 48h, engineered strain MNR M3N2 AD (D) growing amount is 0.23g/L, is original strain
1.28 again；After 96h, engineered strain MNR M3N2AD (D) growing amount is 1.20g/L, is 2.22 times of original strain；After 144h,
Engineered strain MNR M3N2AD (D) growing amount is 2.20g/L, is 2.50 times of original strain；After 192h, engineered strain MNR
M3N2AD (D) growing amount is 2.50g/L, is 2.31 times of original strain.Show to add emulsifying agent in water oil biliquid phase system
After (monoglyceride), the ability of original bacteria and engineering bacteria transformation phytosterin all improves, and when being not added with emulsifying agent compared with embodiment 4,
The expression of nadh oxidase becomes apparent to the effect for improving the ability of mycobacteria transformation phytosterin.
AD (D) yield (g/L) in the tall oil of table 10 and monoglyceride combination system
Embodiment described above only expresses the several embodiments of the present invention, and its description is more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims can not be interpreted as.It should be pointed out that for the person of ordinary skill of the art,
On the premise of not departing from this patent design, the respective embodiments described above can also make some deformations, combination and improve, and these all belong to
In the protection domain of this patent.Therefore, the protection domain of this patent should be determined by the appended claims.
Sequence table
<110>University Of Science and Technology Of Tianjin
<120>Cofactor regeneration mycobacteria and its application in the fermentation of profit Two Liquid Phases
<130> 1
<141> 2017-10-25
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1341
<212> DNA
<213>Lactococcus lactis subsp (Lactococcus lactis subsp. cremorisNZ9000)
<400> 1
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gaacaatatc ccgggcatga aattgtcatg attgaccgta atagcaacat gagttatcta 120
ggttgtggca cagcaatttg ggttggaaga caaattgaaa aaccagatga attattttat 180
gccaaagcag aggattttga ggcaaaaggg gtaaaaattt tgactgaaac agaagtttca 240
gaaattgatt ttgctaataa gaaagtttat gcaaaaacta aatctgatga tgaaataatt 300
gaagcttacg acaagcttgt tttagcaaca ggttcacgtc caattattcc taatctacca 360
ggcaaagacc ttaagggaat tcattttctg aaactttttc aagaaggtca agcaattgac 420
gcagaatttg ccaaagaaaa agtcaagcgt atcgcagtca ttggtgcagg atatatcggt 480
acagagattg cggaagcagc taaacgtcgg ggtaaagaag ttcttctctt tgacgctgaa 540
aatacttcac ttgcatcata ttatgatgaa gaatttgcca aaggaatgga tgaaaacctt 600
gctcaacatg gaattgaact tcattttgga gaactggcca aagaatttaa agcgaatgag 660
gaaggttatg tatcacaaat cgtaaccaac aaggcgactt atgatgttga tcttgtcatc 720
aattgtattg gttttactgc caacagtgcc ttggcaagtg ataagttagc taccttcaaa 780
aatggcgcaa tcaaggtgga taagcatcaa caaagtagtg atccagatgt ttacgcggta 840
ggtgatgttg cgacaattta ttctaatgcc ttgcaagatt ttacttatat cgctcttgcc 900
tcaaacgctg ttcggtcagg aattgtcgca ggacacaata ttggtggaaa agaattagaa 960
tctgttggtg ttcaaggttc taatggtatt tcgatttttg gttacaatat gacttctaca 1020
ggactttctg ttaaagctgc taaaaaatta ggtttagaag tttcatttag tgattttgaa 1080
gataaacaaa aagcttggtt tcttcatgaa aacaacgata gtgtgaaaat tcgtatcgta 1140
tatgagacaa aaagtcgcag aattattgga gcacaacttg ctagtaaaag tgagataatt 1200
gcaggaaata taaatatgtt cagtttagcg attcaagaga aaaaaacaat tgatgaacta 1260
gctttgcttg atttattctt tctcccccac ttcaacagtc catataatta tatgacagtt 1320
gcagctttga atgccaaata a 1341
Claims (7)
1. the genetic engineering bacterium MNR M3N2 of one plant of production androstenedione, it is characterised in that the genetic engineering bacterium is to be to number
CICC21097 new golden mycobacteria (Mycobacterium sp.) MNR M3 are host cell, heterogenous expression nadh oxidase
Obtained by gene nox-2, the nucleotide sequence of the nox-2 is as shown in sequence table SEQ ID NO.1.
2. genetic engineering bacterium MNR M3N2 construction method described in claim 1, it is characterised in that specific as follows：
(1) nox-2 genes and expression plasmid pMV261 connection, are built into pMV261-nox-2 recombinant plasmids by digestion；
(2) recombinant plasmid pMV261-nox-2 is imported in MNR M3 competence bacterial strain, the positive transformant of acquisition is this
The new structure bacterial strain MNR M3N2 of invention.
3. applications of the genetic engineering bacterium MNR M3N2 described in claim 1 in profit Two Liquid Phases fermentation system.
4. genetic engineering bacterium MNR M3N2 application described in claim 3, it is characterised in that the genetic engineering bacterium MNR M3N2
The method of fermenting and producing androstenedione is as follows in profit Two Liquid Phases fermentation system：
MNR M3N2 are inoculated with profit Two Liquid Phases fermentation medium by 5-10% inoculum concentrations, 28-32 DEG C, 130-250r/
Under the conditions of min, pH6.5-7.8, ferment 4-8 days；
The profit Two Liquid Phases fermentation medium composition is as follows：K2HPO40.5g/L, MgSO4·7H2O 0.5g/L, ironic citrate
Ammonium 0.05g/L, citric acid 2g/L, diammonium hydrogen phosphate 3.5g/L, glucose 10g/L, oil phase 10-20%, remaining is water, pH6.5-
7.8。
5. genetic engineering bacterium MNR M3N2 application described in claim 4, it is characterised in that the oil phase is vegetable oil, is selected from
At least one of soybean oil, sunflower oil, rapeseed oil, peanut oil, rice bran oil or tall oil.
6. genetic engineering bacterium MNR M3N2 application described in claim 4, it is characterised in that the profit Two Liquid Phases fermented and cultured
0.5-8g/L emulsifying agent is also added in base.
7. genetic engineering bacterium MNR M3N2 application described in claim 6, it is characterised in that described emulsifying agent be lecithin,
At least one of Tween 80, Span 80, sucrose ester and monoglyceride.
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| CN108913643A (en) * | 2018-08-01 | 2018-11-30 | 天津科技大学 | A method of it improving mycobacteria regenerating coenzyme and androstenedione is promoted to produce simultaneously |
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| CN110643557A (en) * | 2019-04-23 | 2020-01-03 | 天津科技大学 | Construction of coenzyme regeneration system and application of coenzyme regeneration system in efficient catalysis of 5 alpha-AD production |
| CN111454871A (en) * | 2020-03-03 | 2020-07-28 | 天津大学 | Recombinant mycobacterium with high androstenedione yield, construction method and application |
| CN111690586A (en) * | 2019-07-04 | 2020-09-22 | 天津科技大学 | Method for enhancing intracellular propionyl coenzyme A metabolism and improving steroid precursor production |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108913643A (en) * | 2018-08-01 | 2018-11-30 | 天津科技大学 | A method of it improving mycobacteria regenerating coenzyme and androstenedione is promoted to produce simultaneously |
| CN109706107A (en) * | 2019-01-29 | 2019-05-03 | 天津科技大学 | A kind of method of high-efficiency fermenting production steroid medicine precursor |
| CN110643557A (en) * | 2019-04-23 | 2020-01-03 | 天津科技大学 | Construction of coenzyme regeneration system and application of coenzyme regeneration system in efficient catalysis of 5 alpha-AD production |
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| CN111454871A (en) * | 2020-03-03 | 2020-07-28 | 天津大学 | Recombinant mycobacterium with high androstenedione yield, construction method and application |
| CN111454871B (en) * | 2020-03-03 | 2022-06-14 | 天津大学 | Recombinant mycobacterium with high androstenedione yield, construction method and application |
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