CN103756949B - A kind of produce superelevation optical purity R, R-2,3-butyleneglycol genetic engineering bacterium and construction process thereof and application - Google Patents

A kind of produce superelevation optical purity R, R-2,3-butyleneglycol genetic engineering bacterium and construction process thereof and application Download PDF

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CN103756949B
CN103756949B CN201410018001.9A CN201410018001A CN103756949B CN 103756949 B CN103756949 B CN 103756949B CN 201410018001 A CN201410018001 A CN 201410018001A CN 103756949 B CN103756949 B CN 103756949B
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dar
homology arm
paenibacillus polymyxa
cgmcc
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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
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Abstract

The invention discloses and a kind of produce R, R-2,3-butyleneglycol genetic engineering bacterium and construction process thereof and application, the called after restructuring Paenibacillus polymyxa CGMCC3044--dar of this engineering strain -.Dar is dimethyl diketone reductase gene, and by homologous recombination double exchange, the dar gene knocked out in Paenibacillus polymyxa CGMCC3044 obtains bacterial strain restructuring Paenibacillus polymyxa CGMCC3044-dar -.The recombinant bacterium built by aforesaid method can be fermented and synthesize superelevation optical purity R, R-2,3-butyleneglycol, optical purity is up to 100%, without optics by product meso-2,3-butyleneglycol, this bacterial strain can directly utilize the substrates such as jerusalem artichoke inulin, glucose, 3-Hydroxybutanone, and culture condition is extensive, simply easy to operate, optical purity is high, and production cost is low, is conducive to large-scale industrialized production.

Description

A kind of produce 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 produce 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, there are 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 alcohol, has the physico-chemical properties such as unique optics.The R of superelevation optical purity (>=99.9%), R-2,3-butyleneglycol can be used for synthesizing 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 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 the various chiral smectic thermotropic liquid crystalline polyester material of synthesis and chirality natural bioactivity substance.Fermentation technique along with R, R-2,3-butyleneglycol improves constantly and perfect, superelevation optical purity R, R-2,3-butyleneglycol the synthetic route of quiral products improve and novel chiral product the chiral synthesize field such as exploitation in effect increasingly significant.In addition, R, R-2,3-butyleneglycol or excellent antifreezing agent (zero pour of R, R-2,3-butyleneglycol is low reaches-60 DEG C), add in liquid fuel, fuel also can not solidify at-40 DEG C.
The super high-optical-purity chiral R of chemical method synthesis, R-2,3-butyleneglycol, except needing special synthesis path, also need chiral separation, means are particularly complicated, are difficult to realize large-scale industrial production.Some yeast of nature (Saccharomycescerevisiae) is although can synthesize R, R-2,3-butyleneglycol, because fermentation is for extremely low (the about 0.09g L of production peak of R, R-2,3-butyleneglycol output by internal metabolism -1), lack actual application value, therefore, some special Paenibacillus polymyxas (Paenibacillus polymyxa also claims Bacillus polymyxa) uniquely possess suitability for industrialized production R at present, the microorganism of R-2,3-butyleneglycol potentiality.
During P.polymyxa metabolism synthesis R, R-2,3-butyleneglycol, always with the meso-2 that synthesis is a small amount of, 3-butyleneglycol, and the variation of the external source fermentation conditions such as the 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, the optical purity of R, R-2,3-butyleneglycol is about 90-98%, and the optical purity of meso-2,3-butyleneglycol is about 2-10%.Work as R, when R-2,3-butyleneglycol is used for the synthesis of quiral products, general requirement its should reach more than 99.9% superelevation optical purity, otherwise will be not suitable for preparing chiral material.By optimization for fermentation technology condition, although can improve R, the optical purity of R-2,3-butyleneglycol, optical purity still difficulty reaches more than 99.9% this standard, and usually causes R, R-2,3-butyleneglycol production declining.And when extracting R, R-2,3-butyleneglycol by chiral separation method, there is complex operation, difficulty is large, and high in cost of production defect, causes R, R-2, and the market value of 3-butyleneglycol remains high, and significantly limit 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, the construction process of R-2,3-butyleneglycol genetic engineering bacterium.
The technical problem that the present invention finally will solve is to provide above-mentioned product superelevation optical purity R, the application of R-2,3-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 built by following method to obtain: utilize round pcr from Paenibacillus polymyxa CGMCC3044, 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 the oriT replication initiation sequence and apramycin resistance gene Apra of carrying λ Red homologous recombination sequence clone clay SuperCos-1/pIJ790 rthe two ends of sequence, obtain containing " 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 homologous recombination double exchange is there is with Paenibacillus polymyxa CGMCC3044 under λ Red mediates; obtain the Paenibacillus polymyxa of recombinating; namely 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 resistance gene Apra retc. sequence.
This genetic engineering bacterium is built by following method and obtains:
(1) clone of gene dar: the primer according to needed for the sequences Design synthesis PCR of dar gene in the Paenibacillus polymyxa ATCC12321 that Genebank announces:
P1：5’-ATGGAACTTAAAAACAAAACAGCC-3’
P2：5’-CTGTGGGTTGGTTGTCCAAAT-3’
With Paenibacillus polymyxa CGMCC3044 genome for template completes PCR reaction: PCR reaction conditions: 94 DEG C of sex change 5min, through 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 1min, totally 32 circulations, extend 10min through 72 DEG C again, the PCR primer of acquisition confirms through electrophoretic analysis, after PCR primer Purification Kit, be connected with pMD18-T carrier, carry out sequencing, obtain the dimethyl diketone reductase gene deriving 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: introduce EcoR I respectively, Xho I at the two ends of 5 ' the end 39bp base sequence of dar, obtain dar homology arm I; Introduce Not I respectively, BamH I at the two ends of 3 ' the end 20bp base sequence of dar, obtain dar homology arm II; Above-mentioned dar homology arm I and dar homology arm II are connected to respectively oriT replication initiation sequence and the apramycin resistance gene Apra of the clone's clay SuperCos-1/pIJ790 carrying λ Red homologous recombination sequence rthe two ends of sequence, obtain containing " 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 carries out bacterium colony PCR qualification, obtains Paenibacillus polymyxa of recombinating, called after CGMCC3044-dar -.
Wherein, the method comprises the following steps: to utilize round pcr from Paenibacillus polymyxa CGMCC3044, 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 the oriT replication initiation sequence and apramycin resistance gene Apra of carrying λ Red homologous recombination sequence clone clay SuperCos-1/pIJ790 rthe two ends of sequence, obtain containing " 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 mediates with the double exchange of Host Strains generation homologous recombination; obtain the Paenibacillus polymyxa of recombinating; namely produce superelevation optical purity R; R-2,3-butyleneglycol genetic engineering bacterium CGMCC3044-dar -.
Wherein, above-mentioned product superelevation optical purity R, the construction process of R-2,3-butyleneglycol genetic engineering bacterium comprises the following steps: the clone of (1) gene dar: the primer according to needed for the sequences Design synthesis PCR of dar gene in the Paenibacillus polymyxa ATCC12321 that Genebank announces:
P1：5’-ATGGAACTTAAAAACAAAACAGCC-3’
P2：5’-CTGTGGGTTGGTTGTCCAAAT-3’
With Paenibacillus polymyxa CGMCC3044 genome for template completes PCR reaction: PCR reaction conditions: 94 DEG C of sex change 5min, through 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 1min, totally 32 circulations, extend 10min through 72 DEG C again, the PCR primer of acquisition confirms through electrophoretic analysis, after PCR primer Purification Kit, be connected with pMD18-T carrier, carry out sequencing, obtain the dimethyl diketone reductase gene deriving 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: introduce EcoR I respectively, Xho I at the two ends of 5 ' the end 39bp base sequence of dar, obtain dar homology arm I; Introduce Not I respectively, BamH I at the two ends of 3 ' the end 20bp base sequence of dar, obtain dar homology arm II; Above-mentioned dar homology arm I and dar homology arm II are connected to respectively oriT replication initiation sequence and the apramycin resistance gene Apra of the clone's clay SuperCos-1/pIJ790 carrying λ Red homologous recombination sequence rthe two ends of sequence, obtain containing " 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 carries out bacterium colony PCR qualification, obtains 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 the application in R, R-2,3-butyleneglycol.
Above-mentioned application, by the genetic engineering bacterium CGMCC3044-dar built -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 with jerusalem artichoke inulin crude extract for 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 DEG C, shaking speed 120-200rpm, cultivate 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 DEG C, 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 the fermentation of micro-oxygen environment, shake flask fermentation, or batch fermentation, or fed-batch fermentation, flow feeding inulin maintains inulin concentration at 30.0 ± 1.0g/L, substrate about 60-120g/L altogether after feed supplement.
Wherein, the present inventor laboratory seed selection the microorganism strains of preservation is bacillus polymyxa (paenibacillus polymyxa) the CGMCC No.3044 that can produce 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 and a kind of novel gene engineering bacteria is provided, superelevation optical purity (>=99.9%) R, R-2,3-butyleneglycol is produced for fermenting, this bacterium directly utilizes undressed jerusalem artichoke inulin crude extract high-efficiency fermenting to produce superelevation optical purity R under micro-oxygen conditions, R-2,3-butyleneglycol, breaches traditional zymotic and is difficult to obtain superelevation optical purity R, R-2,3-butyleneglycol and inefficient production superelevation optical purity R, the limitation of R-2,3-butyleneglycol.Research discovery metabolism can synthesize R, R-2, precursor compound and carbonyl compound are progressively reduced into R by oxydo-reductase system by the bacterial classification of 3-butyleneglycol in thalline, R-2, in 3-butyleneglycol (optical purity is about 90-98%) process, the by product meso-2 of 2-10% is always about with synthesizing optical purity, 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 more than 99.9% superelevation optical purity, thus cannot for the preparation of quiral products such as chiral liquid crystal materials, and prepare R with chiral separation further, 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 remains high, significantly limit R, R-2, the industrial applications of 3-butyleneglycol.Compared with original Paenibacillus polymyxa, in recombinant bacterial strain fermented liquid, the optical purity of R, R-2,3-butyleneglycol can reach 100%, and output have also been obtained and significantly improves.With utilize chiral separation production superelevation optical purity R after fermentation that current document is reported, 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 the superelevation optical purity R of 100% after fermentation, can be obtained without the need to separation means such as chiral separation, R-2,3-butyleneglycol, reducing separation costs significantly and improve 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.But those skilled in the art will readily understand, the content described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
Embodiment 1:
The preparation method of jerusalem artichoke inulin crude extract:
Fresh jerusalem artichoke cleans peeling, and blanching is gone out after enzyme (100 DEG C, 15min) and thinly sliced, and forced air drying at 70 DEG C, then pulverized 80 mesh sieves and obtain jerusalem artichoke meal, Refrigerator store is for subsequent use.Taking jerusalem artichoke meal according to the ratio of 1:6 puts into after water stirs, and 70 DEG C of heating in water bath lixiviate 4h, regulate pH to be 9 with milk of lime, then 80 DEG C of water bath heat preservation 1h, with after filtered through gauze jerusalem artichoke inulin crude extract.
Embodiment 2: the clone of gene dar:
Primer according to needed for the sequences Design synthesis PCR of dar gene in Paenibacillus polymyxa (paenibacillus polymyxa) ATCC12321 that Genebank announces:
P1：5’-ATGGAACTTAAAAACAAAACAGCC-3’
P2：5’-CTGTGGGTTGGTTGTCCAAAT-3’
With Paenibacillus polymyxa CGMCC3044 genome for template completes PCR reaction; PCR reaction conditions: 94 DEG C of sex change 5min, 94 DEG C of 30s; 56 DEG C of 30s; 72 DEG C of 1min totally 32 circulations, 72 DEG C extend 10min, and the PCR primer of acquisition confirms through electrophoretic analysis, after PCR primer Purification Kit, be connected with pMD18-T carrier, carry out sequencing, obtain the dimethyl diketone reductase gene dar deriving from Paenibacillus polymyxa CGMCC3044.
Embodiment 3: recombinant cosmid " SuperCos-1/pIJ790-dar homology arm I-oriT-Apra r-dar homology arm II " structure:
Hold the two ends of about 39bp base sequence to introduce EcoR I respectively, Xho I at 5 ' of dar, obtain dar homology arm I; Hold the two ends of about 20bp base sequence to introduce Not I respectively, BamH I at 3 ' of dar, obtain dar homology arm II.According to described restriction enzyme site above-mentioned dar homology arm I and dar homology arm II be connected to respectively the oriT replication initiation sequence being connected to clone's clay SuperCos-1/pIJ790 and apramycin resistance gene Apra of carrying λ Red homologous recombination sequence rthe two ends of sequence, obtain containing " 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 " transform 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 carries out bacterium colony PCR qualification, obtains 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: primitive reaction system (the 200 μ L) composition of enzyme activity determination: 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 DEG C of reaction 5min, measure the velocity of variation of 340nm place absorbancy in 1min.An enzyme activity unit (U) is defined as μm oL quantity that 1min NADPH reduces (increase).Being prepared as follows of enzyme extract: be resuspended in pH8.050mmol/L potassiumphosphate after the thalline physiological saline (0.85%NaCl) of centrifugal for fermented liquid gained is washed twice; at 300W; 30min; ultrasonic 2s; stop 4s and carry out ultrasonication; ice bath is protected, and 8500rpm low-temperature centrifugation 30min, supernatant liquor is enzyme extract.Protein concentration Bradford method detects, using bovine serum albumin as standard substance.
Investigate result: restructuring Paenibacillus polymyxa CGMCC3044-dar -middle dimethyl diketone reductase enzyme enzyme work 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: primitive reaction system (the 200 μ L) composition of enzyme activity determination: 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 DEG C of reaction 5min, measure the velocity of variation of 340nm place absorbancy in 1min.An enzyme activity unit (U) is defined as μm oL quantity that 1min NADPH reduces (increase).Being prepared as follows of enzyme extract: be resuspended in pH8.050mmol/L potassiumphosphate after the thalline physiological saline (0.85%NaCl) of centrifugal for fermented liquid gained is washed twice; at 300W; 30min; ultrasonic 2s; stop 4s and carry out ultrasonication; ice bath is protected, and 8500rpm low-temperature centrifugation 30min, supernatant liquor is enzyme extract.Protein concentration Bradford method detects, using 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 contains inulin 2.0, apramycin 30 μ g/mL;
Seed culture: 1000mL triangular flask, liquid amount 200mL, culture temperature 30 DEG C, shaking speed 120rpm, cultivates 22h;
(3) fermentation culture: substratum composition (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%, leavening temperature 30 DEG C, pH6.0, controls the intake of oxygen, and 24h before inoculation secondary fermentation, controls rotating speed 240rpm, after fermentation 24h, controls rotating speed 160rpm, maintains the fermentation of micro-oxygen environment, fermentation time 42h.
Fermentation results: containing the inulin of 60.0g/L in jerusalem artichoke inulin crude extract, obtains the R of 24.4g/L, R-2,3-butyleneglycol, optical purity 100% through fermentation.
Comparative example 2: original bacteria 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, culture temperature 30 DEG C, shaking speed 120rpm, cultivates 22h;
(3) fermentation culture: substratum composition (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%, leavening temperature 30 DEG C, pH6.0, control the intake of oxygen, shaking speed 120rpm, maintains micro-oxygen environment and ferments, fermentation time 42h.
Fermentation results: containing the inulin of 60.0g/L in jerusalem artichoke inulin crude extract, obtains the R of 20.7g/L, R-2,3-butyleneglycol, optical purity 98.0% through fermentation.
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, culture temperature 30 DEG C, shaking speed 120rpm, cultivates 22h;
(3) fermentation culture: substratum composition (g/L): yeast extract paste 6.0, (NH 4) 2hPO 41.0, KCl0.10, MgSO 4.7H 2o0.1, inulin crude extract contains inulin 60.0, apramycin 30 μ g/mL;
Culture condition: 5L fermentor tank (New Brunswick Scientific, NJ), inoculum size 10v/v%, leavening temperature 30 DEG C, pH6.0, control the intake of oxygen, 24h before inoculation secondary fermentation, controls rotating speed 240rpm, after fermentation 24h, control rotating speed 120rpm, maintain the fermentation of micro-oxygen environment, 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, culture temperature 30 DEG C, shaking speed 120rpm, cultivates 22h;
(3) fermentation culture: substratum composition (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%, leavening temperature 30 DEG C, pH6.0, controls the intake of oxygen, 24h before inoculation secondary fermentation, control rotating speed 240rpm, after fermentation 24h, control rotating speed 120rpm, maintain the fermentation of micro-oxygen environment, 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 is recombinated 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, culture temperature 37 DEG C, shaking speed 200rpm, cultivates 12h;
(3) fermentation culture: substratum composition (g/L): yeast extract paste 3.0, (NH 4) 2hPO 40.5, KCl0.2, MgSO 4.7H 2o0.2, inulin crude extract contains inulin 20.0, apramycin 20 μ g/mL;
Culture condition: 5L fermentor tank (New Brunswick Scientific, NJ), inoculum size 10v/v%, leavening temperature 37 DEG C, pH8.0, controls the intake of oxygen, 24h before inoculation secondary fermentation, control rotating speed 320rpm, after fermentation 24h, control rotating speed 80rpm, maintain the fermentation of micro-oxygen environment, start flow feeding when inulin residual concentration is about 5g/L, 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 bacteria 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, culture temperature 37 DEG C, shaking speed 200rpm, cultivates 12h;
(3) fermentation culture: substratum composition (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%, leavening temperature 37 DEG C, pH8.0, controls the intake of oxygen, 24h before inoculation secondary fermentation, control rotating speed 320rpm, after fermentation 24h, control rotating speed 80rpm, maintain the fermentation of micro-oxygen environment, start flow feeding when inulin residual concentration is about 5g/L, 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 is recombinated 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, culture temperature 33 DEG C, shaking speed 160rpm, cultivates 36h;
(3) fermentation culture: substratum composition (g/L): yeast extract paste 8.0, (NH 4) 2hPO 42.0, KCl0.3, MgSO 4.7H 2o0.3, inulin crude extract contains inulin 60.0, apramycin 40 μ g/mL;
Culture condition: 5L fermentor tank (New Brunswick Scientific, NJ), inoculum size 10v/v%, leavening temperature 33 DEG C, pH8.0, controls the intake of oxygen, 24h before inoculation secondary fermentation, control rotating speed 280rpm, after fermentation 24h, control rotating speed 160rpm, maintain the fermentation of micro-oxygen environment, 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 bacteria 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, culture temperature 33 DEG C, shaking speed 160rpm, cultivates 36h;
(3) fermentation culture: substratum composition (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%, leavening temperature 33 DEG C, pH8.0, controls the intake of oxygen, 24h before inoculation secondary fermentation, control rotating speed 280rpm, after fermentation 24h, control rotating speed 160rpm, maintain the fermentation of micro-oxygen environment, 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%.

Claims (6)

1. high-optical-purity r,R-2,3-butanediol genetic engineering bacterium, this project bacterium is built by following method to obtain: utilize round pcr to amplify dimethyl diketone reductase gene from Paenibacillus polymyxa CGMCC 3044 dar, 5 ' end 39bp base sequence forms homology arm I, 3 ' and holds 20 bp base sequences to form homology arms II, homology arm I and homology arm II to be connected in series to respectively to carry λ Red homologous recombination sequence clone clay SuperCos-1/ pIJ790's by multiple clone site orit replication initiation sequence and apramycin resistance gene Apra rthe two ends of sequence, are contained darhomology arm I- orit-Apra r- darthe recombinant cosmid SuperCos-1/ pIJ790-of homology arm II sequence darhomology arm I- orit-Apra r- darhomology arm II, recombinant cosmid transforms Paenibacillus polymyxa CGMCC 3044, and with Paenibacillus polymyxa CGMCC 3044, homologous double-crossover occurs under λ Red mediates, and obtains the Paenibacillus polymyxa of recombinating, and namely produces superelevation optical purity r,R-2,3-butanediol genetic engineering bacterium CGMCC 3044- dar -, this project bacterium is built by following method and obtains:
(1) gene darclone: according to Genebank announce Paenibacillus polymyxa ATCC 12321 in darthe primer of sequences Design synthesis needed for PCR of gene:
P1：5’-ATGGAACTTAAAAACAAAACAGCC-3’
P2：5’-CTGTGGGTTGGTTGTCCAAAT-3’
With Paenibacillus polymyxa CGMCC 3044 genome for template completes PCR reaction: PCR reaction conditions: 94 ° of C sex change 5 min, through 94 DEG C of 30 s, 56 DEG C of 30 s, 72 DEG C of 1 min, totally 32 circulations, extend 10 min through 72 DEG C again, the PCR primer of acquisition confirms through electrophoretic analysis, after PCR primer Purification Kit, be connected with pMD18-T carrier, carry out sequencing, obtain the dimethyl diketone reductase gene deriving from Paenibacillus polymyxa CGMCC 3044, namely dargene;
(2) recombinant cosmid SuperCos-1/ pIJ790- darhomology arm I- orit-Apra r- darthe structure of homology arm II;
(3) acquisition of recombination engineering bacteria: by recombinant cosmid SuperCos-1/ pIJ790- darhomology arm I- orit-Apra r- darhomology arm II transforms Paenibacillus polymyxa CGMCC 3044, and coating is dull and stereotyped containing 40 μ g/mL apramycins, picking positive recombinant, and carries out bacterium colony PCR qualification, obtains Paenibacillus polymyxa of recombinating, called after CGMCC 3044- dar -.
2. product according to claim 1 r,Rthe construction process of-2,3-butanediol genetic engineering bacterium, is characterized in that: the method comprises the following steps: to utilize round pcr to amplify dimethyl diketone reductase gene from Paenibacillus polymyxa CGMCC 3044 dar, 5 ' end 39bp base sequence forms homology arm I, 3 ' and holds 20 bp base sequences to form homology arms II, homology arm I and homology arm II to be connected in series to respectively to carry λ Red homologous recombination sequence clone clay SuperCos-1/ pIJ790's by multiple clone site orit replication initiation sequence and apramycin resistance gene Apra rthe two ends of sequence, are contained darhomology arm I- orit-Apra r- darthe recombinant cosmid SuperCos-1/ pIJ790 of homology arm II sequence, recombinant cosmid transforms Paenibacillus polymyxa CGMCC 3044, and under λ Red mediates with the double exchange of Host Strains generation homologous recombination, obtain the Paenibacillus polymyxa of recombinating, namely produce superelevation optical purity r,R-2,3-butanediol genetic engineering bacterium CGMCC 3044- dar -.
3. product superelevation optical purity according to claim 2 r,Rthe construction process of-2,3-butanediol genetic engineering bacterium, is characterized in that: comprise the following steps: (1) gene darclone: according to Genebank announce Paenibacillus polymyxa ATCC 12321 in darthe primer of sequences Design synthesis needed for PCR of gene:
P1：5’-ATGGAACTTAAAAACAAAACAGCC-3’
P2：5’-CTGTGGGTTGGTTGTCCAAAT-3’
With Paenibacillus polymyxa CGMCC 3044 genome for template completes PCR reaction: PCR reaction conditions: 94 ° of C sex change 5 min, through 94 DEG C of 30 s, 56 DEG C of 30 s, 72 DEG C of 1 min, totally 32 circulations, extend 10 min through 72 DEG C again, the PCR primer of acquisition confirms through electrophoretic analysis, after PCR primer Purification Kit, be connected with pMD18-T carrier, carry out sequencing, obtain the dimethyl diketone reductase gene deriving from Paenibacillus polymyxa CGMCC 3044, namely dargene;
(2) recombinant cosmid SuperCos-1/ pIJ790- darhomology arm I- orit-Apra r- darthe structure of homology arm II: darthe two ends of 5 ' end 39bp base sequence introduce EcoR I respectively, Xho I, obtains darhomology arm I; ? dar3 ' hold the two ends of 20 bp base sequences to introduce Not I respectively, BamH I, obtains darhomology arm II; By above-mentioned darhomology arm I He darhomology arm II is connected to the clone's clay SuperCos-1/ pIJ790's carrying λ Red homologous recombination sequence respectively orit replication initiation sequence and apramycin resistance gene Apra rthe two ends of sequence, are contained darhomology arm I- orit-Apra r- darthe recombinant cosmid SuperCos-1/ pIJ790-of homology arm II sequence and λ Red sequence darhomology arm I- orit-Apra r- darhomology arm II;
(3) acquisition of recombination engineering bacteria: by recombinant cosmid SuperCos-1/ pIJ790- darhomology arm I- orit-Apra r- darhomology arm II transforms Paenibacillus polymyxa CGMCC 3044, and coating is dull and stereotyped containing 40 μ g/mL apramycins, picking positive recombinant, and carries out bacterium colony PCR qualification, obtains Paenibacillus polymyxa of recombinating, called after CGMCC 3044- dar -.
4. product superelevation optical purity according to claim 1 r,R-2,3-butanediol genetic engineering bacterium CGMCC 3044- dar -in production r,Rapplication in-2,3-butanediol.
5. application according to claim 4, is characterized in that: by the genetic engineering bacterium CGMCC 3044-built dar -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-butanediol, wherein, described carbon source is the jerusalem artichoke inulin crude extract without any hydrolysis treatment.
6. application according to claim 4, is characterized in that: this genetic engineering bacterium directly with jerusalem artichoke inulin crude extract for substrate production superelevation optical purity r,R-2,3-butanediol, concrete production technique is:
(1) starting strain: restructuring Paenibacillus polymyxa CGMCC 3044- dar -;
(2) seed culture medium: (NH 4) 2hPO 40.5-2.0 g/L, KCl 0.10-0.3 g/L, MgSO 4.7H 2o 0.1-0.3 g/L, yeast extract paste 0.1-0.3 g/L, glucose 2.0-8.0 g/L, jerusalem artichoke inulin crude extract is containing inulin 1.0-3.0 g/L, apramycin 20-40 μ g/mL;
Seed culture: 1000 mL triangular flasks, liquid amount 200 mL, culture temperature 30-37 DEG C, shaking speed 120-200 rpm, cultivate 12-36 h;
(3) fermentation culture: substratum forms: jerusalem artichoke inulin crude extract is containing inulin 20.0-60.0 g/L, (NH 4) 2hPO 40.5-2.0 g/L, KCl 0.10-0.3 g/L, MgSO 4.7H 2o 0.1-0.3 g/L, yeast extract paste 3.0-8.0 g/L, apramycin 20-40 μ g/mL;
Culture condition: inoculum size 10v/v%, leavening temperature 30-37 DEG C, pH6.0-8.0, control the intake of oxygen, 24 h before inoculation secondary fermentation, control rotating speed 240-320 rpm, ferment after 24 h, control rotating speed 80-160 rpm, maintain the fermentation of micro-oxygen environment, shake flask fermentation, or batch fermentation, or fed-batch fermentation, flow feeding inulin maintains inulin concentration at 30.0 ± 1.0 g/L, substrate about 60-120 g/L altogether after feed supplement.
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