CN108102964B - A kind of trans-4-hydroxy-l-proline synthesis bacterial strain and its L-PROLINE '-hydroxylase gene and application - Google Patents
A kind of trans-4-hydroxy-l-proline synthesis bacterial strain and its L-PROLINE '-hydroxylase gene and application Download PDFInfo
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Abstract
The present invention provides a kind of trans-4-hydroxy-l-proline synthesize bacterial strain, the bacterial strain be bacillus cereus (Bacillus cereus) HBU-AI, depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.14164, and the deposit date is on Mays 17th, 2017.The present invention also provides the L-PROLINE '-hydroxylase gene sequences of above-mentioned bacterial strains, and the L-PROLINE '-hydroxylase gene is applied to the production of trans-4-hydroxy-l-proline.The result shows that, the ability of engineered strain containing L-PROLINE '-hydroxylase gene synthesis of trans -4-hydroxy-L-proline with higher, ferment 52 h when, Hydroxyproline concentration is up to 41.6 g/L in fermentation liquid, and the residual concentration of L-PROLINE is 0.8 g/L in product.
Description
Technical field
The invention belongs to microorganisms and gene engineering technology field, relate in particular to a kind of trans- -4- hydroxyl-L- dried meat ammonia
Acid synthesis bacterial strain and its L-PROLINE '-hydroxylase gene and application.
Background technique
Trans-4-hydroxy-l-proline (trans- 4-Hydroxy-L-proline,trans- Hyp) it is imino acid,
It is the product after L-PROLINE (L-Pro) hydroxylating, is easy to derivative, it is anti-can be used for the southern class of a new generation's training for pharmacological activity multiplicity
The synthesis of a variety of new creating pharmaceuticals such as raw element, antineoplastic, antihypertensive and novel stomach medicine.Due to anti-oxidant, anti-spoke
The effect penetrated, the substance also have important application in cosmetic field.
The production method of trans-4-hydroxy-l-proline mainly has biological extraction method and biological enzyme conversion method.Biology
Extraction method was waited mainly using animal collagen as raw material by strong acid hydrolysis, nitrite-oxidizing and ion-exchange resin purification
Journey preparation, although the method technology maturation, cost of material is high, and recovery rate is low, and " three wastes " discharge amount is big, seriously polluted.Biology
Enzyme catalysis method utilizes the catalysis characteristics of proline hydroxylase, is obtained instead using L-PROLINE or glucose as raw material by microbe conversion
Formula -4-hydroxy-L-proline.Biotransformation method reaction condition compared with biological extraction method is mild, and low energy consumption, pollutes small.
Currently, the bacterial strain with trans-4-hydroxy-l-proline synthesis function has been reported, comprising: helix poly spore category bacterium
(Clonostachys cylindrospora), streptomycete (Streptomycete), ash slightly red streptomyces P-8648
(Streptomyces griseoviridus), refer to sporangiocyst bacterium (Dactylosporangium sp. strain RH1).From finger
The gene of isolated coding proline hydroxylase (P4Hs) is widely used in Escherichia coli in sporangiocyst bacterium
(Escherichia coli) and corynebacterium glutamicum (Corynebacterium glutamicum) genetic engineering bacterium
In building.Such as it is disclosed in patent document (application number 201510899127.6) a kind of for producing trans- -4- hydroxyl
Base-L-PROLINE recombinant bacterial strain, the bacterial strain are by L-PROLINE '-hydroxylase gene (from sporangiocyst bacterium is referred to), wild type L- paddy
Histidine kinase gene, L- paddy ammonia phthalein phosphoric acid reduction enzyme gene and resistant gene are cloned on expression vector respectively, recycle RED weight
Group technology is integrated into genome of E.coli and obtains recombinant bacterial strain.The fermentation that the recombinant bacterial strain is applied to hydroxyproline is raw
It produces, is detected, after the 70h that ferments, the concentration of hydroxyproline is 28.8g/L in fermentation liquid, and the concentration of L-PROLINE is 2.4g/L.By
This using existing method (gene and its recombinant bacterial strain of the coding proline hydroxylase (P4Hs) of originated from fungus) as it can be seen that produced
Efficiency is lower, and the residual quantity of L-PROLINE is higher.Not yet obtaining at present has trans-4-hydroxy-l-proline production capacity
Simple bacterium bacterial strain and novel '-hydroxylase gene separation.
Summary of the invention
It is an object of the invention to provide a kind of simple bacterial strain with trans-4-hydroxy-l-proline production capacity,
To the problems of existing method.
The second object of the present invention is to provide a kind of derived from above-mentioned bacterial strains, raw with trans-4-hydroxy-l-proline
The L-PROLINE '-hydroxylase gene of production capacity power, further to overcome existing trans-4-hydroxy-l-proline production method efficiency
The problems such as L-PROLINE residual quantity low, in synthetic product is high.
The third object of the present invention is to provide a kind of trans-4-hydroxy-l-proline synthesis bacterial strain and its L-PROLINE
The purposes of '-hydroxylase gene.
An object of the present invention is achieved through the following technical solutions: a kind of trans-4-hydroxy-l-proline synthesis
Bacterial strain, the bacterial strain be bacillus cereus (Bacillus cereus) HBU-AI, depositary institution is Chinese microorganism strain guarantor
Administration committee's common micro-organisms center is hidden, deposit number is CGMCC No.14164, and the deposit date is on Mays 17th, 2017.
HBU-AI bacterial strain of the invention is by obtaining in air, and acquisition methods are as follows:
(1) the L-PROLINE standard aqueous solution of 50 g/L is prepared, and under the conditions of 20 DEG C ~ 30 DEG C, is put in air
It sets one week.
(2) by solution sterile water gradient dilution to 10-7, choosing dilution gradient is 10-3、10-4、10-5Dilution 100
μ L is applied on LB plate, 30 DEG C of constant temperature incubation 2d.
(3) it is observed that colonial morphology is similar under different dilutions, picks them separately single bacterium and falls within the LB liquid containing L-PROLINE
In body culture medium, 37 DEG C, 200 rpm are cultivated, and after 48 h are centrifuged 2 min, are obtained phase respectively fermentation liquid in 9000 r/min
The supernatant of corresponding bacterium colony.
(4) the L-PROLINE activity of conversion of each bacterium colony is detected:
It takes the 50 μ L of supernatant of corresponding bacterium colony to be added in 1.5 mL centrifuge tubes respectively, 0.125 is added into centrifuge tube
200 μ L of mol/L sodium tetraborate aqueous solution, 410 μ L of pure water, 320 μ L of acetonitrile, the acetonitrile solution of 0.38 mol/L FMOC-Cl
20 μ L, are placed in after vortex vortex mixer shakes 5 s and stand 10 min, finally cross 0.45 μm of miillpore filter and analyze in RP-HPLC.
Liquid-phase condition is as follows: chromatographic column be Agilent Extend C-18(4.6 mm × 250 mm, 5 μm, Agilent
Company), mobile phase A is 0.1% trifluoroacetic acid aqueous solution, and Mobile phase B is 0.1% trifluoroacetic acid acetonitrile solution, Detection wavelength 263
Nm, 5 μ L of sample volume；
Condition of gradient elution is as follows: 0-5 min(30% B), 5-8 min(30%-40% B), 8-15 min(40% B),
15-20 min(40%-50% B), 20-25 min (50% B), 25-30 min(50%-90% B), 30-38 min(90% B),
38-41min(90%-30% B), 41-45 min(30% B).
(5) the higher bacterial strain of activity of conversion is chosen as candidate strain and verifies the activity of conversion of each bacterial strain, L-PROLINE
For the detection method of activity of conversion with (4) step, testing result is as shown in table 1.
Table 1:
The result shows that: No. 3 bacterial strains obtained from air be one plant can Efficient Conversion L-PROLINE be trans- -4- hydroxyl
Base-L-PROLINE bacterial strain, changing effect is as shown in Figure 1, the L-PROLINE of bacterial strain conversion 3g/L generates trans- -4- hydroxyl
Base-L-PROLINE concentration is 1.86 g/L, and obtaining this bacterial strain conversion L-PROLINE is turning for trans-4-hydroxy-l-proline
Rate is 62.0 %.
The identification of No. 3 bacterial strains
Thalli morphology observation, physiological and biochemical analysis are carried out to the single colonie with hydroxylase activity of acquisition, and to bacterial strain
Carry out the analysis of 16S rDNA sequence.
Strain classification feature is respectively as follows:
2 ~ 3 millimeters of colony diameter, white colony, relatively dry, edge is irregular, when being cultivated in LB liquid medium, bacterium
Body is in short and small straight-bar under an optical microscope；
When being detected using API50 CH kit, bacterial strain can utilize D-ribose, glucose, fructose, mannose, N- acetyl-
Aminoglucose, salicin, maltose, sucrose, trehalose, starch, glycogen, aesculin ferrum citricum.
According to the strain construction phylogenetic tree that 16 srDNA sequences obtain above-mentioned screening, as shown in Fig. 2, utilizing
API50 CH kit carries out API detection to above-mentioned bacterial strains, and the bacterial strain is finally accredited as bacillus cereusBacillus Cereus,Number is HBU-AI.Preservation is carried out to the bacterial strain, depositary institution is that China Committee for Culture Collection of Microorganisms is general
Logical microorganism center, deposit number are CGMCC No.14164, and the deposit date is on Mays 17th, 2017.
A kind of L-PROLINE '-hydroxylase gene from above-mentioned trans-4-hydroxy-l-proline synthesis bacterial strain, the L-
The nucleotide sequence of proline hydroxylase gene is named as Bp4h as shown in SEQ ID NO.1.
A kind of recombinant vector containing above-mentioned L-PROLINE '-hydroxylase gene.Further, the recombinant vector is above-mentioned
The recombination of L-PROLINE '-hydroxylase gene, Pidolidone kinase gene and L- glutamyl phosphate reductase gene coexpression carries
Body；Wherein, Pidolidone kinase gene (ProB) and L- glutamyl phosphate reductase gene (ProA) derive from Escherichia coli
DH5a bacterial strain.
A kind of engineering colon bacillus BL21(DE3 by above-mentioned construction of recombinant vector).
The construction method of engineering bacteria containing above-mentioned L-PROLINE '-hydroxylase gene, comprising the following steps:
(1) amplification of L-PROLINE '-hydroxylase gene Bp4h: to L-PROLINE '-hydroxylase gene Bp4h primers
Right, F-Bp4h, as shown in SEQ ID NO.2 and R-Bp4h expands to obtain Bp4h using round pcr as shown in SEQ ID NO.3
Segment；
(2) acquisition of Pidolidone kinase gene ProB and L- glutamyl phosphate reductase gene ProA gene: with large intestine
The genomic DNA of bacillus DH5a bacterial strain is template, with primers F-proB, shown in SEQ ID NO.4 and R-proA, such as SEQ ID
Shown in NO.5, expand to obtain proBA segment using round pcr；
(3) building of recombinant bacterial strain: Bp4h segment and proBA segment are building up in expression vector pTRC99a, obtained
Recombinant vector is transformed into e. coli bl21 (DE3) bacterial strain, and induces its table by pTRC99a-Bp4h-proBA recombinant vector
It reaches.
A kind of application of above-mentioned L-PROLINE '-hydroxylase gene in production trans-4-hydroxy-l-proline.
The beneficial effects of the present invention are: obtaining from air one plant novel has synthesis of trans -4- hydroxyl-L- dried meat ammonia
The bacterium bacterial strain of sour ability, and the L-PROLINE '-hydroxylase gene sequence of the bacterial strain is obtained, with existing finger sporangiocyst bacterium
(DactylosporangiumSp.) p4h gene order (GeneBank accession number: D78338.1) similitude only has 84%, expands
The big source of '-hydroxylase gene.
Gene (ProBA gene), which is generated, using the L-PROLINE '-hydroxylase gene and L-PROLINE constructs bifunctional enzyme jointly
Expression vector, and further construct the engineering large intestine bar from one step Efficient Conversion of glucose for trans-4-hydroxy-l-proline
Bacterium.The engineering colon bacillus is applied to the production of trans-4-hydroxy-l-proline, (after fermentation 70h, is fermented with the prior art
The concentration of hydroxyproline is 28.8g/L in liquid, and the concentration of L-PROLINE is 2.4g/L) it compares, production efficiency of the present invention significantly mentions
Height, ferment 52 h when, Hydroxyproline concentration is up to 41.6 g/L in fermentation liquid, and the residual concentration of L-PROLINE is 0.8 in product
g/L。
Detailed description of the invention
Fig. 1 is the Chromatographic Comparison of fermentation front and back fermented and cultured based specimen when verifying bacterial strain HBU-AI activity of conversion of the present invention
Figure；Wherein, a is L-PROLINE and trans-4-hydroxy-l-proline standard chromatogram；B is fermentation primary fermentation bouillon-like
Product chromatogram；C is fermentation post-fermentation liquid sample chromatogram figure.
Fig. 2 is the phylogenetic tree of bacterial strain HBU-AI of the present invention.
Fig. 3 is the conversion Dynamic Graph that engineering bacteria fermentation of the present invention produces trans-4-hydroxy-l-proline.
Specific embodiment
Below by embodiment, the present invention will be described in detail, and experimental method used in following embodiments is unless otherwise specified
For conventional method.The materials, reagents and the like used in the following examples, unless otherwise specified, commercially
It obtains.
Embodiment 1:
Bacterial strain HBU-AI construction of gene library and extraction '-hydroxylase gene
(1) genomic DNA for extracting bacillus cereus HBU-AI, using the total of Sau3AI digestion bacterial strain HBU-AI
DNA digests system are as follows: restriction endonuclease 2 uL, DNA 5 uL of 1.5 uL, 10 X Buffer, total volume 50 uL, remaining dd
H2O is supplied, and 37 DEG C, reacts 60min.
(2) it is connected using BamHI digested vector pUC19, digests system are as follows: restriction endonuclease 1.5 uL of 2 uL, DNA,
10 X Buffer, 5 uL, 50 uL of total volume, remaining dd H2O is supplied, and 37 DEG C, reacts 15min.
(3) DNA fragmentation that digestion obtains is connected with the carrier pUC19 through digesting, connection product electrotransformation recipient bacterium E
.coli DH5a, bacterium colony are coated on the LB plate containing ampicillin, and 16-20 h is cultivated in 37 DEG C of inversions, will be grown
Single colonie be inoculated into 96 orifice plates of the LB fluid nutrient medium accordingly containing 3 g/L L-PROLINEs, in 37 DEG C cultivate
The bacterial strain with trans-4-hydroxy-l-proline activity of conversion is checked with chloramine-t method after 24 h.
(4) bacterial strain for choosing Xiang Yingyou trans-4-hydroxy-l-proline activity of conversion is transferred to 5 mL LB(containing 50 mg/
ML Amp) in, 37 DEG C, 200 rpm overnight incubations take broth extraction plasmid, and plasmid is sent to Beijing Hua Da by digestion verification
Sequencing is carried out, used method is the sequencing of Sanger method.It analyses and compares to obtain L-PROLINE hydroxylase through sequence B last
Gene (Bp4h gene) sequence is as shown in SEQ ID NO.1.
(5) activity of above-mentioned L-PROLINE '-hydroxylase gene is verified:
To the '-hydroxylase gene design primer pair after sequence verification: F-Bp4h:ATGCTGACCCCGAC(SEQ ID NO.2
It is shown), shown in R-Bp4h:CTAGACGGGCTGGGCCAGCGCGAAG(SEQ ID NO.3)；Amplification obtains Bp4h segment.Its
In, PCR reaction system are as follows: 10 X Buffer 5uL, dNTP 1uL, DNA profiling 1uL, primers F 2uL, primer R 2uL,
Taq enzyme 0.5uL, ddH2O 38.5uL；Response procedures are as follows: 95 DEG C of 3min；95℃ 15s, 56℃ 15s, 72℃ 60s,
35 circulations；72℃ 5min；The Bp4h segment of amplification is connected with cloning vector PMD19, obtains PMD19-Bp4h.
PMD19-Bp4h is transferred to escherichia coli DH5a bacterial strain, screens positive transformant, after sequence verification is errorless, extracts matter
Grain, carries out double digestion, the expression vector pTRC99a phase of the target fragment of recycling and same double digestion with EcoRI and HindIII
Even, wherein digestion system are as follows: plasmid 15uL, 10 X M Buffer 5uL, EcoRI 2uL, HindIII 2uL, ddH2O
26uL, 37 DEG C of digestions are stayed overnight；Linked system are as follows: target fragment 7uL, carrier 1uL, 10 X Buffer 1uL, T4 ligases
1uL, 16 DEG C of connection 3h, obtains pTRC99a-Bp4h.
PTRC99a-Bp4h is converted into e. coli bl21 (DE3), SDS-PAGE analysis is carried out after IPTG is induced,
And fermenting experiment is carried out to thallus, the activity that L-PROLINE is converted to it carries out HPLC analysis (liquid-phase condition is with embodiment 1).
The result shows that: it obtains more than 1000 transformants altogether according to library constructing method, all transformants is subjected to L- dried meat ammonia
Acid activity detection finally found that a transformant has trans-4-hydroxy-l-proline activity of conversion, through sequencing analysis, the hydroxyl
Change enzyme gene sequence and refer to sporangiocyst bacterium (Dactylosporangium sp.) p4h gene order (GeneBank accession number:
D78338.1) similitude only has 84%.'-hydroxylase gene after sequence verification is cloned into expression vector pTRC99a, conversion is extremely
E. coli bl21 (DE3) has protein expression band, fermented L- dried meat in molecular weight through SDS-PAGE analysis for 30 KDa or so
The verifying of propylhomoserin activity, the recombination bacillus coli of the hydroxylase sequence construct have the energy of production trans-4-hydroxy-l-proline
Power.
The building of the engineering colon bacillus of 2 L-PROLINE '-hydroxylase gene of embodiment and ProBA gene co-expressing
(1) acquisition of ProBA gene
Using the genomic DNA of escherichia coli DH5a bacterial strain as template, with primers F-proB:TATGGTAC
Shown in CAACTGCCGCTAGGCTTGCTG(SEQ ID NO.4) and R-proA:GTAGGATCCCGTCAATGGCCTTGTGAATC
(shown in SEQ ID NO.5) amplification obtains proBA genetic fragment；It is connected with cloning vector PMD19, converts Escherichia coli
DH5a bacterial strain screens positive transformant, the correctness for verifying sequence is sequenced.
(2) recombinant bacterial strain constructs
It is template with plasmid PMD19-Bp4h above-mentioned, carries out double digestion with EcoRI and BamHI respectively, recycles purpose piece
Section；Double digestion is carried out to expression vector pTRC99a with EcoRI and BamHI, makes its linearisation, recycles target fragment；By above-mentioned two
A segment connects 1 h with T4 ligase at 16 DEG C.Connection product converts e. coli strain bl21, screens positive transformant,
PTRC99a-Bp4h(other conditions are obtained with embodiment 2).
It is carried out respectively with BamHI and HindIII with the operation of similar embodiment 2 using plasmid PMD19-proBA as template
Double digestion, the target fragment of recycling；PTRC99a-Bp4h is subjected to double digestion with BamHI and HindIII, recycles target fragment；
Above-mentioned two segment is connected, connection product converts e. coli strain bl21, screens positive transformant, obtains recombinant bacterial strain.
(3) recombinant bacterial strain is verified by the activity of one step Synthesis trans-4-hydroxy-l-proline of glucose:
Seed liquor preparation method: taking out the glycerol tube of the bacterial strain frozen from -80 DEG C of refrigerators, crosses on LB solid medium, and 37
DEG C incubator culture, picking single colonie access in 20 mL LB liquid, and 37 DEG C of cultures, cultured bacterium solution is as seed liquor.
Shake-flask seed is forwarded in the 5 L fermentors that liquid amount is 1.5 L with the inoculum concentration of 1vol%, fermentation medium
Ingredient are as follows: 20.0 g/L of glucose, 3.0 g/L of dikalium phosphate, 0.5 g/L of sodium chloride, 1.0 g/L of ammonium chloride, ferrous sulfate 0.3
G/L, 5.0 g/L of peptone, 5.0 g/L of yeast extract, ammonia benzyl mycin 0.1g/L.
The cultivation temperature of fermentor is set as 28 DEG C, initial to stir 200 r/min, DO automatic control is closed in 10-30%(and revolving speed
Connection), 1.8 L/min of ventilatory capacity；Maintain the concentration of glucose in fermentor in 20.0 g/L, fermentation process by continuously adding
In every 2h sampling, measure its pH value, thallus OD value, the content of L-PROLINE and hydroxyproline, thallus OD600And dry cell weight
(CDW) reduction formula is as follows: OD600 4.24=1 gCDWL-1。
The result shows that: proBA gene, sequencing knot are obtained by template amplification of the genomic DNA of escherichia coli DH5a bacterial strain
Fruit identification is errorless, and the size of the genetic fragment is 2358bp.By after sequence verification '-hydroxylase gene and proBA gene lead to respectively
The mode for crossing digestion connection is connected in expression vector pTRC99a, conversion to e. coli bl21 (DE3).Through SDS-PAGE points
Analysing in molecular weight is the protein band that 30 KDa or so have hydroxylase；Molecular weight be 40 KDa or so have Pidolidone kinases and
The protein band of L-Glutamine phosphate dehydrogenase.
It is verified through 5L ferment tank glucose activity, as shown in figure 3, the recombination bacillus coli of the hydroxylase sequence construct
Ability with production trans-4-hydroxy-l-proline, and when fermenting 52 h, Hydroxyproline concentration is reachable in fermentation liquid
41.6 g/L, the residual concentration of L-PROLINE are 0.8 g/L.It follows that L-PROLINE '-hydroxylase gene (nucleotide of the present invention
Sequence is as shown in SEQ ID NO.1) relative to existing originated from fungus '-hydroxylase gene (referring to sporangiocyst bacterium '-hydroxylase gene) in hydroxyl
There is advantage outstanding in proline production application.
SEQUENCE LISTING
<110>University Of Hebei
<120>a kind of trans-4-hydroxy-l-proline synthesis bacterial strain and its L-PROLINE '-hydroxylase gene and application
<130>
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 819
<212> DNA
<213> Bp4h
<400> 1
atgctgaccc cgaccgaacc gaaacagtat cgtgaagcgg gctatctgct gattgaagat 60
ggcctgggcc cgcgtgaagt tgactgcctg cgtcgtgctg ctgctgctct gtacgctcag 120
gactctccgg accgtaccct ggaaaaagac ggtcgtaccg ttcgtgctgt tcacggttgc 180
caccgtcgtg acccggtttg ccgtgacctg gttcgtcacc cgcgtctgct gggtccggct 240
atgcagatcc tgtctggtga cgtttacgtt caccagttca aaatcaacgc taaagctccg 300
atgaccggtg acgtttggcc gtggcaccag gactacatct tctgggctcg tgaagacggt 360
atggaccgtc cgcacgttgt taacgttgct gttctgctgg acgaagctac ccacctgaac 420
ggtccgctgc tgttcgttcc gggtacccac gaactgggtc tgatcgacgt tgaacgtcgt 480
gctccggctg gtgacggtga cgctcagtgg ctgccgcagc tgtctgctga cctggactac 540
gctatcgacg ctgacctgct ggctcgtctg accgctggtc gtggtatcga atctgctacc 600
ggtccggctg gttctatcct gctgttcgac tctcgtatcg ttcacggttc tggtaccaac 660
atgtctccgc acccgcgtgg tgttgttctg gttacctaca accgtaccga caacgctctg 720
ccggctcagg ctgctccgcg tccggaattc ctggctgctc gtgacgctac cccgctggtt 780
ccgctgccgg ctggcttcgc gctggcccag cccgtctag 819
<210> 2
<211> 14
<212> DNA
<213> F-Bp4h
<400> 2
atgctgaccc cgac 14
<210> 3
<211> 25
<212> DNA
<213> R-Bp4h
<400> 3
ctagacgggc tgggccagcg cgaag 25
<210> 4
<211> 29
<212> DNA
<213> F-proB
<400> 4
tatggtacca actgccgcta ggcttgctg 29
<210> 5
<211> 29
<212> DNA
<213> R-proA
<400> 5
gtaggatccc gtcaatggcc ttgtgaatc 29
Claims (1)
1. a kind of trans-4-hydroxy-l-proline synthesizes bacterial strain, characterized in that the bacterial strain is bacillus cereus
(Bacillus cereus) HBU-AI, depositary institution is China Committee for Culture Collection of Microorganisms's common micro-organisms center,
Deposit number is CGMCC No.14164, and the deposit date is on Mays 17th, 2017.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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