CN106282085A - A kind of method utilizing Corynebacterium crenatum resting cell glucose synthesis acetoin - Google Patents
A kind of method utilizing Corynebacterium crenatum resting cell glucose synthesis acetoin Download PDFInfo
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- CN106282085A CN106282085A CN201610979546.5A CN201610979546A CN106282085A CN 106282085 A CN106282085 A CN 106282085A CN 201610979546 A CN201610979546 A CN 201610979546A CN 106282085 A CN106282085 A CN 106282085A
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Abstract
The invention discloses a kind of method utilizing Corynebacterium crenatum resting cell glucose synthesis acetoin, belong to genetic engineering field.The present invention will derive from bacillus subtilis alsSD gene transformation in Corynebacterium crenatum Corynebacterium crenatum SDNN403, knock out its lactate dehydrogenase gene (ldh) and 2 simultaneously, 3 butanediol dehydrogenase genes (butA), build C.crenatum Δ butA Δ ldh/pXMJ19 alsSD.Based on the genetic engineering bacterium obtained, resting cell 100g/L glucose synthesis acetoin, yield reaches 30 ± 1.2g/L, and molar yield reaches the 61% of theoretical value.This bacterium has certain industrialization potential in terms of utilizing cheap glucose by one step to convert production acetoin.
Description
Technical field
The present invention relates to a kind of method utilizing recombinant corynebacterium crematum resting cell glucose synthesis acetoin, belong to
Gene engineering technology field.
Background technology
Acetoin (3-hydroxy-2-butanone) is naturally occurring in some fruit, is a kind of important flavorant, it is possible to make
For synthesizing the precursor substance of other compound.At present the main production process of acetoin be divided into chemical synthesis, enzyme transforming process and
Microbe fermentation method.Along with the day by day exhausted of fossil resource and the increase of ambient pressure, utilize microorganism to convert cheap raw material and send out
Ferment produces large chemical products more and more to be favored；But owing to Production by Microorganism Fermentation acetoin can be along with more
The synthesis of by-product, microbial metabolism approach the most reasonable in design, it is achieved microbe whole-cell transforming glucose is directly synthesized
Acetoin is significant and using value.
Summary of the invention
The invention provides a kind of structure recombinant corynebacterium crematum, it is achieved its resting cell glucose synthesis acetoin
Method.Bacillus subtilis alsSD gene transformation will be derived to Corynebacterium crenatum Corynebacterium crenatum
In SDNN403, (this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number CGMCC
NO.0890；Declare Patents: ZL 03112896.3), and knock out its lactate dehydrogenase gene (ldh) and 2,3-butanediol and take off
Hydrogen enzyme (butA), builds C.crenatum Δ butA Δ ldh/pXMJ19-alsSD.Based on the genetic engineering bacterium obtained, full cell
The yield of transforming glucose synthesis acetoin reaches 30 ± 1.2g/L, and molar yield reaches the 61% of theoretical value.
Following research method and microorganism culturing condition is used during invention:
1. culture medium and condition of culture
(1) escherichia coli:
Condition of culture: rotary shaker 37 DEG C, 160r/min.
LB culture medium (g/L): yeast extract 5, tryptone 10, NaCl 10, pH 7.0；Solid LB media need to add
The agar adding 2%；
(2) Corynebacterium crenatum:
Condition of culture: cultivation temperature 30 DEG C, rotating speed 180r/min.
Seed culture medium is LBG culture medium, i.e. LB culture medium adds the glucose of 0.5%；
The competence culture medium converted for electricity: LB culture medium adds the glycine of 3%, the Tween 80 of 0.1%, is used for
The cultivation of C.crenatum competent cell；
Fermentation medium (g/L): yeast powder 8, (NH4)2SO440, MgSO4·7H2O 0.5, KCl 1, KH2PO41.5,
MnSO4·H2O 0.02, FeSO4·7H2O 0.02, CaCO330；
2. the extraction of chromogene group DNA and the extraction of plasmid DNA
C.crenatum and B.subtilis chromogene group is according to bacterial chromosomal genes group DNA extraction kit phase
Close operating instruction to extract.The extraction of E.coli plasmid DNA uses test kit to extract, and the recombiant plasmid of C.crenatum is carried out
After need to being initially charged lysozyme function cells wall a period of time during extraction, re-use test kit and extract recombiant plasmid.
3. the preparation of competent escherichia coli cell and conversion
CaCl2Method prepares E.coli competent cell；42 DEG C of thermal shock Transformed E .coli JM109, resist through ampicillin
Property plate screening obtain positive transformant.
The preparation of 4.C.crenatum competent cell and electricity method for transformation
(1) inoculation on the fresh inclined-plane of picking is in the LB culture medium containing 0.5% glucose, 30 DEG C, 200r/min
Cultivate 12h；(2) transferring into competence cultivation based on 30 DEG C by inoculum concentration 1%, 200r/min cultivates 3-5h to OD562It is about 0.9；
(3) after cell is cultivated and terminated, first by bacterium solution ice bath 20min, centrifugal 10min, 4 DEG C, 8000r/min；(4) 10% glycerol of pre-cooling
Washed cell, finally with the 10% glycerol Eddy diffusion cell of 500 μ L, 1.5mL centrifuge tube subpackage, often pipe 70 μ L, competence is thin
Born of the same parents can be used for electricity conversion；(5), when electricity turns, add the recombiant plasmid of 3 μ L to the competent cell in often pipe (4), place on ice
10min, adds in the pole cup of pre-cooling and shocks by electricity, voltage: 1.8kV, time: 5ms；(6), after, add containing 0.5% glucose
LB culture medium 800 μ L, and it is positioned over water-bath 6min in 46 DEG C of water-baths, after completing, 30 DEG C, 200r/min cultivates 2h；(7) train afterwards
Support after terminating, 8000r/min, centrifugal 10min, remove major part supernatant, leave and take 200 μ L, suspension bacteria liquid, be coated with Km resistant panel
Or Cm resistant panel, 30 DEG C of incubators are cultivated 60h, are observed transformant growing state.
Detailed description of the invention
The acquisition of embodiment 1 excalation type gene Δ butA and Δ ldh
(1) Δ butA and the acquisition of Δ ldh: with C.crenatum SDNN403 chromosomal DNA as template, respectively with
PbutA1F, PbutA3R and PbutA2R, PbutA4F are that primer carries out first round PCR, it is thus achieved that two kinds of PCR primer；Secondly, by upper
State and add second according to the ratio of 1:1 after two kinds of PCR primer glue reclaim and take turns PCR reaction system as template, with butA gene
Upstream and downstream primer PbutA1F, PbutA2R be primer carry out two take turns PCR reaction, it is thus achieved that gene deletion type fragment Δ butA.With
C.crenatum SDNN403 chromosomal DNA is template, enters with Pldh1F, Pldh3R and Pldh2R, Pldh4F for primer respectively
Row first round PCR, it is thus achieved that two kinds of PCR primer；Secondly, the is added according to the ratio of 1:1 after being reclaimed by above two PCR primer glue
Two take turns PCR reaction system as template, and carrying out two with upstream and downstream primer Pldh1F, Pldh2R of ldh gene for primer, to take turns PCR anti-
Should, it is thus achieved that gene deletion type fragment Δ ldh.Primer sequence is as follows:
PbutA1F:ACCGGAATTCATGAGCAAAGTTGCAATGGT
PbutA2R:ACCGAAGCTTCTAGTTGTAGAGCATGCCGC
PbutA3R:TAGGCATTGACGGTGTGACCCTTGGCATCAATTGCACTGTCGAAATTA GC
PbutA4F:GCTAATTTCGACAGTGCAATTGATGCCAAGGGTCACACCGTCAATGCC TA
Pldh1F:ACCGGAATTCATGAAAGAAACCGTCGGT
Pldh2R:ACCGAAGCTTTTAGAAGAACTGCTTCTG
Pldh3R:GTAGGATTGCGCGGGTGATGCGAGCCTTCGCAGTCAGCGTAGGTTCCCT T
Pldh4F:AAGGGAACCTACGCTGACTGCGAAGGCTCGCATCACCCGCGCAATCCTA C
The structure of embodiment 2 butA Yu ldh Loss-of-function recombinant bacterium C.crenatum Δ butA Δ ldh
Gene deletion type fragment Δ butA Yu pK18mobsacB embodiment 1 obtained is through EcoR I and Hind III double digestion
It is attached afterwards, Transformed E .coli JM109, construction recombination plasmid pK18-Δ butA；The Homologous integration plasmid that will build
PK18-Δ butA electroporated C.crenatum SDNN403, the finally coating resistant panel containing kanamycin, 30 DEG C of cultivations
48-60h, son to be transformed grows, and this is homologous recombination transformant for the first time, it is characterized by that pK18mobsacB-Δ butA recombinates
Plasmid integration is in C.crenatum SDNN403 chromogene group；Then by homologous recombination transformant for the first time containing 10%
Carrying out second time homologous recombination on the LBG solid medium (LBGS) of sucrose, the sucrose in high concentration is coerced under pressure, sacB base
Because integrating from chromogene group with Km resistant gene, the butA in C.crenatum SDNN403 chromogene group
Gene is replaced by Δ butA, successfully builds Gene Deletion bacterial strain C.crenatum Δ butA.Single bacterium on picking LBGS flat board
Fall, streak culture on LBG flat board, the bacterial strain after twice homologous recombination is carried out Gene Deletion bacterium colony PCR identify (with
The upstream and downstream primer of butA gene).To verify that correct butA Gene Deletion Strain Designation is C.crenatum Δ butA.
The construction method of ldh and butA dual-gene deletion form bacterial strain ibid, the gene deletion type fragment that embodiment 1 is obtained
Δ ldh with pK18mobsacB is attached after EcoR I and Hind III double digestion, Transformed E .coli JM109, builds restructuring
Plasmid pK18mobsacB-Δ ldh；The Homologous integration plasmid electroporated C.crenatum Δ butA that will build, to through two
Bacterial strain after secondary homologous recombination carries out Gene Deletion bacterium colony PCR and identifies (with the upstream and downstream primer of ldh gene).By correct for checking
ButA gene and ldh Gene Double deletion form Strain Designation be C.crenatum Δ butA Δ ldh.
The structure of embodiment 3 recombinant bacterium C.crenatum Δ butA Δ ldh/pXMJ19-alsSD
With Bacillus subtilis168 chromosome as template, carry out with PalsSDF and PalsSDR for primer respectively
PCR, genetic fragment alsSD that PCR is obtained (SEQ ID NO.1) and plasmid pXMJ19 Sma I and Sac I double digestion, then
Glue reclaims, and connects pXMJ19 Yu alsSD fragment, Transformed E .coli JM109, construction recombination plasmid with T4DNA ligase
pXMJ19-alsSD.Primer sequence is as follows:
PalsSDF:ACCGCCCGGGAAAGGAGGGAAATCATGACAAAAGCAACAAAAG
PalsSDR:ACCGGAGCTCTTATTCAGGGCTTCCTTC
The recombiant plasmid pXMJ19-alsSD electroporated C.crenatum Δ butA Δ ldh that will build, at Km flat board
On filter out positive bacterium colony, be recombinant bacterium C.crenatum Δ butA Δ ldh/pXMJ19-alsSD.
Embodiment 4 recombinant bacterium C.crenatum Δ butA Δ ldh/pXMJ19-alsSD transforming glucose synthesis acetoin
(1) seed activation: C.crenatum Δ butA Δ ldh/pXMJ19-alsSD is seeded to according to the inoculum concentration of 1%
In 10mLLBG culture medium, 30 DEG C, 180r/min cultivates about 12h；
(2) yeast culture: seed liquor be forwarded in fermentation medium according to the inoculum concentration of 5%, 30 DEG C, 180r/min trains
Support about 24h；
(3) resting cell: the C.crenatum Δ butA Δ ldh/pXMJ19-alsSD fermentation liquid of 24h will be cultivated
10000r/min is centrifuged 30min, collects somatic cells, according to thalline weight in wet base: conversional solution volume is the ratio of 1:20, is hanged by cell
Float on resting cell culture fluid (g/L): KH2PO40.5, K2HPO4·3H2O 0.5, MgSO4·7H2O 0.5, FeSO4·
7H2O 6, MnSO4·H2O 4.2, glucose 100, conversion process adds the CaCO of 30g/L3For maintaining pH.Convert 60h,
The acetoin of available 30 ± 1.2g/L and a small amount of 2,3-butanediol (1.2 ± 0.2g/L), molar yield reaches theoretical value
61%.
Although the present invention is open the most as above with preferred embodiment, but it is not limited to the present invention, any is familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention
Enclosing should be with being as the criterion that claims are defined.
Sequence table
<110>Southern Yangtze University
<120>a kind of method utilizing Corynebacterium crenatum resting cell glucose synthesis acetoin
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 2587
<212> DNA
<213>PCR obtains
<400> 1
1 CCCGGGAAAG GAGGGAAATC ATGACAAAAG CAACAAAAGA TGACAAAAGC AACAAAAGAA
61 CAAAAATCCC TTGTGAAAAA CAGAGGGGCG GAGCTTGTTG TTGATTGCTT AGTGGAGCAA
121 GGTGTCACAC ATGTATTTGG CATTCCAGGT GCAAAAATTG ATGCGGTATT TGACGCTTTA
181 CAAGATAAAG GACCTGAAAT TATCGTTGCC CGGCACGAAC AAAACGCAGC ATTCATGGCC
241 CAAGCAGTCG GCCGTTTAAC TGGAAAACCG GGAGTCGTGT TAGTCACATC AGGACCGGGT
301 GCCTCTAACT TGGCAACAGG CCTGCTGACA GCGAACACTG AAGGAGACCC TGTCGTTGCG
361 CTTGCTGGAA ACGTGATCCG TGCAGATCGT TTAAAACGGA CACATCAATC TTTGGATAAT
421 GCGGCGCTAT TCCAGCCGAT TACAAAATAC AGTGTAGAAG TTCAAGATGT AAAAAATATA
481 CCGGAAGCTG TTACAAATGC ATTTAGGATA GCGTCAGCAG GGCAGGCTGG GGCCGCTTTT
541 GTGAGCTTTC CGCAAGATGT TGTGAATGAA GTCACAAATA CGAAAAACGT GCGTGCTGTT
601 GCAGCGCCAA AACTCGGTCC TGCAGCAGAT GATGCAATCA GTGCGGCCAT AGCAAAAATC
661 CAAACAGCAA AACTTCCTGT CGTTTTGGTC GGCATGAAAG GCGGAAGACC GGAAGCAATT
721 AAAGCGGTTC GCAAGCTTTT GAAAAAGGTT CAGCTTCCAT TTGTTGAAAC ATATCAAGCT
781 GCCGGTACCC TTTCTAGAGA TTTAGAGGAT CAATATTTTG GCCGTATCGG TTTGTTCCGC
841 AACCAGCCTG GCGATTTACT GCTAGAGCAG GCAGATGTTG TTCTGACGAT CGGCTATGAC
901 CCGATTGAAT ATGATCCGAA ATTCTGGAAT ATCAATGGAG ACCGGACAAT TATCCATTTA
961 GACGAGATTA TCGCTGACAT TGATCATGCT TACCAGCCTG ATCTTGAATT GATCGGTGAC
1021 ATTCCGTCCA CGATCAATCA TATCGAACAC GATGCTGTGA AAGTGGAATT TGCAGAGCGT
1081 GAGCAGAAAA TCCTTTCTGA TTTAAAACAA TATATGCATG AAGGTGAGCA GGTGCCTGCA
1141 GATTGGAAAT CAGACAGAGC GCACCCTCTT GAAATCGTTA AAGAGTTGCG TAATGCAGTC
1201 GATGATCATG TTACAGTAAC TTGCGATATC GGTTCGCACG CCATTTGGAT GTCACGTTAT
1261 TTCCGCAGCT ACGAGCCGTT AACATTAATG ATCAGTAACG GTATGCAAAC ACTCGGCGTT
1321 GCGCTTCCTT GGGCAATCGG CGCTTCATTG GTGAAACCGG GAGAAAAAGT GGTTTCTGTC
1381 TCTGGTGACG GCGGTTTCTT ATTCTCAGCA ATGGAATTAG AGACAGCAGT TCGACTAAAA
1441 GCACCAATTG TACACATTGT ATGGAACGAC AGCACATATG ACATGGTTGC ATTCCAGCAA
1501 TTGAAAAAAT ATAACCGTAC ATCTGCGGTC GATTTCGGAA ATATCGATAT CGTGAAATAT
1561 GCGGAAAGCT TCGGAGCAAC TGGCTTGCGC GTAGAATCAC CAGACCAGCT GGCAGATGTT
1621 CTGCGTCAAG GCATGAACGC TGAAGGTCCT GTCATCATCG ATGTCCCGGT TGACTACAGT
1681 GATAACATTA ATTTAGCAAG TGACAAGCTT CCGAAAGAAT TCGGGGAACT CATGAAAACG
1741 AAAGCTCTCT AGCACTCTGC GCATCACGAC ACTGTTTTAT GAACAGCACT AAATAAAAGG
1801 AGTGAAGGGA AATATGAAAC GAGAAAGCAA CATTCAAGTG CTCAGCCGTG GTCAAAAAGA
1861 TCAGCCTGTG AGCCAGATTT ATCAAGTATC AACAATGACT TCTCTATTAG ACGGAGTATA
1921 TGACGGAGAT TTTGAACTGT CAGAGATTCC GAAATATGGA GACTTCGGTA TCGGAACCTT
1981 TAACAAGCTT GACGGAGAGC TGATTGGGTT TGACGGCGAA TTTTACCGTC TTCGCTCAGA
2041 CGGAACCGCG ACACCGGTCC AAAATGGAGA CCGTTCACCG TTCTGTTCAT TTACGTTCTT
2101 TACACCGGAC ATGACGCACA AAATTGATGC GAAAATGACA CGCGAAGACT TTGAAAAAGA
2161 GATCAACAGC ATGCTGCCAA GCAGAAACTT ATTTTATGCA ATTCGCATTG ACGGATTGTT
2221 TAAAAAGGTG CAGACAAGAA CAGTAGAACT TCAAGAAAAA CCTTACGTGC CAATGGTTGA
2281 AGCGGTCAAA ACACAGCCGA TTTTCAACTT CGACAACGTG AGAGGAACGA TTGTAGGTTT
2341 CTTGACACCA GCTTATGCAA ACGGAATCGC CGTTTCTGGC TATCACCTGC ACTTCATTGA
2401 CGAAGGACGC AATTCAGGCG GACACGTTTT TGACTATGTG CTTGAGGATT GCACGGTTAC
2461 GATTTCTCAA AAAATGAACA TGAATCTCAG ACTTCCGAAC ACAGCGGATT TCTTTAATGC
2521 GAATCTGGAT AACCCTGATT TTGCGAAAGA TATCGAAACA ACTGAAGGAA GCCCTGAATA
2581 AGAGCTC
Claims (4)
1. one kind can resting cell glucose synthesis acetoin recombinant corynebacterium crematum C.crenatum Δ butA Δ
Ldh/pXMJ19-alsSD, it is characterised in that described recombinant bacterium have expressed the alsSD gene (sequence deriving from bacillus subtilis
Row are as shown in SEQ ID NO.1), knock out himself lactate dehydrogenase gene (ldh) and 2,3-butanediol dehydrogenase base simultaneously
Because of (butA).
Recombinant corynebacterium crematum C.crenatum Δ butA Δ ldh/pXMJ19-alsSD the most according to claim 1 is the thinnest
The method that born of the same parents are applied to synthesize acetoin, it is characterised in that directly carry out converting production with cheap glucose for substrate
Acetoin.
3. the method that the strategy described in claim 2 is applied to resting cell glucose production acetoin, is characterized in that: will live
Change seed liquor be forwarded in fermentation medium according to the inoculum concentration of 5%, 30 DEG C, 180r/min cultivates about 24h, by thalline from
According to thalline weight in wet base after heart collection: conversional solution volume is the ratio of 1:20, cell suspension is maintained in resting cell culture fluid
60h, adds the CaCO of 30g/L in conversion process3For maintaining pH.
Conversional solution composition the most according to claim 4, it is characterised in that conversional solution composition is (g/L): KH2PO40.5,
K2HPO4·3H2O 0.5, MgSO4·7H2O 0.5, FeSO4·7H2O 6, MnSO4·H2O 4.2, glucose 100.
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| CN109486871A (en) * | 2018-12-07 | 2019-03-19 | 山东大学深圳研究院 | A method of utilizing bacillus licheniformis engineered strain fermenting and producing 3-hydroxy-2-butanone |
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| CN108728389A (en) * | 2017-04-19 | 2018-11-02 | 北京工商大学 | One plant of colibacillus engineering and its application for producing 2,3,5,6- Tetramethylpyrazines |
| CN108728389B (en) * | 2017-04-19 | 2020-05-15 | 北京工商大学 | Escherichia coli engineering bacterium for producing 2,3,5, 6-tetramethylpyrazine and application thereof |
| CN109486871A (en) * | 2018-12-07 | 2019-03-19 | 山东大学深圳研究院 | A method of utilizing bacillus licheniformis engineered strain fermenting and producing 3-hydroxy-2-butanone |
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Application publication date: 20170104 |