CN108949760A - A kind of combined promoter and its application in the 3- hydracrylic acid yield for improving klepsiella pneumoniae - Google Patents
A kind of combined promoter and its application in the 3- hydracrylic acid yield for improving klepsiella pneumoniae Download PDFInfo
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Abstract
The present invention provides a combined promoter, and the combined promoter is arranged by 2-5 tac promoter core sequence.The present invention also provides the preparation methods of carrier, engineering recombinant bacterium and the engineering recombinant bacterium with the combined promoter.This with combined promoter engineering recombinant bacterium can high efficient expression aldehyde dehydrogenase, thus quick catalysis 3-HPA produce 3-HP, on the one hand prevent weakening of the accumulation of 3-HPA to cell growth metabolism, the yield of 3-HP on the other hand can be improved.
Description
Technical field
The invention belongs to biochemical industry production field, it is related to a kind of combined promoter and its is improving e coil k 1 pneumonia bar
Application in the 3- hydracrylic acid yield of bacterium.
Background technique
3- hydracrylic acid (3-Hydroxypropionic acid, 3-HP) is important platform chemicals, has wide city
Field prospect.Chemical synthesis 3-HP difficulty is big, isolates and purifies difficulty, and pollute environment, therefore constrains its large-scale application.
Bioanalysis, which produces 3-HP, has the advantages such as inexpensive, easy to operate, mild condition, environmental-friendly.Klepsiella pneumoniae
(Klebsiella pneumoniae) glycerol tolerance with higher, stronger glycerol metabolism ability, and itself can synthesize auxiliary
Enzyme B12The features such as, therefore become the advantage host strain using glycerol bioanalysis production 3-HP.K.pneumoniae is de- using glycerol
Water enzyme (DhaB) catalyzing glycerol generates 3-HPA (3-HPA), and 3-HPA generates 3- under the catalysis of aldehyde dehydrogenase (ALDH)
HP。
Since the activity of glycerol dehydratase is higher than aldehyde dehydrogenase, leading to the accumulation of 3-HPA, 3-HPA has toxicity to cell,
Therefore the growth metabolism of impairing cellular.For high yield 3-HP, need to improve the activity of aldehyde dehydrogenase.Promoter is as important base
Because of expression regulation element, can activity directly influences foreign gene high efficient expression.K.pneumoniae is non-mode biology,
Lacking suitable strong promoter makes aldehyde dehydrogenase high efficient expression, causes the production process conversion ratio of 3-HP lower.
Summary of the invention
To solve the above-mentioned problems, the purpose of the present invention is to provide a kind of combined promoter, which can efficient table
On the one hand prevent the accumulation of 3-HPA from cutting to cell growth metabolism up to aldehyde dehydrogenase so that quick catalysis 3-HPA produces 3-HP
It is weak, the yield of 3-HP on the other hand can be improved.
Second object of the present invention is to provide a kind of recombinant vector.
Third object of the present invention is to provide a kind of preparation method of above-mentioned recombinant vector.
Fourth object of the present invention is to provide a kind of klepsiella pneumoniae recombinant bacterium for producing 3- hydracrylic acid.
Of the invention the 5th is designed to provide a kind of klepsiella pneumoniae weight of above-mentioned production 3- hydracrylic acid
The preparation method of group bacterium.
Of the invention the 6th is designed to provide a kind of above-mentioned combined promoter or the recombination with combined promoter carries
Application of the body in the 3- hydracrylic acid yield for improving klepsiella pneumoniae.
To achieve the goals above, the present invention provides a kind of combined promoter, and the combined promoter contains 2-5 tac
Promoter core sequence；
Wherein, when the combined promoter contains 2 tac promoter core sequences, sequence is as shown in Seq ID No.2；
When the combined promoter contains 3 tac promoter core sequences, sequence is as shown in Seq ID No.3；
When the combined promoter contains 4 tac promoter core sequences, sequence is as shown in Seq ID No.4；
When the combined promoter contains 5 tac promoter core sequences, sequence is as shown in Seq ID No.5.
The present invention also provides a kind of recombinant vectors, by above-mentioned combined promoter, puuC aldehyde dehydrogenase gene and pET-28a
Carrier composition, wherein puuC aldehyde dehydrogenase gene sequence is as shown in Seq ID No.6；The recombinant vector is expression vector.
The present invention also provides the preparation methods of above-mentioned recombinant vector: including the following steps:
1) compound starting subsequence and pET-28a carrier are carried out respectively with restriction enzyme Bgl II/EcoR I double
Digestion；
2) electrophoresis, Ago-Gel recycle compound starting sub-piece and pET-28a carrier segments after digestion；
3) compound starting sub-piece is connected using ligase with pET-28a carrier segments, is obtained containing combined promoter
The carrier of segment；
4) PCR amplification puuC gene order；
5) with restriction enzyme Sac I/EcoR I respectively to the load containing compound starting sub-piece made from step 3)
PuuC gene order made from body and step 4) carries out double digestion；
6) electrophoresis, Ago-Gel recycle the carrier segments containing combined promoter and puuC genetic fragment after digestion；
7) carrier segments containing combined promoter are connected using ligase with puuC genetic fragment, obtains recombination and carries
Body.
The present invention also provides a kind of klepsiella pneumoniae recombinant bacterium for producing 3- hydracrylic acid, the kerekou pneumonias primary
Family name's bacillus recombinant bacterium includes above-mentioned combined promoter or above-mentioned recombinant vector.
The present invention also provides the preparation methods of the klepsiella pneumoniae recombinant bacterium of above-mentioned production 3- hydracrylic acid: including
Following steps:
1) competent cell processed: by wild type klepsiella pneumoniae culture activation, ice bath 30min, refrigerated centrifuge is gone
Twice, competent cell is made in supernatant, washing；
2) it is electroporated that electric shock cup progress is put into after being sufficiently mixed recombinant vector with competent cell；
3) electricity after will be electroporated turns to be applied to after liquid is mixed with culture medium in resistance solid medium, obtains through screening
Produce the klepsiella pneumoniae recombinant bacterium of 3- hydracrylic acid.
The present invention also provides a kind of for cultivating the fermentation medium of above-mentioned klepsiella pneumoniae recombinant bacterium, the fermentation
The formula of culture medium are as follows: KH2PO42.7g/L；NH4NO32.4g/L；K2HPO4·3H2O 4.2g/L；(NH4)2SO42g/L；
CaCO30.15g/L；MgSO4·7H2O 0.12g/L；NaH2PO41.6g/L；EDTANa20.001g/L；Citric acid 0.06g/
L；Glycerol 40g/L；Yeast powder 4g/L；Microelement 1.25mL/L；Trace element solution: Na2MO4·2H2O 0.045g/L；
FeSO4·7H2O 1.7g/L；CoCl2·2H2O 0.12g/L；ZnCl20.05g/L；NiCl2·6H2O 0.025g/L；H3BO3
0.16g/L；MnCl2·4H2O 0.1g/L；CuCl2·2H2O 0.06g/L；HCl (37%) 3.6mL.
The present invention also provides above-mentioned combined promoters or above-mentioned recombinant vector in the 3- hydroxyl for improving klepsiella pneumoniae
Application in base propionic acid yield.
Tac promoter used in the present invention is spliced by trp promoter and lacUV5, and tac is as stronger induction type
Promoter promotor gene can be expressed in klepsiella pneumoniae (K.pneumoniae).To obtain higher transcriptional activity,
It meets the needs of production, tac promoter core sequence is connected, design obtains a series of combined promoter Pntac (n
=2~5), and with the ALDH encoding gene puuC of its overexpression K.pneumoniae itself (KPN_01018 is (in KEGG database
Accession number), to construct 3-HP high production bacteria.Combined promoter and ALDH encoding gene are subjected to PCR amplification.Sequencing is just
Promoter and PuuC are overexpressed in K.pneumoniae after really, obtain recombination K.pneumoniae (Pntac-puuC),
And measure its catalytic capability to 3-HPA.The result shows that being recombinated when the series connection of the core sequence of 3 sections of tac promoters
The ALDH activity highest of K.pneumoniae (P3tac-puuC), reaches 34.36U/mg, is K.pneumoniae wild mushroom
8.16 times of (4.21U/mg).Cultivate the engineering bacteria in 5L fermentor, 3-HP yield is up to 99.8g/L when 96h.Glycerol at this time
Conversion ratio is 87.7%, and space-time yield (productivity) is 1.04gL-1·h-1。
The beneficial effects of the present invention are:
The present invention provides a kind of combined promoter, recombinant vector and its high yield 3- hydracrylic acid engineering bacteria of building, this is multiple
Close promoter can high efficient expression aldehyde dehydrogenase, thus quick catalysis 3-HPA produce 3-HP, on the one hand prevent the accumulation pair of 3-HPA
On the other hand the yield of 3-HP can be improved in the weakening of cell growth metabolism, and utilize the K.pneumoniae of promoter building
Efficient expression system, the high yield, it can be achieved that 3-HP is transformed to K.pneumoniae glycerol catabolic pathway.
Detailed description of the invention
Fig. 1 is the electrophoresis result figure of puuC gene in the present invention.
Fig. 2A is the electrophoresis result figure of the PCR of recombinant bacterium K.p (Ptac-puuC) in the present invention.
Fig. 2 B is the electrophoresis result figure of the PCR of recombinant bacterium K.p (P2tac-puuC) in the present invention.
Fig. 2 C is the electrophoresis result figure of the PCR of recombinant bacterium K.p (P3tac-puuC) in the present invention.
Fig. 2 D is the electrophoresis result figure of the PCR of recombinant bacterium K.p (P4tac-puuC) in the present invention.
Fig. 2 E is the electrophoresis result figure of the PCR of recombinant bacterium K.p (P5tac-puuC) in the present invention.
Fig. 3 is that the ALDH enzyme activity of recombinant bacterium provided by the invention is analyzed.
Fig. 4 A is the 96h fermentation results of wild-type strain K.p-WT.
Fig. 4 B is the 96h fermentation results of recombinant bacterium K.p (P2tac-puuC) provided by the invention.
Fig. 4 C is the 96h fermentation results of recombinant bacterium K.p (P3tac-puuC) provided by the invention.
Fig. 4 D is the 96h fermentation results of recombinant bacterium K.p (P4tac-puuC) provided by the invention.
Fig. 4 E is the 96h fermentation results of recombinant bacterium K.p (P4tac-puuC) provided by the invention.
Specific embodiment
Following specific method can make those skilled in the art that the present invention be more fully understood, but not limit in any way
The system present invention.
1. material:
1) competent escherichia coli cell (E.coli BMTop10) is purchased from Beijing Bo Maide Bioisystech Co., Ltd.
2) 2026 (K.pneumoniae of wild type klepsiella pneumoniae Klebsiella pneumoniae DSM
DSM 2026) it is purchased from Germany Microbiological Culture Collection Center (DSMZ), culture presevation number: DSM 2026, in embodiment
It can be abbreviated as K.p-WT, K.pneumoniae-WT or K.pneumoniae.
3) expression vector pET-28a is purchased from Invitrogen company.
2. culture medium:
LB culture medium (liquid): yeast extract 5g/L, peptone 10g/L, NaCl 10g/L, pH value 7.0.The training of LB solid
Supporting base is the agar (mass volume ratio) that 1.5% is added in liquid medium.Kanamycin sulfate need to be added in resistance culture base
To 50 μ g/mL of final concentration.
The fermentation medium (basis) of klepsiella pneumoniae: KH2PO41.4g/L；NH4NO31.7g/L；K2HPO4·
3H2O 3.6g/L；(NH4)2SO42.9g/L；CaCO30.15g/L；MgSO4·7H2O 0.5g/L；NaH2PO41.6g/L；
EDTANa20.001g/L；Citric acid 0.06g/L；Glycerol 40g/L；Yeast powder 4.2g/L；Microelement 1.25mL/L；
Trace element solution: Na2MO4·2H2O 0.045g/L；FeSO4·7H2O 1.7g/L；CoCl2·2H2O0.12g/
L；ZnCl20.05g/L；NiCl2·6H2O 0.025g/L；H3BO30.16g/L；MnCl2·4H2O0.1g/L；CuCl2·2H2O
0.06g/L；HCl (37%) 3.6mL.
3. sequent synthesis is completed by Beijing Bo Maide gene technology Co., Ltd.
4. restriction enzyme Bgl II/EcoR I/Sac I, T4Ligase ligase are purchased from New England
Biolabs(Beijing,China)。
5. agarose QIAquick Gel Extraction Kit is purchased from Beijing Bo Maide gene technology Co., Ltd.
The preparation of 1 recombinant vector pET-Pntac of embodiment
Combined promoter is designed according to the tac promoter core sequence (29bp) of report, and carries out chemical synthesis, is contained
There is the genetic fragment Pntac (n=2~5) of 2-5 tac promoter core sequence.Since combined promoter contains more repetition sequence
Column, and molecular weight is smaller, it is more difficult to control using condition when PCR progress gene magnification, therefore Bgl II/EcoR I is introduced in design
Restriction enzyme site, connect after the direct double digestion of the gene of synthesis to obtain with carrier pET-28a recombinant vector pET-Pntac (n=2~
5), wherein the part of underscore mark is restriction enzyme site, and black overstriking font is labeled as tac promoter core sequence.Two cores
It is intervening sequence (TGTGGAATTGTG) between heart sequence, gives the enough spaces of RNA polymerase and be combined, sequence reference
Partial sequence after carrier pET28a promoter；It is intervening sequence after the last one tac promoter core sequence
It (TGTGGAATTGTG), is inducible promoter tac repressor followed by lac operon sequence (lac operator)
Binding site also contains ribosome recognition site AAGGAG after operon sequence, and needing to reserve space later makes ribosomes
In conjunction with, 6~15bp is divided between general carrier, wherein 9bp effect is preferable, thus before restriction enzyme site add 9bp sequence.
The Pntac (n=2) when the combined promoter contains 2 tac promoter core sequences, sequence such as Seq ID No.2
It is shown；
The Pntac (n=3) when the combined promoter contains 3 tac promoter core sequences, sequence such as Seq ID No.3
It is shown；
The Pntac (n=4) when the combined promoter contains 4 tac promoter core sequences, sequence such as Seq ID No.4
It is shown；
The Pntac (n=5) when the combined promoter contains 5 tac promoter core sequences, sequence such as Seq ID No.5
It is shown:
In order to compare the difference containing different tac promoter core sequences, also devised here containing 1 tac promoter
The sequence of core sequence Pntac (n=1), sequence is as shown in Seq ID No.1:
The genetic fragment containing different tac core sequences is inserted respectively into pET-28a carrier first.
1) digestion
Double enzymes are carried out to compound starting sub-piece and carrier pET-28a respectively with restriction enzyme Bgl II/EcoR I
It cuts.Digestion temperature is 37 DEG C, reaction time 2h.After reaction, system is through 65 DEG C of heat inactivation 15min, to terminate reaction.
Endonuclease reaction system
2) electrophoresis
Digestion products are carried out to electrophoresis according to a conventional method respectively.
Select big offset plate.
0.60g agarose is weighed, 80mL TAE buffer is poured into, is dissolved by heating.
Glue enters offset plate, and 2 μ l Goldview dye liquors are added, and stands gel 20min.
Loading comb carefully is pulled up, offset plate is put into electrophoresis tank, supplement buffer to not having offset plate completely.
By the reaction solution after 10 μ l digestions, 2 μ l Loading buffer are added, are uniformly mixed, are added in loading hole.
10 μ l BM 5000 are added+DNA Marker。
Electrophoresis tank is covered, is powered on, electrophoresis is started.
To after, moves under dark box type ultraviolet projectoscope and observe result.
3) agarose gel extraction
It is completed using agarose QIAquick Gel Extraction Kit, specific steps are as follows: the electrophoretic band cut is put into small size EP and managed by (a)
In, the sol solutions of 300 μ L are added, warm bath to colloid becomes liquid in 65 DEG C of metal baths；(b) cross adsorption column after, at a high speed from
The heart loses waste liquid；(c) adsorption column is rinsed with 500 μ L, 75% ethyl alcohol, rinsing is twice；(d) after ethyl alcohol is dry, with 30 μ L
Molecule manipulation washes the segment of De contamination, and -20 DEG C save backup.
4) it connects
The target gene obtained after gel extraction is uniformly mixed with plasmid vector with 1:1 (v:v), using ligase by the two
Connection.65 DEG C of temperature of connection, reaction time 1h.
Coupled reaction system:
5) chemical method thermal transition
In aseptic operating platform, above-mentioned linked system is added to competent escherichia coli cell (E.coli BMTop10)
In and be uniformly mixed, 890 μ L LB culture mediums (liquid) are added, 37 DEG C, resistant panel, 37 DEG C of trainings are coated with after 200rpm recovery 1h
15h is supported, the dientification of bacteria is chosen, respectively obtains recombinant vector pET-Pntac (n=1~5).
The preparation of the recombination klepsiella pneumoniae of embodiment 2
According to puuC gene order (as shown in Seq ID No.6) design primer that GenBank is announced, pass through primer sequence
Sac I and EcoR I restriction enzyme site is introduced between base by (puuC-F (Seq ID No.7) and puuC-R (Seq ID No.8))
The both ends of cause carry out PCR amplification using 2026 genome of K.pneumoniae DSM as template.PuuC gene is connected to carrier
In pET-Pntac, recombinant plasmid (pET-Pntac-puuC) is obtained, is transferred in Escherichia coli BMTop10 and carries out sequence verification.It mentions
The correct plasmid electroporation of sequencing is taken to convert to wild type klepsiella pneumoniae K.pneumoniae DSM 2026, culture
And extract genomic DNA, carry out PCR, rear electrophoresis verifying puuC gene whether successful conversion is entered, the primer of PCR is
The universal primer of pET28a carrier, as a result as shown in Fig. 2A~Fig. 2 E.It saves respectively and connects correct recombinant bacterium
K.pneumoniae (Pntac-puuC) (n=1~5).
The specific method is as follows:
1) PCR amplification
PCR primer are as follows: underscore mark is restriction enzyme site
PCR reaction system:
PCR reaction condition:
PCR product is subjected to electrophoresis, confirms whether PCR product is puuC gene by clip size, as a result such as Fig. 1 institute
Show, wherein M is DNA marker DL2000plus；1 is puuC gene, is really puuC gene from electrophoresis size
(1488bp)。
Ago-Gel recycling, is respectively connected to carrier pET- for gene puuC by restriction enzyme site EcoR I/Sac I
In Pntac (n=1~5), recombinant plasmid (pET-Pntac-puuC (n=1~5)) are obtained, E. coli is transferred to
Sequence verification is carried out in BMTop10.The correct plasmid electroporation of sequencing is extracted to convert to klepsiella pneumoniae and tested
Card.It saves respectively and connects correct recombinant bacterium K.pneumoniae (Pntac-puuC) (n=1~5).In addition, will not connect
The carrier pET-Ptac of puuC gene also carries out electrotransformation, obtains one plant of recombinant bacterium K.pneumoniae (Ptac) as containing free
The control strain of carrier is used for enzyme activity determination.
Specific step is as follows by carrier Electroporation Transformation K.pneumoniae:
(a) by wild type K.pneumoniae in 4mL LB culture medium, the isothermal vibration in 37 DEG C, the shaking table of 200rpm
Cultivate the activation that 12h is used for bacterial strain；(b) by the bacterium solution of activation with the amount of 1% (V/V), sterile working is connected in 10mL LB, in
37 DEG C of constant temperature, 200rpm concussion, cultivate 90min；(c) by bacterium solution ice bath 30min；(d) height is carried out to thallus with refrigerated centrifuge
Fast refrigerated centrifuge removes supernatant and cleans competent cell with aseptic double-distilled water, is repeated twice；(e) by recombinant plasmid with prepare
Competent cell be sufficiently mixed be placed on electric revolving cup carry out it is electroporated；(f) electricity is turned into liquid and 890 μ L LB culture mediums is abundant
Mixing, is put into small size centrifuge tube, 37 DEG C, 200rpm, cultivates 1h, is coated in LB resistance solid medium, 37 DEG C of inversions,
12h is about cultivated, whether successful conversion enters host strain to the carrier of progress PCR verifying carrying puuC gene.
PCR is carried out using preceding method, as a result electrophoresis is respectively to the carrier in conversion bacterium colony as shown in Fig. 2A-Fig. 2 E
Middle puuC segment carries out PCR, and M is 1 to extract from K.p in DNA marker DL2000plus, Fig. 2A in Fig. 2A-Fig. 2 E
(Ptac-puuC), 1 K.p (P2tac-puuC) is extracted from Fig. 2 B；1 extracts from K.p (P3tac-puuC) in Fig. 2 C；1 in Fig. 2 D
Extract from K.p (P4tac-puuC)；1 extracts from K.p (P5tac-puuC) in Fig. 2 E.It can be seen from the results above that occurring
Correct band, illustrates the carrier successful conversion into klebsiella.
The vitality test of 3 aldehyde dehydrogenase of embodiment
Recombinant bacterium is subjected to shake flask fermentation respectively, culture medium uses the fermentation medium (base of klepsiella pneumoniae
Plinth): the bottled fermentation medium 100mL of 250mL taper, tampon sealing, inoculum concentration are 1% (volume fraction), 37 DEG C of 150r/min
Shake culture recombinant bacterium.Thalline were collected by centrifugation by fermentation liquid 10mL, 12000r/min when taking for 24 hours, with 5mL PBS buffer solution
(pH7.0) thallus is resuspended, sonicated cells, operating condition 80W, work 3s, is spaced 2s, recycles 80 times to get crude enzyme liquid.
Enzyme activity reaction system is 2mL, including positive 200 μ L, the 20mM NAD of propionic aldehyde of 150mM+200 μ L, 200 μ L of crude enzyme liquid, PBS buffer solution
(pH7.0)1.4mL.37 DEG C of water-bath 5min determine the life of NADH in reaction system by the increment of light absorption value at detection 340nm
Cheng Liang.Enzyme-activity unit definition: at 37 DEG C, the enzyme amount that catalysis generates 1 μm of ol NADH per minute is an enzyme activity unit.It
The total protein content in 200 μ L crude enzyme liquids is demarcated with 5mL Coomassie brilliant blue G250 reagent afterwards, obtains the Rate activity of recombinase.
Test result is as shown in Figure 3, wherein K.p-WT is klepsiella pneumoniae wild type；K.p (Ptac) be containing
The control strain of empty carrier；K.p (Pntac-puuC) (n=1~5) is combined promoter recombinant bacterium, from figure 3, it can be seen that
Recombinant bacterium has different degrees of promotion compared to control bacterium, enzymatic activity, it was demonstrated that ALDH realizes greater efficiency in recombinant bacterium
Expression, and have biology catalytic activity, and wherein n=3 when recombinant bacterium K.pneumoniae (P3tac-puuC) enzyme activity
Highest.
The determination of 4 optimal culture condition of embodiment
The fermentation medium (basis) of klepsiella pneumoniae: KH2PO41.4g/L；NH4NO31.7g/L；K2HPO4·
3H2O 3.6g/L；(NH4)2SO42.9g/L；CaCO30.15g/L；MgSO4·7H2O 0.5g/L；NaH2PO41.6g/L；
EDTANa20.001g/L；Citric acid 0.06g/L；Glycerol 40g/L；Yeast powder 4.2g/L；Microelement 1.25mL/L.
Trace element solution: Na2MO4·2H2O 0.045g/L；FeSO4·7H2O 1.7g/L；CoCl2·2H2O
0.12g/L；ZnCl20.05g/L；NiCl2·6H2O 0.025g/L；H3BO30.16g/L；MnCl2·4H2O 0.1g/L；
CuCl2·2H2O 0.06g/L；HCl (37%) 3.6mL.
Since above-mentioned culture medium is the common culture medium of klepsiella pneumoniae fermentation in the prior art, and cultivating
Klepsiella pneumoniae recombinant bacterium produce 3-HP when, can to carbon source, nitrogen source generate consumption and basal medium can not
Together, therefore, it is optimized on above-mentioned formula technique.
Determine that recombinant bacterium produces the best culture item of 3-HP according to the variation of different culture medium ingredient additive amount in shake flask fermentation
Part (with the highest K.pneumoniae of ALDH enzyme activity (P3tac-puuC) for Object of Development).
The bottled fermentation medium 100mL of 250mL taper, tampon sealing, inoculum concentration are 1% (volume fraction), 37 DEG C of 150r/
Min shake culture recombinant bacterium.It is sampled every 3h, high performance liquid chromatography measures 3- hydracrylic acid yield.Specific analytical method is as follows:
Chromatographic column is the reversed column of C18, and 25 DEG C of column temperature, mobile phase is ultrapure water and adds 0.05% phosphoric acid, working flow rate 0.8mL/
Min, using UV detector.It takes 2mL fermentation liquid 12000rpm to be centrifuged 5min, takes supernatant after 0.22 μm of filtering with microporous membrane
It retains, is used for high performance liquid chromatography detection.
Wherein the concentration of fermentation medium used is as shown in table 1.When one of component content changes, other at
Point content is remained unchanged according to the component content of basal medium.
Influence of the 1 various concentration medium component of table to 3-HP yield
According to the above shake flask fermentation as a result, to obtain optimal medium component prescription as shown in table 2:
2 optimal medium ingredient of table
Condition of culture after advanced optimizing:
The fermentation medium of klepsiella pneumoniae: KH2PO42.7g/L；NH4NO32.4g/L；K2HPO4·3H2O
4.2g/L；(NH4)2SO42g/L；CaCO30.15g/L；MgSO4·7H2O 0.12g/L；NaH2PO41.6g/L；EDTANa2
0.001g/L；Citric acid 0.06g/L；Glycerol 40g/L；Yeast powder 4g/L；Microelement 1.25mL/L.
Trace element solution: Na2MO4·2H2O 0.045g/L；FeSO4·7H2O 1.7g/L；CoCl2·2H2O
0.12g/L；ZnCl20.05g/L；NiCl2·6H2O 0.025g/L；H3BO30.16g/L；MnCl2·4H2O 0.1g/L；
CuCl2·2H2O 0.06g/L；HCl (37%) 3.6mL.
Using the condition of culture after optimization, the pH value optimization of tank fermentation is carried out, the pH value constant shape 6,7 and 8 respectively
The fermentation that 36h is carried out under state, measures the yield of 3-HP and associated metabolites, with the highest K.pneumoniae of ALDH enzyme activity
(P3tac-puuC) it is object, the results are shown in Table 3.
The carbon sulphur content cloth of glycerol under the different pH value of table 3
Wherein, GCR is glycerol conversion yield；3-HP is 3- hydracrylic acid；LA is lactic acid；AC is acetic acid；1,3-PD is 1,3-
Propylene glycol；2,3-BD is 2,3- butanediol；Gly is glycerol.
Since glycerol provides necessary substrate as the sole carbon source in culture medium for thalli growth in fermentation medium.
And 3-HP is the primary product of glycerol metabolism in klebsiella, and during producing 3-HP, lactic acid, acetic acid, 1,3-PD,
2,3- butanediol becomes the by-product of competition glycerol resource.
As can be seen from Table 3, in the state that pH value is 7, the 3-HP yield of K.pneumoniae (P3tac-puuC) is most
Height, it is thus determined that pH7 is optimal fermented and cultured pH value.
The upper tank of 5 recombinant bacterium of embodiment ferments
In 5L fermentor (Shanghai protect emerging) respectively to wild type and comprising combined promoter (Pntac-puuC (n=2~
5) different recombination engineerings) carry out the upper tank fermentation of 96h, and liquid amount 3L maintains the temperature at 37 DEG C, and auto-feeding soda acid makes
PH maintains 7.0, and biomass, glycerol surplus, 3- is measured by sampling in ventilatory capacity 1.5vvm, speed of agitator 400rpm, every 3h
Hydracrylic acid and associated metabolites yield, make glycerol concentration maintain 20g/L or more.The same shake flask fermentation of analysis method.
Used medium formula is as follows:
The fermentation medium of klepsiella pneumoniae: KH2PO42.7g/L；NH4NO32.4g/L；K2HPO4·3H2O
4.2g/L；(NH4)2SO42g/L；CaCO30.15g/L；MgSO4·7H2O 0.12g/L；NaH2PO41.6g/L；EDTANa2
0.001g/L；Citric acid 0.06g/L；Glycerol 40g/L；Yeast powder 4g/L；Microelement 1.25mL/L.
Trace element solution: Na2MO4·2H2O 0.045g/L；FeSO4·7H2O 1.7g/L；CoCl2·2H2O
0.12g/L；ZnCl20.05g/L；NiCl2·6H2O 0.025g/L；H3BO30.16g/L；MnCl2·4H2O 0.1g/L；
CuCl2·2H2O 0.06g/L；HCl (37%) 3.6mL.
Experimental result as shown in figs. 4a-4e, can be seen that the yield of the 3-HP of wild type K.pWT very from Fig. 4 A
It is low, as can be seen that work as n=2 from Fig. 4 B, Fig. 4 D, Fig. 4 E, recombinant bacterium K.pneumoniae (P2tac-puuC) when 4,5, recombinate
Bacterium K.pneumoniae (P4tac-puuC), recombinant bacterium K.pneumoniae (P5tac-puuC) yield of 3-HP after 54h become
In steady, and from Fig. 4 C as can be seen that as n=3, recombinant bacterium K.pneumoniae (P3tac-puuC) 3-HP after 54h
Yield continue for rising, maximum output appear in fermentation 96h when, yield 99.8g/L.Glycerol conversion yield at this time is timely
Empty yield is respectively 87.7%, 1.04gL-1·h-1。
Specific 3-HP yield and glycerol conversion yield are as shown in table 4:
It can be seen from the results above that recombinant bacterium is greatly improved than the 3-HP yield of wild type, wherein recombinant bacterium
The yield highest of K.pneumoniae (P3tac-puuC), the 3-HP yield of other recombinant bacteriums are minimum also at 10 times of wild-type bacteria
More than.
From above-described embodiment shake flask fermentation and upper tank fermentation the result shows that, combined promoter Pntac (n=2~5) protect
Aldehyde dehydrogenase gene puuC has been demonstrate,proved in the intracorporal high efficient expression of Klebsiella Pneumoniae, has improved the yield of 3- hydracrylic acid, therefore
The present invention successfully constructs a series of Producing Strain K.pneumoniae (Pntac-puuC) (n=2~5) of 3- hydracrylic acids.
SEQUENCE LISTING
<110>Beijing University of Chemical Technology
<120>a kind of combined promoter and its application in the 3- hydracrylic acid yield for improving klepsiella pneumoniae
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 111
<212> DNA
<213>artificial sequence
<400> 1
agatctttga caattaatca tcggctcgta taatgtgtgg aattgtgagc ggataacaat 60
tcccctctag aaataatttt gtttaacttt aaggagaagg atatagaatt c 111
<210> 2
<211> 152
<212> DNA
<213>artificial sequence
<400> 2
agatctttga caattaatca tcggctcgta taatgtgtgg aattgtgttg acaattaatc 60
atcggctcgt ataatgtgtg gaattgtgag cggataacaa ttcccctcta gaaataattt 120
tgtttaactt taaggagaag gatatagaat tc 152
<210> 3
<211> 193
<212> DNA
<213>artificial sequence
<400> 3
agatctttga caattaatca tcggctcgta taatgtgtgg aattgtgttg acaattaatc 60
atcggctcgt ataatgtgtg gaattgtgtt gacaattaat catcggctcg tataatgtgt 120
ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaaggagaa 180
ggatatagaa ttc 193
<210> 4
<211> 234
<212> DNA
<213>artificial sequence
<400> 4
agatctttga caattaatca tcggctcgta taatgtgtgg aattgtgttg acaattaatc 60
atcggctcgt ataatgtgtg gaattgtgtt gacaattaat catcggctcg tataatgtgt 120
ggaattgtgt tgacaattaa tcatcggctc gtataatgtg tggaattgtg agcggataac 180
aattcccctc tagaaataat tttgtttaac tttaaggaga aggatataga attc 234
<210> 5
<211> 275
<212> DNA
<213>artificial sequence
<400> 5
agatctttga caattaatca tcggctcgta taatgtgtgg aattgtgttg acaattaatc 60
atcggctcgt ataatgtgtg gaattgtgtt gacaattaat catcggctcg tataatgtgt 120
ggaattgtgt tgacaattaa tcatcggctc gtataatgtg tggaattgtg ttgacaatta 180
atcatcggct cgtataatgt gtggaattgt gagcggataa caattcccct ctagaaataa 240
ttttgtttaa ctttaaggag aaggatatag aattc 275
<210> 6
<211> 1488
<212> DNA
<213> Klebsiella pneumoniae
<400> 6
atgaattttc agcacctggc ttactggcag gaaaaagcga aaaacctggc cattgaaacg 60
cgcttattta ttaacggcga atattgcgcc gcggccgata ataccacctt tgagactatc 120
gaccccgccg cgcagcagac attagcccag gtcgcccgcg gtaaaaaagc cgacgtcgaa 180
cgggcggtga aagccgcgcg ccaggctttt gataacggcg actggtcgca ggcctccccc 240
gcacagcgta aagcgatcct cactcgcttt gctaatctga tggaggccca tcgtgaagag 300
ctggcgctgc tggaaacgct ggataccggc aagccgattc gccacagcct gcgcgacgat 360
attcccggcg ccgcccgcgc cattcgctgg tatgccgaag cgctggataa agtctatggc 420
gaagtggccc ccaccggcag caacgagctg gcgatgatcg ttcgcgaacc aattggcgtg 480
atcgccgcgg tggtgccgtg gaacttcccg ctgctgctgg cctgctggaa actcggcccg 540
gcgctggcgg caggcaatag cgtaatcctc aaaccctcgg aaaaatcgcc gcttaccgcc 600
ctgcgtctgg ccgggctggc gaaagaggcc ggcctgccgg acggcgtgtt gaacgtggtc 660
agcggctttg gccacgaggc cgggcaggcg ctggccctgc atcctgatgt tgaagtcatc 720
accttcaccg gctccacccg caccggcaag cagctgctga aagacgccgg cgacagcaat 780
atgaagcgcg tgtggctgga agcgggcggc aagagcgcca acattgtctt cgccgattgc 840
ccggatctgc aacaagcggt tcgcgccacc gccggcggca tcttctacaa ccagggacag 900
gtgtgcatcg ccgggacccg tctgctgctc gaggagagca tcgctgacga gttcctggcg 960
cggctgaaag ctgaggcgca acactggcag ccgggcaacc cgctcgatcc ggacaccacc 1020
atgggcatgc tgattgacaa tacccatgcc gacaacgtgc atagctttat tcgcggcggc 1080
gaaagccaaa gcaccctgtt cctcgacgga cggaaaaacc cgtggcctgc cgccgttggc 1140
ccgaccattt tcgttgacgt cgacccggca tcaaccctca gccgggaaga gatcttcggc 1200
ccggtgctgg tggtgacccg cttcaaaagc gaagaagagg cgctaaagct cgccaatgac 1260
agcgactacg gcttgggcgc cgcggtgtgg acccgcgatc tctcccgcgc ccaccgcatg 1320
agccgccgcc tgaaggccgg ctcggtcttc gtcaacaact ataacgatgg tgatatgacc 1380
gttccgttcg gcggctacaa gcagagcggc aacgggcgcg ataaatcgct gcacgcgctg 1440
gaaaaattca ccgaactgaa aaccatctgg attgccctgg agtcttga 1488
<210> 7
<211> 30
<212> DNA
<213>artificial sequence
<400> 7
ccggaattca tgatgaattt tcagcacctg 30
<210> 8
<211> 27
<212> DNA
<213>artificial sequence
<400> 8
tccgagctct caagactcca gggcaat 27
Claims (8)
1. a kind of combined promoter, which is characterized in that the combined promoter contains 2-5 tac promoter core sequence；
Wherein, when the combined promoter contains 2 tac promoter core sequences, sequence is as shown in Seq ID No.2；
When the combined promoter contains 3 tac promoter core sequences, sequence is as shown in Seq ID No.3；
When the combined promoter contains 4 tac promoter core sequences, sequence is as shown in Seq ID No.4；
When the combined promoter contains 5 tac promoter core sequences, sequence is as shown in Seq ID No.5.
2. a kind of recombinant vector, which is characterized in that by combined promoter as described in claim 1, puuC aldehyde dehydrogenase gene
It is formed with pET-28a carrier, wherein puuC aldehyde dehydrogenase gene sequence is as shown in Seq ID No.6.
3. recombinant vector as claimed in claim 2, which is characterized in that the recombinant vector is expression vector.
4. the preparation method of recombinant vector as claimed in claim 2 or claim 3, characterized by the following steps:
1) double digestion is carried out to compound starting subsequence and pET-28a carrier respectively with restriction enzyme Bgl II/EcoR I；
2) electrophoresis, Ago-Gel recycle compound starting sub-piece and pET-28a carrier segments after digestion；
3) compound starting sub-piece is connected using ligase with pET-28a carrier segments, is obtained containing compound starting sub-piece
Carrier；
4) PCR amplification puuC gene order；
5) with restriction enzyme Sac I/EcoR I respectively to the carrier containing compound starting sub-piece made from step 3) and
PuuC gene order made from step 4) carries out double digestion；
6) electrophoresis, Ago-Gel recycle the carrier segments containing combined promoter and puuC genetic fragment after digestion；
7) carrier segments containing combined promoter are connected using ligase with puuC genetic fragment, obtains recombinant vector.
5. a kind of klepsiella pneumoniae recombinant bacterium for producing 3- hydracrylic acid, which is characterized in that the e coil k 1 pneumonia
Bacillus recombinant bacterium includes combined promoter as described in claim 1 or recombinant vector described in claim 2 or 3.
6. a kind of preparation method of the klepsiella pneumoniae recombinant bacterium of production 3- hydracrylic acid as claimed in claim 5,
It is characterized by comprising the following steps:
1) prepare competent cell: by wild type klepsiella pneumoniae culture activation, ice bath 30min, refrigerated centrifuge is gone
Clearly, twice, competent cell is made in washing；
2) electricity is put into after being sufficiently mixed the competent cell of recombinant vector as claimed in claim 2 or claim 3 and step 1) preparation
It is electroporated to hit cup progress；
3) electricity after will be electroporated turns to be applied to after liquid is mixed with culture medium in resistance solid medium, is produced through screening
The klepsiella pneumoniae recombinant bacterium of 3- hydracrylic acid.
7. a kind of for cultivating the fermentation medium of klepsiella pneumoniae recombinant bacterium as claimed in claim 5, feature exists
In the formula of the fermentation medium are as follows: KH2PO42.7g/L；NH4NO32.4g/L；K2HPO4·3H2O 4.2g/L；(NH4)2SO42g/L；CaCO30.15g/L；MgSO4·7H2O 0.12g/L；NaH2PO41.6g/L；EDTANa20.001g/L；Lemon
Sour 0.06g/L；Glycerol 40g/L；Yeast powder 4g/L；Microelement 1.25mL/L；
Trace element solution: Na2MO4·2H2O 0.045g/L；FeSO4·7H2O 1.7g/L；CoCl2·2H2O 0.12g/L；
ZnCl20.05g/L；NiCl2·6H2O 0.025g/L；H3BO30.16g/L；MnCl2·4H2O 0.1g/L；CuCl2·2H2O
0.06g/L；HCl (37%) 3.6mL.
8. a kind of combined promoter as described in claim 1 or recombinant vector as claimed in claim 2 or claim 3 are improving pneumonia gram
Application in the 3- hydracrylic acid yield of thunder Bai Shi bacillus.
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