CN103266161B - Fermentation strategy for producing androstenedione (ADD) by using recombinant bacillus subtilis - Google Patents
Fermentation strategy for producing androstenedione (ADD) by using recombinant bacillus subtilis Download PDFInfo
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Abstract
The invention discloses a high-yield fermentation strategy for producing androstenedione (ADD) by using recombinant bacillus subtilis. A single factor experiment and an orthogonal experiment show that fermentation medium components, culturing conditions and a full-cell transformation technology of recombinant bacteria bacillus subtilis 168/pMA5-ksdD are optimized, concentration and pH values of maltose, soy peptone to yeast extract (2 to 1), ammonium chloride, potassium chloride, corn steep liquid, cosolvent methyl-beta-cyclodextrin and a substrate androstane (AD) in an optimal medium are determined; and the optimal inoculum size and the culturing conditions are determined. The ADD yield after optimization reaches 1.80g/L which is greatly improved by 177 percent compared with 0.65g/L before optimization. According to the fermentation strategy, based on the research on the fermentation strategy of the recombinant bacteria, the ability of producing ADD by using an AD full-cell transformation technology is greatly improved, and a feasible fermentation strategy is provided for highly yielding ADD in an industrial microorganism one-step fermentation process.
Description
Technical field
Recombined bacillus subtilis Bacillus subtilis produces the fermentation strategies of ADD, and especially a kind of fermentation condition optimization strategy that increases substantially ADD output in recombinant bacterium, belongs to biotechnology and enzyme engineering field.
Technical background
Steroid drugs (steroid hormone drugs) refers to the medicine that contains steroidal structure in molecular structure, and it has anti-infective, antianaphylaxis, antiviral and antishock pharmacological action very by force, is the important medicine of a class clinically.Mainly be divided into two class medicines: glucocorticosteroid and sexual hormoue.Chinese Pharmacopoeia reaches 97 kinds to including of steroid drugs and preparation thereof.The main manufacturer of domestic steroid drugs has: Xian Ju pharmacy group, black bamboo medicine company group, Jia Erke medicine company group, Divine Land medicine company group etc.The Aging Problem along with people, raising day by day and the society of quality of life, healthy demand being increased the weight of day by day in recent years, the value that steroid drugs embodies is day by day remarkable.
Androstenedione (androstenedione), has another name called Δ 4-androstene-3, and 17 diketone are a kind of steroids with male hormone effect, is to produce the important intermediate of steroid drugs.Its proterties is near-white crystalline powder, is dissolved in ethanol, water insoluble.Modal is 4-AD (AD) and Androsta-1,4-diene-3,17-dione (ADD).Microbiological deterioration plant sterol produces many important steroidal drug intermediates, and wherein AD/ADD is most important steroidal drug intermediate, can be used for the synthetic of cortex steroid drugs, salt cortex steroidal drug, conventional contraceptive bian and other steroid class medicine.The demand in the annual market of AD/ADD overbalances 1,000,000,000 dollars.
3-sterone-Δ
1-desaturase (3-ketosteroid-Δ
1-dehydrogenase, KSDD or KSTD) also claim C1,2 desaturases, it is the key enzyme of steroidal parent nucleus degraded, belongs to flavoprotein enzyme, is positioned on cytolemma, can between steroidal parent nucleus C1 and C2, introduce two keys, the anti-inflammatory activity and the economic worth that improve original substrate, realize AD and transform to ADD, in steroid drugs exploitation, plays an important role.To 3-sterone-Δ
1the further investigation of-desaturase (KSDD) is of far-reaching significance to the development of whole steroid drugs industry.Producing the more direct effective means of ADD of high added value is exactly taking 4-AD as substrate, by microorganisms 3-sterone-Δ
1-desaturase is realized 4-AD and is transformed to a step of ADD.
This laboratory has successfully built a strain recombined bacillus subtilis Bacillus subtilis168/pMA5-ksdD in earlier stage, realize the expression of the KSDD gene that derives from new golden mycobacterium Mycobacterium neoaurum in type strain subtilis 168, and the KSDD enzyme in this recombinant bacterium has been carried out to Function Identification and analysis (patent: a kind of method of utilizing restructuring Bacillus subtilis resting cell AD to generate ADD; Application number: 201210210431.1).Through enzyme activity determination, enzyme work reaches 1.75U/mg, resting cell AD, and after 12h, transformation efficiency reaches 65.7%.Mycobacterium KSDD expresses in withered grass system and resting cell AD is reported first, and the enzyme transformation efficiency alive and AD that recon is expressed is all higher.Subtilis is as the industrial producing strain of safety and stability, and culture condition is simple, and fermentation period is short, is the potential production bacterial strain of microorganism sterol fermentation industry.
The present invention tests and orthogonal experiment by single-factor, and these three aspects of fermention medium component, culture condition and resting cell technique to this recombinant bacterium Bacillus subtilis168/pMA5-ksdD are optimized.Best medium is maltose 35g/L, soy peptone: yeast extract paste (2: 1) 35g/L, ammonium chloride 1g/L, Repone K 3.5g/L, corn steep liquor 17.5g/L, pH7.0; Optimum inoculation amount is 3%; Optimal culture conditions is 37 DEG C, 160r/min; Best solubility promoter first group-beta-cyclodextrin, concentration is 2g/L; Substrate A D concentration 2g/L.After optimizing, ADD output reaches 1.80g/L, and the 0.65g/L before optimizing has improved 177%.By the research to this recombinant bacterium fermentation strategies, the ability that makes its resting cell AD produce ADD has obtained raising by a relatively large margin, thereby makes its industrialization further become possibility.
Summary of the invention
The object of the present invention is to provide: a kind of microbe whole-cell that makes transforms the fermentation strategies that AD product ADD ability increases substantially.The recombinant bacterial strain Bacillus subtilis168/pMA5-ksdD building has been carried out to the research of fermentation strategies, and after optimizing, ADD output has improved 177%, has obtained desirable result.For the industrialization of microorganism fermentative production ADD provides useful guidance.
Technical scheme of the present invention: be to test and orthogonal experiment by single-factor, fermention medium component, culture condition and the resting cell technique of a strain energy resting cell AD who builds being produced to the recombined bacillus subtilis Bacillus subtilis168/pMA5-ksdD of ADD are optimized.The present invention has successfully obtained a kind of fermentation optimization strategy that makes subtilis engineering strain resting cell AD high yield ADD, and its ADD output improves a lot before optimizing.
Main agents: AD, ADD is purchased from SIGMA company of the U.S.
The optimization of recombinant bacterial strain fermentation strategies:
1. recombinant bacterial strain resting cell AD produces the medium component optimization of ADD
(1) different carbon sources transforms the impact of AD product ADD on recombined bacillus subtilis B.subtilis168/pMA5-ksdD
The impact of carbon source on biomass and AD transformation efficiency: investigated six kinds of different carbon sources---the impact on thalline biomass and AD transformation efficiency of glucose, sucrose, lactose, Zulkovsky starch, maltose and glycerine, carbon source concentration is 1%.Initial fermention medium is LB substratum.Owing to there is not proper carbon source in LB substratum, the carbon source of choosing directly adds as sole carbon source, and other component all remains unchanged.Inoculum size 1%, 37 DEG C of culture temperature, shaking speed 160rpm, cultivates 24h, sampling, the biomass that suitably light absorption value under dilution metering 600nm is bacterium.Carry out resting cell experiment, get the bacterium liquid of cultivating 24h, 4 DEG C, the centrifugal 10min of 8000r/min collects thalline, with the 50mM Tris-HCl damping fluid washing secondary of 100mL pH7.0, is resuspended in isopyknic this damping fluid.In this system, drop into 0.1% (w/v) AD and 0.1% (v/v) tween-80, put in 30 DEG C of shaking tables and continue to cultivate, point different time sampling, the conversion fluid after sampling extracts by ethyl acetate, and suitably dilution, crosses film, and HPLC analyzes.Be beneficial to define the optimum carbon source that AD transformation efficiency improves.
HPLC analyzes: AD and ADD all have charateristic avsorption band under 254nm ultraviolet wavelength, so adopt HPLC method to measure.Chromatographic column: dimosoil C
18(5 μ l, 250mm × 4.6mm), moving phase: methanol-water (V/V=70: 30), detector: UVDetector, detects wavelength: 254nm, column temperature: 30 DEG C, sample size: 10 μ L, flow velocity: 1.0ml/min.
LB substratum: peptone 10g/L, yeast extract paste 5g/L, NaCl10g/L (solid medium adds 2% agar powder)
In every group of test, the relative transformation efficiency of peak rate of conversion is defined as 100%, and the transformation efficiency of surveying during by experiment with computing is relative transformation efficiency with the ratio of this batch of peak rate of conversion.
The impact of the carbon source of different concns on biomass and AD transformation efficiency: investigating on optimum carbon source basis, the impact of the optimum carbon source of continuation investigation different concns on biomass and AD transformation efficiency, concentration is set as 1%, 2%, 3%, 4%, 5%, culture condition is described above, carry out biomass estimation and transformation experiment, be beneficial to define the optimum carbon source concentration that AD transformation efficiency improves.
(2) impact of organic nitrogen source on biomass and AD transformation efficiency
The impact of organic nitrogen source on biomass and AD transformation efficiency: on the basis of the suitableeest carbon source, nine kinds of different organic nitrogen sources or organic nitrogen source combination---soy peptone, peptone fish meal, Tryptones, extractum carnis, yeast extract paste, soy peptone: extractum carnis (1: 1), extractum carnis: yeast extract paste (1: 1), soy peptone: yeast extract paste (1: 1) and soy peptone: the impact of yeast extract paste (2: 1) on thalline biomass and AD transformation efficiency, nitrogen concentration is 1.5% are investigated.Initial fermention medium is LB substratum.Owing to there being nitrogenous source soy peptone in LB substratum: yeast extract paste (2: 1), so the nitrogenous source of choosing replaces original nitrogenous source, other component all remains unchanged.Inoculum size 1%, 37 DEG C of culture temperature, shaking speed 160rpm, cultivates, and measures its biomass and carries out transformation experiment (method is the same), is beneficial to define the best organic nitrogen source that AD transformation efficiency improves.
The impact of the organic nitrogen source of different concns on biomass and AD transformation efficiency: investigating on best organic nitrogen source basis, the impact of the best organic nitrogen source of continuation investigation different concns on biomass and AD transformation efficiency, concentration is set as 1.5%, 3.0%, 4.5%, 6.0%, 7.5%, culture condition is described above, carry out biomass estimation and transformation experiment, be beneficial to define the best organic nitrogen source concentration that AD transformation efficiency improves.
(3) the inorganic nitrogen-sourced impact on biomass and AD transformation efficiency
The inorganic nitrogen-sourced impact on biomass and AD transformation efficiency: in the suitableeest carbon source, on the basis of organic nitrogen source, investigated four kinds inorganic nitrogen-sourced---the impact on thalline biomass and AD transformation efficiency of urea, ammonium nitrate, ammonium chloride and ammonium sulfate, concentration is set as 0.2%.Initial fermention medium is LB substratum.Inorganic nitrogen-sourced owing to not existing in LB substratum, so the inorganic nitrogen-sourced direct interpolation of choosing, other component all remains unchanged.Inoculum size 1%, 37 DEG C of culture temperature, shaking speed 160rpm, cultivate, measure its biomass and carry out transformation experiment (method is the same), with define be beneficial to transformation efficiency improve the best inorganic nitrogen-sourced.
The impact of the inorganic nitrogen-sourced concentration of different concns on biomass and AD transformation efficiency: on the inorganic nitrogen-sourced basis of the best, continue to investigate the inorganic nitrogen-sourced impact on biomass and AD transformation efficiency of the best of different concns, concentration is set as 0%, 0.1%, 0.2%, 0.3%, 0.4% and 0.5%, culture condition is described above, carry out biomass estimation and transformation experiment, be beneficial to define the inorganic nitrogen-sourced concentration of the best that AD transformation efficiency improves.
(4) impact of inorganic salt on biomass and AD transformation efficiency
The impact of inorganic salt on biomass and AD transformation efficiency: this is on the suitableeest carbon source, organic nitrogen source and inorganic nitrogen-sourced basis, seven kinds of inorganic salt or inorganic salt combination---sodium-chlor, Repone K, SODIUMNITRATE, saltpetre, Trisodium Citrate, SODIUM PHOSPHATE, MONOBASIC: Sodium phosphate dibasic (1: 1) and potassium primary phosphate: the impact of dipotassium hydrogen phosphate (1: 1) on thalline biomass and transformation efficiency, concentration 0.25% are investigated.Initial fermention medium is LB substratum.Owing to there is inorganic salt sodium-chlor in LB substratum, so the inorganic salt of choosing directly replace original inorganic salt, other component all remains unchanged.Inoculum size 1%, 37 DEG C of culture temperature, shaking speed 160rpm, cultivates, and measures its biomass and carries out transformation experiment (method is the same), is beneficial to define the optimal inorganic salts that AD transformation efficiency improves.
The impact of the inorganic salt of different concns on biomass and AD transformation efficiency: on optimal inorganic salts basis, the impact of the optimal inorganic salts of continuation investigation different concns on biomass and AD transformation efficiency, concentration is set as 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% and 0.6%, culture condition is described above, carry out biomass estimation and transformation experiment, be beneficial to define the optimal inorganic salts concentration that AD transformation efficiency improves.
(5) impact of corn steep liquor on biomass and AD transformation efficiency
Somatomedin be a class to the requisite material of microorganism, although demand is little, the effect in biochemical metabolism is very important.Somatomedin does not generally need independent interpolation, and general much nutritious natural matters that add, as corn steep liquor.In corn steep liquor, contain a large amount of VITAMIN, vitamin H and abundant amino acid etc.Therefore optimizing on the basis of carbon source, organic nitrogen source, inorganic nitrogen-sourced and inorganic salt, the impact of the corn steep liquor of consideration interpolation different concns on biomass and AD transformation efficiency, concentration is set as 0%, 0.5%, 1.0%, 1.5%, 2.0% and 2.5%, culture condition is described above, carry out biomass estimation and transformation experiment, to define the corn steep liquor of the optimum concn that is beneficial to the raising of AD transformation efficiency.
(6) orthogonal Optimal Medium composition
On above monofactorial basis, design orthogonal test is carried out the composition of Optimal Medium." four factor three levels " scheme that experiment adopts, the optimum carbon source of choosing, optimum nitrogenous source, optimum inorganic salt and corn steep liquor are investigation factor, test.
2. recombined bacillus subtilis B.subtilis168/pMA5-ksdD transforms the culture condition optimization of AD
(1) impact of initial pH on biomass and AD transformation efficiency
On the basis of Optimal Medium composition, investigate the impact of initial pH on cellular biomass and AD transformation efficiency, initial substratum pH is set respectively to pH5.0, pH6.0, pH7.0, pH8.0 and pH9.0, culture condition is described above, carry out biomass estimation and transformation experiment, be beneficial to define the best initial pH of substratum that AD transformation efficiency improves.
(2) impact of inoculum size on biomass and AD transformation efficiency
On the basis of Optimal Medium composition and initial pH, investigate the impact of different vaccination amount on cellular biomass and AD transformation efficiency, inoculum size is set respectively to 1%, 2%, 3%, 4%, 5% and 6%, culture condition is described above, carry out biomass estimation and transformation experiment, be beneficial to define the optimum inoculation amount that transformation efficiency improves.
3. the solubility promoter optimization of recombined bacillus subtilis B.subtilis168/pMA5-ksdD resting cell AD
(1) impact of different substrate solubility promoters on AD solubleness
Substrate A D charging capacity 2g/L, adds ethanol, tween 80, Yelkin TTS and the first group-beta-cyclodextrin of different concns, 160rpm, and 2h concussion mixes, and reaches balance, centrifugal, filters, and measures the light absorption value of filtrate under 242nnm.Because substrate A D has distinctive absorption peak under 242nm, the size of light absorption value, has reflected that in filtrate, the dissolving of substrate is how many, i.e. the effect quality of substrate solubility promoter.Determine best solubility promoter.
(2) impact of different substrate solubility promoter concentration on resting cell AD
In the transformation system of substrate A D charging capacity 2g/L, conversion condition is described above, adds the best solubility promoter of different concns, and concentration is set as 1g/L, 2g/L, 3g/L, 4g/L, transforms, and measures ADD output.To define the best solubility promoter concentration that is beneficial to substrate conversion.
Beneficial effect of the present invention: test and orthogonal experiment by single-factor, fermention medium component, culture condition and the resting cell technique of a strain energy resting cell AD who builds being produced to the recombined bacillus subtilis Bacillus subtilis168/pMA5-ksdD of ADD are optimized.Successfully obtained a kind of fermentation strategies that makes subtilis engineering strain resting cell AD high yield ADD, its ADD output improves a lot before optimizing.Best medium is maltose 35g/L, soy peptone: yeast extract paste (2: 1) 35g/L, ammonium chloride 1g/L, Repone K 3.5g/L, corn steep liquor 17.5g/L, pH7.0; Optimum inoculation amount is 3%; Optimal culture conditions is 37 DEG C, 160r/min; Best solubility promoter first group-beta-cyclodextrin, concentration is 2g/L; Substrate A D concentration 2g/L.After optimizing, ADD output is up to 1.80g/L, and the 0.65g/L before optimizing has improved 177%.
Substrate A D mono-step is changed into and has the more ADD of high additive value, and it is important medicine intermediate; Produce ADD with microbial method simultaneously, its than chemical production method there is reaction conditions gentleness, raw material availability is high, product purity is high and technique is simple, be easy to the advantages such as control, is conducive to environment protection simultaneously, be easy to apply.
Brief description of the drawings
Figure 1A D/ADD standard specimen HPLC detects figure
The carbon source that Fig. 2 is different transforms the impact of AD product ADD on recombined bacillus subtilis B.subtilis168/pMA5-ksdD
The impact of the different solubility promoters of Fig. 3 on AD solubleness
The impact of the concentration of the best solubility promoter of Fig. 4 on ADD output
Specific implementation method
Embodiment 1: different carbon sources transforms the impact of AD product ADD on recombined bacillus subtilis B.subtilis168/pMA5-ksdD
The impact of carbon source on biomass and AD transformation efficiency: this study tour six kinds of different carbon sources---the impact on thalline biomass and AD transformation efficiency of glucose, sucrose, lactose, Zulkovsky starch, maltose and glycerine, carbon source concentration is 1%.Initial fermention medium is LB substratum.Owing to there is not proper carbon source in LB substratum, the carbon source of choosing directly adds as sole carbon source, and other component all remains unchanged.Inoculum size 1%, 37 DEG C of culture temperature, shaking speed 160rpm, cultivates 24h, measures its biomass and carries out resting cell experiment.(accompanying drawing 2) as seen from the figure, the biomass during using maltose as carbon source is higher, and the transformation efficiency of AD is the highest.
Embodiment 2: the impact of different substrate solubility promoters on AD solubleness
Substrate A D charging capacity 2g/L, adds ethanol, tween 80, Yelkin TTS and the first group-beta-cyclodextrin of different concns, 160rpm, and 2h concussion mixes, and reaches balance, centrifugal, filters, and measures the light absorption value of filtrate under 242nm.Shown in result accompanying drawing 3, the light absorption ratio of first group-beta-cyclodextrin is the highest, proves that its solubilization-aid effect to AD is best.
Embodiment 3: the impact of different substrate solubility promoter concentration on resting cell AD
In the transformation system of substrate A D charging capacity 2g/L, conversion condition is described above, adds the best solubility promoter of different concns, and concentration is set as 1g/L, 2g/L, 3g/L, 4g/L, transforms, and measures ADD output.As shown in Figure 4, under the condition of substrate 2g/L, the output of ADD does not increase along with the increase of solubility promoter concentration result always, illustrates that the inversion quantity of recombinant bacterial strain maintains 1.8g/L.Determine substrate 2g/L, solubility promoter 2g/L, carry out resting cell, ADD output is up to 1.80g/L, and the output 0.65g/L before optimizing has improved 177%.
Claims (1)
1. an optimization method of ADD is produced in recombined bacillus subtilis fermentation, it is characterized in that: these three aspects of fermention medium component, culture condition and the resting cell technique of recombinant bacterium Bacillus subtilis168/pMA5-ksdD are optimized; Best medium is maltose 35g/L, the soy peptone yeast extract paste 35g/L of 2: 1 ratios, ammonium chloride 1g/L, Repone K 3.5g/L, corn steep liquor 17.5g/L, pH7.0; Optimum inoculation amount is 3%; Optimal culture conditions is 37 DEG C, 160r/min; Best solubility promoter first group-beta-cyclodextrin, concentration is 2g/L; Substrate A D concentration 2g/L.
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