CN103756949A - Gene engineering bacteria for producing ultrahigh-optical purity R,R-2,3-butanediol as well as construction method and application thereof - Google Patents

Gene engineering bacteria for producing ultrahigh-optical purity R,R-2,3-butanediol as well as construction method and application thereof Download PDF

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CN103756949A
CN103756949A CN201410018001.9A CN201410018001A CN103756949A CN 103756949 A CN103756949 A CN 103756949A CN 201410018001 A CN201410018001 A CN 201410018001A CN 103756949 A CN103756949 A CN 103756949A
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dar
homology arm
cgmcc3044
paenibacillus polymyxa
sequence
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CN103756949B (en
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高健
徐虹
徐尤勇
薛锋
丁鸽
李莎
冯小海
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Yangcheng Institute of Technology
Yancheng Institute of Technology
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Abstract

The invention discloses gene engineering bacteria for producing ultrahigh-optical purity R,R-2,3-butanediol as well as a construction method and application thereof. The gene engineering bacteria is named recombinant paenibacillus polymyxa CGMCC3044--dar-, wherein dar is a butanedione reductase gene. Due to homologous recombination double exchange, strain-recombined paenibacillus polymyxa CGMCC3044-dar- is obtained by removing the dar gene from the paenibacillus polymyxa CGMCC3044. The recombinant bacteria constructed by the method can be used for synthesizing the ultrahigh-optical purity R,R-2,3-butanediol through fermentation, the optical purity is as high as 100%, and an optical side product meso-2,3-butanedio is not generated; substrates including jerusalem artichoke inulin, glucose and 3-hydroxyl butanone can be directly utilized by the strain, the condition of culture is extensive, the operation is convenient and simple, the optical purity is high, the production cost is low, and the gene engineering bacteria is benefit for large-scale industrial production.

Description

A product superelevation optical purity R, R-2,3-butyleneglycol genetic engineering bacterium and construction process thereof and application
Technical field
The invention belongs to biological chemical field, be specifically related to a kind of product superelevation optical purity (>=99.9%) R, R-2,3-butyleneglycol genetic engineering bacterium and construction process thereof and application.
Background technology
2,3-butanediol belongs to typical bio-based chemical, has 3 optically active isomers, is respectively R, R type (D type), S, S type (L-type) and meso type (meso-form).R, R-2,3-butyleneglycol is a kind of emerging microorganism four carbon chiral alcohols, has unique physico-chemical properties such as optics.The R of superelevation optical purity (>=99.9%), R-2,3-butyleneglycol can be used for synthetic trihydroxybutane-p-toluenesulfonic esters, and trihydroxybutane-p-toluenesulfonic esters is the key intermediate of synthetic drugs acetylsalicylic acid.The R of superelevation optical purity, R-2,3-butyleneglycol can also be for the synthesis of optically active 2-methyl isophthalic acid, 4-butyleneglycol, 2-methyl isophthalic acid, 4-butyleneglycol and derivative thereof are the important intermediate of synthetic various chiral smectic type thermotropic liquid crystalline polyester materials and chirality natural bioactivity substance.Along with R, R-2, the fermentation technique of 3-butyleneglycol improves constantly and is perfect, superelevation optical purity R, R-2, the effect increasingly significant of 3-butyleneglycol in the synthetic route improvement of quiral products and the synthetic field of the chiralitys such as exploitation of novel chiral product.In addition, R, R-2,3-butyleneglycol or good antifreezing agent (R, R-2, zero pour low the reaching-60 ℃ of 3-butyleneglycol), add in liquid fuel, and fuel can not solidify at-40 ℃ yet.
Chemical method synthesizes super high-optical-purity chiral R, R-2, and 3-butyleneglycol, except needing special synthesis path, also needs chiral separation, and means are particularly complicated, are difficult to realize large-scale industrial production.Some yeast of nature (Saccharomyces cerevisiae) although can internal metabolism synthetic R, R-2,3-butyleneglycol, due to fermentation preparation R, R-2, extremely low (the about 0.09g L of production peak of 3-butyleneglycol output -1), lack actual application value, therefore, some special Paenibacillus polymyxas (Paenibacillus polymyxa also claims Bacillus polymyxa) are at present unique possess suitability for industrialized production R, R-2, microorganisms of 3-butyleneglycol potentiality.
R is synthesized in P.polymyxa metabolism, and R-2, during 3-butyleneglycol, always follow synthetic a small amount of meso-2,3-butyleneglycol, and the change of the external source fermentation conditions such as stream rate of acceleration of pH, dissolved oxygen, substrate often causes the meso-2 of by product, the content generation considerable change of 3-butyleneglycol.In fermented liquid, R, R-2, the optical purity of 3-butyleneglycol is about 90-98%, meso-2, the optical purity of 3-butyleneglycol is about 2-10%.Work as R, R-2,3-butyleneglycol is during for quiral products synthetic, and it should reach more than 99.9% superelevation optical purity general requirement, otherwise will be not suitable for preparing chiral material.By optimization for fermentation technology condition, although can improve R, R-2, the optical purity of 3-butyleneglycol, optical purity still difficulty reaches more than 99.9% this standard, and usually causes R, R-2,3-butyleneglycol production declining.And extract R by chiral separation method, and, during 3-butyleneglycol, there is complex operation in R-2, and difficulty is large, and high in cost of production defect causes R, R-2, the market value of 3-butyleneglycol is high, has greatly limited R, R-2, the industrial applications of 3-butyleneglycol.
Summary of the invention
Goal of the invention: in order to overcome the deficiencies in the prior art, first technical problem to be solved by this invention is to provide a kind of product superelevation optical purity (>=99.9%) R, R-2,3-butyleneglycol genetic engineering bacterium.
Second technical problem to be solved by this invention is to provide above-mentioned product superelevation optical purity R, R-2, the construction process of 3-butyleneglycol genetic engineering bacterium.
The technical problem that the present invention finally will solve is to provide above-mentioned product superelevation optical purity R, R-2, the application of 3-butyleneglycol genetic engineering bacterium.
Technical scheme: for solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of product superelevation optical purity R, R-2,3-butyleneglycol genetic engineering bacterium, this project bacterium is to be built and obtained by following method: utilize round pcr from Paenibacillus polymyxa CGMCC3044, to amplify dimethyl diketone reductase gene dar, 5 ' end 39bp base sequence forms homology arm I, 3 ' end 20bp base sequence forms homology arm II, by multiple clone site, homology arm I and homology arm II is connected in series to respectively to oriT replication initiation sequence and the apramycin resistant gene Apra that carries λ Red homologous recombination sequence clone clay SuperCos-1/pIJ790 rthe two ends of sequence, are contained " dar homology arm I-oriT-Apra r-dar homology arm II " recombinant cosmid " the SuperCos-1/pIJ790-dar homology arm I-oriT-Apra of sequence r-dar homology arm II "; recombinant cosmid transforms Paenibacillus polymyxa CGMCC3044; and there is homologous recombination double exchange with Paenibacillus polymyxa CGMCC3044 under λ Red mediation; obtain the Paenibacillus polymyxa of restructuring; produce superelevation optical purity R; R-2,3-butyleneglycol genetic engineering bacterium CGMCC3044-dar -.
Wherein, in SuperCos-1/pIJ790, SuperCos-1 is clay; Plasmid pIJ790 carries λ Red homologous recombination sequence, oriT replication initiation sequence and apramycin resistant gene Apra retc. sequence.
This genetic engineering bacterium is built and is obtained by following method:
(1) clone of gene dar: the synthetic required primer of PCR of the sequences Design of dar gene in the Paenibacillus polymyxa ATCC12321 announcing according to Genebank:
P1：5’-ATGGAACTTAAAAACAAAACAGCC-3’
P2：5’-CTGTGGGTTGGTTGTCCAAAT-3’
The Paenibacillus polymyxa CGMCC3044 genome of take reacts as template completes PCR: PCR reaction conditions: 94 ℃ of sex change 5min, through 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 1min, totally 32 circulations, through 72 ℃, extend 10min again, the PCR product of acquisition is confirmed through electrophoretic analysis, after PCR product purification test kit purifying, be connected with pMD18-T carrier, carry out sequencing, obtain the dimethyl diketone reductase gene that derives from Paenibacillus polymyxa CGMCC3044, i.e. dar gene;
(2) recombinant cosmid " SuperCos-1/pIJ790-dar homology arm I-oriT-Apra r-dar homology arm II " structure: EcoR I is introduced respectively at the two ends at 5 ' the end 39bp base sequence of dar, and Xho I, obtains dar homology arm I; At 3 ' of dar, hold the two ends of 20bp base sequence to introduce respectively Not I, BamH I, obtains dar homology arm II; Above-mentioned dar homology arm I and dar homology arm II are connected to respectively to oriT replication initiation sequence and the apramycin resistant gene Apra of the clone's clay SuperCos-1/pIJ790 that carries λ Red homologous recombination sequence rthe two ends of sequence, are contained " dar homology arm I-oriT-Apra r-dar homology arm II " the recombinant cosmid SuperCos-1/pIJ790 of sequence;
(3) acquisition of recombination engineering bacteria: by recombinant cosmid " SuperCos-1/pIJ790-dar homology arm I-oriT-Apra r-dar homology arm II " transform Paenibacillus polymyxa CGMCC3044, coating is dull and stereotyped containing 40 μ g/mL apramycins, picking positive recombinant, and carry out bacterium colony PCR evaluation, obtain the Paenibacillus polymyxa of recombinating, called after CGMCC3044-dar -.
Wherein, the method comprises the following steps: to utilize round pcr from Paenibacillus polymyxa CGMCC3044, to amplify dimethyl diketone reductase gene dar, 5 ' end 39bp base sequence forms homology arm I, 3 ' end 20bp base sequence forms homology arm II, by multiple clone site, homology arm I and homology arm II is connected in series to respectively to oriT replication initiation sequence and the apramycin resistant gene Apra that carries λ Red homologous recombination sequence clone clay SuperCos-1/pIJ790 rthe two ends of sequence, are contained " dar homology arm I-oriT-Apra r-dar homology arm II " the recombinant cosmid SuperCos-1/pIJ790 of sequence; recombinant cosmid transforms CGMCC3044; and under λ Red mediation with the double exchange of Host Strains generation homologous recombination; obtain the Paenibacillus polymyxa of restructuring; produce superelevation optical purity R; R-2,3-butyleneglycol genetic engineering bacterium CGMCC3044-dar -.
Wherein, above-mentioned product superelevation optical purity R, R-2, the construction process of 3-butyleneglycol genetic engineering bacterium comprises the following steps: the clone of (1) gene dar: the synthetic required primer of PCR of the sequences Design of dar gene in the Paenibacillus polymyxa ATCC12321 announcing according to Genebank:
P1：5’-ATGGAACTTAAAAACAAAACAGCC-3’
P2：5’-CTGTGGGTTGGTTGTCCAAAT-3’
The Paenibacillus polymyxa CGMCC3044 genome of take reacts as template completes PCR: PCR reaction conditions: 94 ℃ of sex change 5min, through 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 1min, totally 32 circulations, through 72 ℃, extend 10min again, the PCR product of acquisition is confirmed through electrophoretic analysis, after PCR product purification test kit purifying, be connected with pMD18-T carrier, carry out sequencing, obtain the dimethyl diketone reductase gene that derives from Paenibacillus polymyxa CGMCC3044, i.e. dar gene;
(2) recombinant cosmid " SuperCos-1/pIJ790-dar homology arm I-oriT-Apra r-dar homology arm II " structure: EcoR I is introduced respectively at the two ends at 5 ' the end 39bp base sequence of dar, and Xho I, obtains dar homology arm I; At 3 ' of dar, hold the two ends of 20bp base sequence to introduce respectively Not I, BamH I, obtains dar homology arm II; Above-mentioned dar homology arm I and dar homology arm II are connected to respectively to oriT replication initiation sequence and the apramycin resistant gene Apra of the clone's clay SuperCos-1/pIJ790 that carries λ Red homologous recombination sequence rthe two ends of sequence, are contained " dar homology arm I-oriT-Apra r-dar homology arm II " the recombinant cosmid SuperCos-1/pIJ790 of sequence;
(3) acquisition of recombination engineering bacteria: by recombinant cosmid " SuperCos-1/pIJ790-dar homology arm I-oriT-Apra r-dar homology arm II " transform Paenibacillus polymyxa CGMCC3044, coating is dull and stereotyped containing 40 μ g/mL apramycins, picking positive recombinant, and carry out bacterium colony PCR evaluation, obtain the Paenibacillus polymyxa of recombinating, called after CGMCC3044-dar -.
Above-mentioned product superelevation optical purity R, R-2,3-butyleneglycol genetic engineering bacterium CGMCC3044-dar -producing R, R-2, the application in 3-butyleneglycol.
Above-mentioned application, by the genetic engineering bacterium CGMCC3044-dar building -be inoculated in the aseptic culture medium of carbonaceous sources, nitrogenous source and inorganic salt and cultivate, fermentative production superelevation optical purity R, R-2,3-butyleneglycol, wherein, described carbon source is the jerusalem artichoke inulin crude extract without any hydrolysis treatment.
Above-mentioned application, this genetic engineering bacterium directly be take jerusalem artichoke inulin crude extract as substrate production superelevation optical purity R, R-2,3-butyleneglycol, concrete production technique is:
(1) starting strain: restructuring Paenibacillus polymyxa CGMCC3044-dar -;
(2) seed culture medium: (NH 4) 2hPO 40.5-2.0g/L, KCl0.10-0.3g/L, MgSO 4.7H 2o0.1-0.3g/L, yeast extract paste 0.1-0.3g/L, glucose 2.0-8.0g/L, jerusalem artichoke inulin crude extract is containing inulin 1.0-3.0g/L, apramycin 20-40 μ g/mL;
Seed culture: 1000mL triangular flask, liquid amount 200mL, culture temperature 30-37 ℃, shaking speed 120-200rpm, cultivates 12-36h;
(3) fermentation culture: substratum forms: jerusalem artichoke inulin crude extract is containing inulin 20.0-60.0g/L, (NH 4) 2hPO 40.5-2.0g/L, KCl0.10-0.3g/L, MgSO 4.7H 2o0.1-0.3g/L, yeast extract paste 3.0-8.0g/L, apramycin 20-40 μ g/mL;
Culture condition: inoculum size 10v/v%, leavening temperature 30-37 ℃, pH6.0-8.0, control the intake of oxygen, 24h before inoculation secondary fermentation, controls rotating speed 240-320rpm, after fermentation 24h, control rotating speed 80-160rpm, maintain micro-oxygen environment fermentation, shake flask fermentation, or batch fermentation, or fed-batch fermentation, flow feeding inulin maintains inulin concentration at 30.0 ± 1.0g/L, and after feed supplement, substrate is total to about 60-120g/L.
Wherein, the microorganism strains of the seed selection of inventor laboratory preservation is for producing bacillus polymyxa (paenibacillus polymyxa) the CGMCC No.3044 of inulinase, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) at present, depositary institution address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, the numbering of registering on the books is CGMCC No.3044, and preservation date is: on April 29th, 2009.Using this bacterium as starting strain.The applying date of the corresponding patent of this bacterial strain is on June 1st, 2009, and the patent No. is ZL200910026807.1.
Beneficial effect:
The invention reside in a kind of novel gene engineering bacteria is provided, be used for fermenting and produce superelevation optical purity (>=99.9%) R, R-2,3-butyleneglycol, this bacterium directly utilizes undressed jerusalem artichoke inulin crude extract high-efficiency fermenting to produce superelevation optical purity R under micro-oxygen condition, R-2,3-butyleneglycol, has broken through traditional zymotic and has been difficult to obtain superelevation optical purity R, R-2,3-butyleneglycol and inefficient production superelevation optical purity R, R-2, the limitation of 3-butyleneglycol.Research is found metabolism to synthesize R, R-2, the bacterial classification of 3-butyleneglycol is to be that carbonyl compound is progressively reduced into R by precursor compound by oxydo-reductase in thalline, R-2, in 3-butyleneglycol (optical purity is about 90-98%) process, always follow synthesizing optical purity to be about the by product meso-2 of 2-10%, 3-butyleneglycol, by controlling dissolved oxygen, add reducing substances, regulate the optimization for fermentation technology conditions such as pH, R, R-2, 3-butyleneglycol still difficulty reaches 99.9% above superelevation optical purity, thereby cannot be for the preparation of quiral products such as chiral liquid crystal materials, and further use chiral separation legal system for R, R-2, 3-butyleneglycol, there is complex operation, difficulty is large, cost is high, the defect such as yield poorly, above-mentioned factor causes R, R-2, the market value of 3-butyleneglycol is high, greatly limited R, R-2, the industrial applications of 3-butyleneglycol.Compare with original Paenibacillus polymyxa, R in recombinant bacterial strain fermented liquid, R-2, the optical purity of 3-butyleneglycol can reach 100%, and output is also significantly improved.With after fermentation that current document is reported, utilize chiral separation production superelevation optical purity R, R-2,3-butyleneglycol method is compared, not only recombinant bacterium produces superelevation optical purity R, R-2,3-butyleneglycol stability and production concentration are all higher, and without separation means such as chiral separation, can obtain 100% superelevation optical purity R after fermentation, R-2,3-butyleneglycol, has lowered significantly separation costs and has improved productivity effect, is microbe fermentation method suitability for industrialized production superelevation optical purity R, R-2,3-butyleneglycol is had laid a good foundation.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand, the described content of embodiment is only for the present invention is described, and should also can not limit the present invention described in detail in claims.
Embodiment 1:
The preparation method of jerusalem artichoke inulin crude extract:
Fresh jerusalem artichoke is cleaned peeling, blanching go out enzyme (100 ℃, thinly slice after 15min), forced air drying at 70 ℃, then pulverized 80 mesh sieves and made jerusalem artichoke meal, Refrigerator store is standby.After taking jerusalem artichoke meal and put into water and stir according to the ratio of 1:6,70 ℃ of heating in water bath lixiviate 4h, with milk of lime, regulating pH is 9,80 ℃ of water bath heat preservation 1h then, with getting final product to obtain jerusalem artichoke inulin crude extract after filtered through gauze.
Embodiment 2: the clone of gene dar:
The synthetic required primer of PCR of the sequences Design of dar gene in Paenibacillus polymyxa (paenibacillus polymyxa) ATCC12321 announcing according to Genebank:
P1：5’-ATGGAACTTAAAAACAAAACAGCC-3’
P2：5’-CTGTGGGTTGGTTGTCCAAAT-3’
The Paenibacillus polymyxa CGMCC3044 genome of take reacts as template completes PCR; PCR reaction conditions: 94 ℃ of sex change 5min, 94 ℃ of 30s; 56 ℃ of 30s; 72 ℃ of 1min totally 32 circulations, 72 ℃ are extended 10min, and the PCR product of acquisition is confirmed through electrophoretic analysis, after PCR product purification test kit purifying, be connected with pMD18-T carrier, carry out sequencing, obtain the dimethyl diketone reductase gene dar that derives from Paenibacillus polymyxa CGMCC3044.
Embodiment 3: recombinant cosmid " SuperCos-1/pIJ790-dar homology arm I-oriT-Apra r-dar homology arm II " structure:
At 5 ' of dar, hold the two ends of about 39bp base sequence to introduce respectively EcoR I, Xho I, obtains dar homology arm I; At 3 ' of dar, hold the two ends of about 20bp base sequence to introduce respectively Not I, BamH I, obtains dar homology arm II.According to described restriction enzyme site, above-mentioned dar homology arm I and dar homology arm II are connected to respectively to the oriT replication initiation sequence and the apramycin resistant gene Apra that are connected to clone's clay SuperCos-1/pIJ790 that carries λ Red homologous recombination sequence rthe two ends of sequence, are contained " dar homology arm I-oriT-Apra r-dar homology arm II " recombinant cosmid " the SuperCos-1/pIJ790-dar homology arm I-oriT-Apra of sequence and λ Red sequence r-dar homology arm II ".
Embodiment 4: recombinant cosmid " SuperCos-1/pIJ790-dar homology arm I-oriT-Apra r-dar homology arm II " conversion Paenibacillus polymyxa CGMCC3044.
By recombinant cosmid " SuperCos-1/pIJ790-dar homology arm I-oriT-Apra r-dar homology arm II " transform Paenibacillus polymyxa CGMCC3044, coating is dull and stereotyped containing 40 μ g/mL apramycins, picking positive recombinant, and carry out bacterium colony PCR evaluation, obtain the Paenibacillus polymyxa of recombinating, called after CGMCC3044-dar -.
Embodiment 5: restructuring Paenibacillus polymyxa CGMCC3044-dar -middle dimethyl diketone reductase enzyme enzyme activity determination
(1) starting strain: restructuring Paenibacillus polymyxa CGMCC3044-dar -
(2) used medium (g/L): peptone 10, yeast extract paste 5, sodium-chlor 10, apramycin 40 μ g/mL, pH6.0
(3) enzyme activity determination method: the primitive reaction system of enzyme activity determination (200 μ L) forms: 200mmol/L potassiumphosphate pH6.0177 μ L, enzyme liquid 10 μ L, 5.3mmol/L dimethyl diketone 10 μ L, 14.2mmol/L NADPH (NADP +) 3 μ L, after 37 ℃ of reaction 5min, measure the velocity of variation of 340nm place absorbancy in 1min.An enzyme activity unit (U) is defined as the μ moL quantity that 1min NADPH reduces (increase).Being prepared as follows of enzyme extract: will be resuspended in pH8.050mmol/L potassiumphosphate after physiological saline for thalline (0.85%NaCl) washed twice of the centrifugal gained of fermented liquid; at 300W; 30min; ultrasonic 2s; stop 4s and carry out ultrasonication; ice bath protection, 8500rpm low-temperature centrifugation 30min, supernatant liquor is enzyme extract.Protein concentration detects by Bradford method, usings bovine serum albumin as standard substance.
Investigate result: restructuring Paenibacillus polymyxa CGMCC3044-dar -the work of middle dimethyl diketone reductase enzyme enzyme is 0.
Comparative example 1: dimethyl diketone reductase enzyme enzyme activity determination in original strain Paenibacillus polymyxa CGMCC3044
(1) starting strain: Paenibacillus polymyxa CGMCC3044
(2) used medium (g/L): peptone 10, yeast extract paste 5, sodium-chlor 10, pH6.0
(3) enzyme activity determination method: the primitive reaction system of enzyme activity determination (200 μ L) forms: 200mmol/L potassiumphosphate pH6.0177 μ L, enzyme liquid 10 μ L, 5.3mmol/L dimethyl diketone 10 μ L, 14.2mmol/L NADPH (NADP +) 3 μ L, after 37 ℃ of reaction 5min, measure the velocity of variation of 340nm place absorbancy in 1min.An enzyme activity unit (U) is defined as the μ moL quantity that 1min NADPH reduces (increase).Being prepared as follows of enzyme extract: will be resuspended in pH8.050mmol/L potassiumphosphate after physiological saline for thalline (0.85%NaCl) washed twice of the centrifugal gained of fermented liquid; at 300W; 30min; ultrasonic 2s; stop 4s and carry out ultrasonication; ice bath protection, 8500rpm low-temperature centrifugation 30min, supernatant liquor is enzyme extract.Protein concentration detects by Bradford method, usings bovine serum albumin as standard substance.
Investigate result: in original strain Paenibacillus polymyxa CGMCC3044, dimethyl diketone reductase enzyme enzyme is lived as 0.239U/mg.
Embodiment 6: restructuring Paenibacillus polymyxa CGMCC3044-dar -directly utilize jerusalem artichoke inulin crude extract to ferment in shaking flask and produce superelevation optical purity R, R-2,3-butyleneglycol
(1) starting strain: paenibacillus polymyxa CGMCC3044-dar -;
(2) seed culture medium (g/L): (NH 4) 2hPO 41.0, KCl0.2, MgSO 4.7H 2o0.1, yeast extract paste 0.2, glucose 5.0, jerusalem artichoke inulin crude extract is containing inulin 2.0, apramycin 30 μ g/mL;
Seed culture: 1000mL triangular flask, liquid amount 200mL, 30 ℃ of culture temperature, shaking speed 120rpm, cultivates 22h;
(3) fermentation culture: substratum forms (g/L): inulin crude extract is containing inulin 60.0, (NH 4) 2hPO 41.0, KCl0.10, MgSO 4.7H 2o0.1, yeast extract paste 6.0, apramycin 30 μ g/mL;
Culture condition: inoculum size 10v/v%, 30 ℃ of leavening temperatures, pH6.0, the intake of control oxygen, 24h before inoculation secondary fermentation, controls rotating speed 240rpm, and after fermentation 24h, control rotating speed 160rpm, maintains micro-oxygen environment fermentation, fermentation time 42h.
Fermentation results: containing the inulin of 60.0g/L, obtain the R of 24.4g/L through fermentation, R-2,3-butyleneglycol, optical purity 100% in jerusalem artichoke inulin crude extract.
Comparative example 2: original bacterium Paenibacillus polymyxa CGMCC directly utilizes jerusalem artichoke inulin crude extract to ferment in shaking flask and produces R, R-2,3-butyleneglycol
(1) starting strain: Paenibacillus polymyxa CGMCC3044;
(2) seed culture medium (g/L): (NH 4) 2hPO 41.0, KCl0.2, MgSO 4.7H 2o0.1, yeast extract paste 0.2, glucose 5.0, inulin 2.0;
Seed culture: 1000mL triangular flask, liquid amount 200mL, 30 ℃ of culture temperature, shaking speed 120rpm, cultivates 22h;
(3) fermentation culture: substratum forms (g/L): inulin crude extract is containing inulin 60.0, (NH 4) 2hPO 41.0, KCl0.10, MgSO 4.7H 2o0.1, yeast extract paste 6.0;
Culture condition: inoculum size 10v/v%, 30 ℃ of leavening temperatures, pH6.0, the intake of control oxygen, shaking speed 120rpm, maintains micro-oxygen environment fermentation, fermentation time 42h.
Fermentation results: containing the inulin of 60.0g/L, obtain the R of 20.7g/L through fermentation, R-2,3-butyleneglycol, optical purity 98.0% in jerusalem artichoke inulin crude extract.
Embodiment 7: restructuring Paenibacillus polymyxa CGMCC3044-dar -directly utilize jerusalem artichoke inulin crude extract batch fermentation production superelevation optical purity R in 5L fermentor tank, R-2,3-butyleneglycol
(1) starting strain: restructuring Paenibacillus polymyxa CGMCC3044-dar -;
(2) seed culture medium (g/L): (NH 4) 2hPO 41.0, KCl0.2, MgSO 4.7H 2o0.1, yeast extract paste 0.2, glucose 5.0, inulin 2.0, apramycin 30 μ g/mL;
Seed culture: 1000mL triangular flask, liquid amount 200mL, 30 ℃ of culture temperature, shaking speed 120rpm, cultivates 22h;
(3) fermentation culture: substratum forms (g/L): yeast extract paste 6.0, (NH 4) 2hPO 41.0, KCl0.10, MgSO 4.7H 2o0.1, inulin crude extract is containing inulin 60.0, apramycin 30 μ g/mL;
Culture condition: 5L fermentor tank (New Brunswick Scientific, NJ), inoculum size 10v/v%, 30 ℃ of leavening temperatures, pH6.0, control the intake of oxygen, 24h before inoculation secondary fermentation, controls rotating speed 240rpm, after fermentation 24h, control rotating speed 120rpm, maintain micro-oxygen environment fermentation, fermentation time 42 ± 1h.
Fermentation results: substrate is about the inulin of 60.0g/L, obtains the R of 26.2g/L, R-2,3-butyleneglycol, optical purity 100% through fermentation.
Comparative example 3: original Paenibacillus polymyxa CGMCC3044 directly utilizes jerusalem artichoke inulin crude extract batch fermentation in 5L fermentor tank to produce R, R-2,3-butyleneglycol
(1) starting strain: Paenibacillus polymyxa CGMCC3044;
(2) seed culture medium (g/L): (NH 4) 2hPO 41.0, KCl0.2, MgSO 4.7H 2o0.1, yeast extract paste 0.2, glucose 5.0, inulin 2.0;
Seed culture: 1000mL triangular flask, liquid amount 200mL, 30 ℃ of culture temperature, shaking speed 120rpm, cultivates 22h;
(3) fermentation culture: substratum forms (g/L): yeast extract paste 6.0, (NH 4) 2hPO 41.0, KCl0.10, MgSO 4.7H 2o0.1, inulin crude extract is containing inulin 60.0;
Culture condition: 5L fermentor tank (New Brunswick Scientific, NJ), inoculum size 10v/v%, 30 ℃ of leavening temperatures, pH6.0, the intake of control oxygen, 24h before inoculation secondary fermentation, control rotating speed 240rpm, after fermentation 24h, control rotating speed 120rpm, maintain micro-oxygen environment fermentation, fermentation time 42 ± 1h.
Fermentation results: substrate is about the inulin of 60.0g/L, obtains the R of 22.1g/L, R-2,3-butyleneglycol, optical purity 98.8% through fermentation.
Embodiment 8 restructuring Paenibacillus polymyxa CGMCC3044-dar -directly utilize jerusalem artichoke inulin crude extract fed-batch fermentation production superelevation optical purity R in 5L fermentor tank, R-2,3-butyleneglycol
(1) starting strain: restructuring Paenibacillus polymyxa CGMCC3044-dar -;
(2) seed culture medium (g/L): (NH 4) 2hPO 40.5, KCl0.1, MgSO 4.7H 2o0.3, yeast extract paste 0.1, glucose 2.0, inulin 1.0, apramycin 30 μ g/mL;
Seed culture: 1000mL triangular flask, liquid amount 200mL, 37 ℃ of culture temperature, shaking speed 200rpm, cultivates 12h;
(3) fermentation culture: substratum forms (g/L): yeast extract paste 3.0, (NH 4) 2hPO 40.5, KCl0.2, MgSO 4.7H 2o0.2, inulin crude extract is containing inulin 20.0, apramycin 20 μ g/mL;
Culture condition: 5L fermentor tank (New Brunswick Scientific, NJ), inoculum size 10v/v%, 37 ℃ of leavening temperatures, pH8.0, the intake of control oxygen, 24h before inoculation secondary fermentation, control rotating speed 320rpm, after fermentation 24h, control rotating speed 80rpm, maintain micro-oxygen environment fermentation, when inulin residual concentration is about 5g/L, start flow feeding, feed supplement stream adds inulin and maintains inulin concentration at 30.0 ± 1.0g/L, fermentation time 72 ± 1h.
Fermentation results: after feed supplement, substrate is about the inulin of 60g/L altogether, obtains the R of 28.6g/L, R-2,3-butyleneglycol, optical purity 100% through fermentation.
Comparative example 4: original bacterium Paenibacillus polymyxa CGMCC3044 directly utilizes jerusalem artichoke inulin crude extract fed-batch fermentation in 5L fermentor tank to produce R, R-2,3-butyleneglycol
(1) starting strain: Paenibacillus polymyxa CGMCC3044;
(2) seed culture medium (g/L): (NH 4) 2hPO 40.5, KCl0.1, MgSO 4.7H 2o0.3, yeast extract paste 0.1, glucose 2.0, inulin 1.0;
Seed culture: 1000mL triangular flask, liquid amount 200mL, 37 ℃ of culture temperature, shaking speed 200rpm, cultivates 12h;
(3) fermentation culture: substratum forms (g/L): yeast extract paste 3.0, (NH 4) 2hPO 40.5, KCl0.2, MgSO 4.7H 2o0.2, inulin crude extract is containing inulin 20.0;
Culture condition: 5L fermentor tank (New Brunswick Scientific, NJ), inoculum size 10v/v%, 37 ℃ of leavening temperatures, pH8.0, the intake of control oxygen, 24h before inoculation secondary fermentation, control rotating speed 320rpm, after fermentation 24h, control rotating speed 80rpm, maintain micro-oxygen environment fermentation, when inulin residual concentration is about 5g/L, start flow feeding, feed supplement stream adds inulin and maintains inulin concentration at 30.0 ± 1.0g/L, fermentation time 72 ± 1h.
Fermentation results: after feed supplement, substrate is about the inulin of 60g/L altogether, obtains the R of 24.3g/L, R-2,3-butyleneglycol, optical purity 98.9% through fermentation.
Embodiment 9 restructuring Paenibacillus polymyxa CGMCC3044-dar -directly utilize jerusalem artichoke inulin crude extract fed-batch fermentation production superelevation optical purity R in 5L fermentor tank, R-2,3-butyleneglycol
(1) starting strain: restructuring Paenibacillus polymyxa CGMCC3044-dar -;
(2) seed culture medium (g/L): (NH 4) 2hPO 42.0, KCl0.3, MgSO 4.7H 2o0.2, yeast extract paste 0.3, glucose 8.0, inulin 3.0, apramycin 40 μ g/mL;
Seed culture: 1000mL triangular flask, liquid amount 200mL, 33 ℃ of culture temperature, shaking speed 160rpm, cultivates 36h;
(3) fermentation culture: substratum forms (g/L): yeast extract paste 8.0, (NH 4) 2hPO 42.0, KCl0.3, MgSO 4.7H 2o0.3, inulin crude extract is containing inulin 60.0, apramycin 40 μ g/mL;
Culture condition: 5L fermentor tank (New Brunswick Scientific, NJ), inoculum size 10v/v%, 33 ℃ of leavening temperatures, pH8.0, the intake of control oxygen, 24h before inoculation secondary fermentation, control rotating speed 280rpm, after fermentation 24h, control rotating speed 160rpm, maintain micro-oxygen environment fermentation, feed supplement stream adds inulin and maintains inulin concentration at 30.0 ± 1.0g/L, fermentation time 96 ± 1h.
Fermentation results: after feed supplement, substrate is about the inulin of 120g/L altogether, obtains the R of 58.5g/L, R-2,3-butyleneglycol, optical purity 100% through fermentation.
Comparative example 5: original bacterium Paenibacillus polymyxa CGMCC3044 directly utilizes jerusalem artichoke inulin crude extract fed-batch fermentation in 5L fermentor tank to produce R, R-2,3-butyleneglycol
(1) starting strain: Paenibacillus polymyxa CGMCC3044;
(2) seed culture medium (g/L): (NH 4) 2hPO 42.0, KCl0.3, MgSO 4.7H 2o0.2, yeast extract paste 0.3, glucose 8.0, inulin 3.0;
Seed culture: 1000mL triangular flask, liquid amount 200mL, 33 ℃ of culture temperature, shaking speed 160rpm, cultivates 36h;
(3) fermentation culture: substratum forms (g/L): yeast extract paste 8.0, (NH 4) 2hPO 42.0, KCl0.3, MgSO 4.7H 2o0.3, inulin crude extract is containing inulin 60.0;
Culture condition: 5L fermentor tank (New Brunswick Scientific, NJ), inoculum size 10v/v%, 33 ℃ of leavening temperatures, pH8.0, the intake of control oxygen, 24h before inoculation secondary fermentation, control rotating speed 280rpm, after fermentation 24h, control rotating speed 160rpm, maintain micro-oxygen environment fermentation, feed supplement stream adds inulin and maintains inulin concentration at 30.0 ± 1.0g/L, fermentation time 96 ± 1h.
Fermentation results: after feed supplement, substrate is about the inulin of 120g/L altogether, through the R of 44.8g/L of fermenting to obtain, R-2,3-butyleneglycol, optical purity 98.6%.
Figure IDA0000457192830000011
Figure IDA0000457192830000031
Figure IDA0000457192830000041

Claims (7)

1. one kind is produced superelevation optical purity R, R-2,3-butyleneglycol genetic engineering bacterium, this project bacterium is to be built and obtained by following method: utilize round pcr from Paenibacillus polymyxa CGMCC3044, to amplify dimethyl diketone reductase gene dar, 5 ' end 39bp base sequence forms homology arm I, 3 ' end 20bp base sequence forms homology arm II, by multiple clone site, homology arm I and homology arm II is connected in series to respectively to oriT replication initiation sequence and the apramycin resistant gene Apra that carries λ Red homologous recombination sequence clone clay SuperCos-1/pIJ790 rthe two ends of sequence, obtain containing dar homology arm I-oriT-Apra rthe recombinant cosmid SuperCos-1/pIJ790-dar homology arm I-oriT-Apra of-dar homology arm II sequence r-dar homology arm II, recombinant cosmid transforms Paenibacillus polymyxa CGMCC3044, and there is homology double exchange with Paenibacillus polymyxa CGMCC3044 under λ Red mediation, obtain the Paenibacillus polymyxa of restructuring, produce superelevation optical purity R, R-2,3-butyleneglycol genetic engineering bacterium CGMCC3044-dar -.
2. product superelevation optical purity R according to claim 1, R-2,3-butyleneglycol genetic engineering bacterium, is characterized in that: this project bacterium is built and obtained by following method:
(1) clone of gene dar: the synthetic required primer of PCR of the sequences Design of dar gene in the Paenibacillus polymyxa ATCC12321 announcing according to Genebank:
P1：5’-ATGGAACTTAAAAACAAAACAGCC-3’
P2：5’-CTGTGGGTTGGTTGTCCAAAT-3’
The Paenibacillus polymyxa CGMCC3044 genome of take reacts as template completes PCR: PCR reaction conditions: 94 ℃ of sex change 5min, through 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 1min, totally 32 circulations, through 72 ℃, extend 10min again, the PCR product of acquisition is confirmed through electrophoretic analysis, after PCR product purification test kit purifying, be connected with pMD18-T carrier, carry out sequencing, obtain the dimethyl diketone reductase gene that derives from Paenibacillus polymyxa CGMCC3044, i.e. dar gene;
(2) recombinant cosmid SuperCos-1/pIJ790-dar homology arm I-oriT-Apra rthe structure of-dar homology arm II: hold the two ends of 39bp base sequence to introduce respectively EcoR I at 5 ' of dar, Xho I, obtains dar homology arm I; At 3 ' of dar, hold the two ends of 20bp base sequence to introduce respectively Not I, BamH I, obtains dar homology arm II; Above-mentioned dar homology arm I and dar homology arm II are connected to respectively to oriT replication initiation sequence and the apramycin resistant gene Apra of the clone's clay SuperCos-1/pIJ790 that carries λ Red homologous recombination sequence rthe two ends of sequence, obtain containing dar homology arm I-oriT-Apra rthe recombinant cosmid SuperCos-1/pIJ790-dar homology arm I-oriT-Apra of-dar homology arm II sequence and λ Red sequence r-dar homology arm II;
(3) acquisition of recombination engineering bacteria: by recombinant cosmid SuperCos-1/pIJ790-dar homology arm I-oriT-Apra r-dar homology arm II transforms Paenibacillus polymyxa CGMCC3044, and coating is dull and stereotyped containing 40 μ g/mL apramycins, picking positive recombinant, and carry out bacterium colony PCR evaluation, obtain the Paenibacillus polymyxa of recombinating, called after CGMCC3044-dar -.
3. product R claimed in claim 1, R-2, the construction process of 3-butyleneglycol genetic engineering bacterium, it is characterized in that: the method comprises the following steps: to utilize round pcr from Paenibacillus polymyxa CGMCC3044, to amplify dimethyl diketone reductase gene dar, 5 ' end 39bp base sequence forms homology arm I, 3 ' end 20bp base sequence forms homology arm II, by multiple clone site, homology arm I and homology arm II is connected in series to respectively to oriT replication initiation sequence and the apramycin resistant gene Apra that carries λ Red homologous recombination sequence clone clay SuperCos-1/pIJ790 rthe two ends of sequence, obtain containing dar homology arm I-oriT-Apra rthe recombinant cosmid SuperCos-1/pIJ790 of-dar homology arm II sequence, recombinant cosmid transforms Paenibacillus polymyxa CGMCC3044, and under λ Red mediation with the double exchange of Host Strains generation homologous recombination, obtain the Paenibacillus polymyxa of restructuring, produce superelevation optical purity R, R-2,3-butyleneglycol genetic engineering bacterium CGMCC3044-dar -.
4. product superelevation optical purity R according to claim 3, R-2, the construction process of 3-butyleneglycol genetic engineering bacterium, is characterized in that: the clone who comprises the following steps: (1) gene dar: the synthetic required primer of PCR of the sequences Design of dar gene in the Paenibacillus polymyxa ATCC12321 announcing according to Genebank:
P1：5’-ATGGAACTTAAAAACAAAACAGCC-3’
P2：5’-CTGTGGGTTGGTTGTCCAAAT-3’
The Paenibacillus polymyxa CGMCC3044 genome of take reacts as template completes PCR: PCR reaction conditions: 94 ℃ of sex change 5min, through 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 1min, totally 32 circulations, through 72 ℃, extend 10min again, the PCR product of acquisition is confirmed through electrophoretic analysis, after PCR product purification test kit purifying, be connected with pMD18-T carrier, carry out sequencing, obtain the dimethyl diketone reductase gene that derives from Paenibacillus polymyxa CGMCC3044, i.e. dar gene;
(2) recombinant cosmid SuperCos-1/pIJ790-dar homology arm I-oriT-Apra rthe structure of-dar homology arm II: hold the two ends of 39bp base sequence to introduce respectively EcoR I at 5 ' of dar, Xho I, obtains dar homology arm I; At 3 ' of dar, hold the two ends of 20bp base sequence to introduce respectively Not I, BamH I, obtains dar homology arm II; Above-mentioned dar homology arm I and dar homology arm II are connected to respectively to oriT replication initiation sequence and the apramycin resistant gene Apra of the clone's clay SuperCos-1/pIJ790 that carries λ Red homologous recombination sequence rthe two ends of sequence, obtain containing dar homology arm I-oriT-Apra rthe recombinant cosmid SuperCos-1/pIJ790-dar homology arm I-oriT-Apra of-dar homology arm II sequence and λ Red sequence r-dar homology arm II;
(3) acquisition of recombination engineering bacteria: by recombinant cosmid SuperCos-1/pIJ790-dar homology arm I-oriT-Apra r-dar homology arm II transforms Paenibacillus polymyxa CGMCC3044, and coating is dull and stereotyped containing 40 μ g/mL apramycins, picking positive recombinant, and carry out bacterium colony PCR evaluation, obtain the Paenibacillus polymyxa of recombinating, called after CGMCC3044-dar -.
5. product superelevation optical purity R claimed in claim 1, R-2,3-butyleneglycol genetic engineering bacterium CGMCC3044-dar -producing R, R-2, the application in 3-butyleneglycol.
6. application according to claim 5, is characterized in that: by the genetic engineering bacterium CGMCC3044-dar building -be inoculated in the aseptic culture medium of carbonaceous sources, nitrogenous source and inorganic salt and cultivate, fermentative production superelevation optical purity R, R-2,3-butyleneglycol, wherein, described carbon source is the jerusalem artichoke inulin crude extract without any hydrolysis treatment.
7. application according to claim 5, is characterized in that: this genetic engineering bacterium directly be take jerusalem artichoke inulin crude extract as substrate production superelevation optical purity R, R-2, and 3-butyleneglycol, concrete production technique is:
(1) starting strain: restructuring Paenibacillus polymyxa CGMCC3044-dar -;
(2) seed culture medium: (NH 4) 2hPO 40.5-2.0g/L, KCl0.10-0.3g/L, MgSO 4.7H 2o0.1-0.3g/L, yeast extract paste 0.1-0.3g/L, glucose 2.0-8.0g/L, jerusalem artichoke inulin crude extract is containing inulin 1.0-3.0g/L, apramycin 20-40 μ g/mL;
Seed culture: 1000mL triangular flask, liquid amount 200mL, culture temperature 30-37 ℃, shaking speed 120-200rpm, cultivates 12-36h;
(3) fermentation culture: substratum forms: jerusalem artichoke inulin crude extract is containing inulin 20.0-60.0g/L, (NH 4) 2hPO 40.5-2.0g/L, KCl0.10-0.3g/L, MgSO 4.7H 2o0.1-0.3g/L, yeast extract paste 3.0-8.0g/L, apramycin 20-40 μ g/mL;
Culture condition: inoculum size 10v/v%, leavening temperature 30-37 ℃, pH6.0-8.0, control the intake of oxygen, 24h before inoculation secondary fermentation, controls rotating speed 240-320rpm, after fermentation 24h, control rotating speed 80-160rpm, maintain micro-oxygen environment fermentation, shake flask fermentation, or batch fermentation, or fed-batch fermentation, flow feeding inulin maintains inulin concentration at 30.0 ± 1.0g/L, and after feed supplement, substrate is total to about 60-120g/L.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104830851A (en) * 2015-05-11 2015-08-12 盐城工学院 Recombinant bacterium of formate dehydrogenase and application of recombinant bacterium
CN111004807A (en) * 2019-11-29 2020-04-14 西南大学 Cotton fiber specific expression gene GhFSE1 and application thereof
CN111718965A (en) * 2020-06-17 2020-09-29 宁波酶赛生物工程有限公司 Preparation method of (2S,3S) -2, 3-butanediol
CN113337546A (en) * 2021-06-04 2021-09-03 上海启讯医药科技有限公司 Preparation method of (S) -1,2, 4-butanetriol

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101016530A (en) * 2007-01-29 2007-08-15 山东省食品发酵工业研究设计院 Bacillus subtilis capable of producing high purity 3-hydroxy butanone
CN102071174A (en) * 2010-11-24 2011-05-25 天津工业生物技术研究所 (2R, 3R)-2,3-butanediol dehydrogenase and coding gene and application thereof
CN103060255A (en) * 2013-01-24 2013-04-24 盐城工学院 Genetically engineering bacterium capable of producing S-3-hydroxy-butanone as well as construction method and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101016530A (en) * 2007-01-29 2007-08-15 山东省食品发酵工业研究设计院 Bacillus subtilis capable of producing high purity 3-hydroxy butanone
CN102071174A (en) * 2010-11-24 2011-05-25 天津工业生物技术研究所 (2R, 3R)-2,3-butanediol dehydrogenase and coding gene and application thereof
CN103060255A (en) * 2013-01-24 2013-04-24 盐城工学院 Genetically engineering bacterium capable of producing S-3-hydroxy-butanone as well as construction method and application thereof

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
王冠凤: "重组肺炎克雷伯氏菌及代谢产物分析", 《大连理工大学硕士论文》 *
纪晓俊等: "2,3-丁二醇发酵过程中葡萄糖碳流分配数学模型", 《化学与生物工程》 *
纪晓俊等: "微生物发酵法生产2,3-丁二醇的研究进展", 《现代化工》 *
胡文效等: "微生物发酵生产2,3-丁二酮的研究", 《香料香精化妆品》 *
韩丽等: "3-羟基丁酮的研究现状", 《食品与发酵工业》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104830851A (en) * 2015-05-11 2015-08-12 盐城工学院 Recombinant bacterium of formate dehydrogenase and application of recombinant bacterium
CN111004807A (en) * 2019-11-29 2020-04-14 西南大学 Cotton fiber specific expression gene GhFSE1 and application thereof
CN111718965A (en) * 2020-06-17 2020-09-29 宁波酶赛生物工程有限公司 Preparation method of (2S,3S) -2, 3-butanediol
CN113337546A (en) * 2021-06-04 2021-09-03 上海启讯医药科技有限公司 Preparation method of (S) -1,2, 4-butanetriol
CN113337546B (en) * 2021-06-04 2023-09-12 上海启讯医药科技有限公司 Preparation method of (S) -1,2, 4-butanetriol

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