CN107988128A - A kind of genetic engineering bacterium of production D-1,2,4- butantriols and its application - Google Patents
A kind of genetic engineering bacterium of production D-1,2,4- butantriols and its application Download PDFInfo
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- 238000010353 genetic engineering Methods 0.000 title claims abstract description 30
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- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims abstract description 22
- 235000014897 Streptococcus lactis Nutrition 0.000 claims abstract description 18
- 101150110790 xylB gene Proteins 0.000 claims abstract description 15
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims abstract description 12
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims abstract description 11
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- NNDIXBJHNLFJJP-UHFFFAOYSA-N 20-Hydroxyeicosatetraenoic acid Chemical compound OCCCCCC=CCC=CCC=CCC=CCCCC(O)=O NNDIXBJHNLFJJP-UHFFFAOYSA-N 0.000 description 2
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- PKAUICCNAWQPAU-UHFFFAOYSA-N 2-(4-chloro-2-methylphenoxy)acetic acid;n-methylmethanamine Chemical compound CNC.CC1=CC(Cl)=CC=C1OCC(O)=O PKAUICCNAWQPAU-UHFFFAOYSA-N 0.000 description 1
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Abstract
Genetic engineering bacterium and its application the invention discloses a kind of production 1,2,4 butantriols of D, host strain BL21 is transferred to by 2 keto-acid decarboxylase enzyme genes, xylose dehydrogenase gene XylB, xylonate dehydratase YjhG and alcohol dehydrogenase gene YqhD（DE3）Genetic engineering bacterium is obtained, the 2 keto-acid decarboxylase enzyme gene is from Lactococcus lactis（Lactococcus lactis）α keto-acid decarboxylase enzyme genes KdcA.Fermented culture production 1,2,4 butantriols of D.The present invention can effectively improve the ability of D xyloses synthesis 1,2,4 butantriols of D, final reachable 6.82g/L by screening provided α keto-acid decarboxylase enzyme gene KdcA.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to one kind production D-1, the genetic engineering bacterium of 2,4- butantriols and
It is applied.
Background technology
D-1,2,4- butantriols（Abbreviation BT）It is a kind of polyalcohol of four carbon, property is similar with glycerine, is widely used in curing
The fields such as medicine, pesticide, cosmetics, papermaking, high molecular material, tobacco, military project.BT and its related derivatives are many natural products
Important substrate in synthesis, and the synthesis precursor of many chipal compounds：In military affairs, since the nitro compound impact of BT is quick
Perception is low, heat endurance is good, hypotoxicity, good hygroscopicity, is used in mixed way, is remarkably improved to nitrify fibre with other energetic plasticisers
Dimension element is the cryogenic mechanics performance of base gunpowder.Therefore, before BT is frequently as the synthesis of 1,2,4-butanetriol trisnitrate (BTTN)
Body, the latter can replace nitroglycerine as propellant and energetic plasticiser, and with more preferably physicochemical property.Medical aspect, BT
The synthesis precursor of cationic-liposome and multi-medicament is used as, BT can also be used to prepare norcholesterol class medicine
Movinolin, cancer therapy drug compatin, treatment dermatosis treating medicine hydroxyeicosatetraenoic acid (12-HETE) and AIDS medicine
Thing 3- dihydroxy-tetrahydro furans.In addition, BT also acts as the murder by poisoning that can eliminate nitro compound to human body, tar ingredients are reduced
The cigarette additive of harm, can be used as the crosslinking agent of high molecular material, increases the intensity and hardness of material, also acts as advanced ink
Antidesiccant, high-grade clothing surface conditioning agent, Ceramic manufacturing auxiliary agent, specific use packaging and accumulating of water etc..BT can suppress micro- life
Thing, is anti-microbial agents（Such as preservative）Part.
2003, Niu etc. first proposed the biological synthesis process of BT.By Escherichia coli (Escherichia coli) in the heterologous metabolic pathway of structure, using D- xyloses or L-arabinose as substrate, realized by four step enzymatics.Entirely
Technique has used Pseudomonas fragiPseudomonas fragi And Escherichia coliEscherichia coli Two kinds of micro- lifes
Thing is as catalyst.Afterwards, researcher exists four step enzymes of BT path for transformationE. coli In expressed, useE. coli The bioconversion of whole BT can be completed, enormously simplify conversion process.But from the point of view of currently reported, with weight
GroupE. coli It is relatively low to produce the catalytic efficiency of BT, causes BT yield can not meet industrial demand.If will be into
One step improves the transformation efficiency of BT it may first have to and BT route of synthesis is systematically optimized, finds out catalysis rate-limiting step,
It is accurate to adjust the expression quantity for being catalyzed each step enzyme, to realize maximum conversion flux.
BT is a kind of polyalcohol with important application prospect, but is used as a kind of non-natural compound, its bioconversion way
Footpath is still faced with the problem of approach efficiency is low, therefore the optimization for BT route of synthesis is most important.
The content of the invention
The problem of efficiency still faced for existing BT biotransformation pathways is low, for the 2- ketone acids in BT route of synthesis
Decarboxylase is screened, to further improve the yield of BT.
The technical solution adopted by the present invention is:
One kind production D-1, the genetic engineering bacterium of 2,4- butantriols, the genetic engineering bacterium is by 2- keto-acid decarboxylases enzyme gene, xylose
Dehydrogenase gene XylB, xylonate dehydratase YjhG and alcohol dehydrogenase gene YqhD are transferred to host strain BL21（DE3）Intracellular
The genetic engineering bacterium arrived.The present invention has investigated the influence of the 2- keto acid decarboxylase gene pairs BT synthesis yield of separate sources, example
Such as：Benzoylformate decarboxylase (pseudomonas putida,Pseudomonas putida), GenBank: AY143338.1、α-
Ketoisovalerate decarboxylase (Lactococcus lactisKF147,Lactococcus lactis subsp. lactis (strain KF147), GenBank:ADA65057.1,2-ketoacid decarboxylase (Lactococcus lactis,Lactococcus lactis),
GenBank:AAS49166.1, phenylpyruvate decarboxylase (saccharomyces cerevisiae,Saccharomyces cerevisiae),
GenBank:KZV12623.1, show that the 2- keto-acid decarboxylases enzyme gene uses the 2-ketoacid decarboxylation from Lactococcus lactis
During enzyme, BT yield highests, considerably beyond the production of current bioanalysis BT.
The host strain is Escherichia coliBL21(DE3)。
Heretofore described xylonate dehydratase YjhG Gene ID: 946829；The xylose dehydrogenase XylB,
Gene ID: 7329904；The alcohol dehydrogenase YqhD, GenBank: ADK47404.1；The benzoyl formic acid decarboxylation
Enzyme MdlC, GenBank: AY143338.1；α-ketoisovaleric acid the decarboxylase KivD, GenBank: ADA65057.1；Institute
2-ketoacid the decarboxylase KdcA, GenBank stated: AAS49166.1；The phenylpyruvate decarboxylase Aro10, GenBank:
KZV12623.1。
Derive fromLactococcus lactis2-ketoacid decarboxylase KdcA gene orders as shown in SEQ.No.1；Source
InLactococcus lactis subsp. lactis (strain KF147)α-ketoisovaleric acid decarboxylase gene KivD sequences
Row are as shown in SEQ.No.2；Derive fromPseudomonas putidaBenzoylformate decarboxylase MdlC sequences such as SEQ.No.3
It is shown；Phenylpyruvate decarboxylase Gene A ro10 sequences are as shown in SEQ.No.4
The production D-1, the construction method of the genetic engineering bacterium of 2,4- butantriols, includes the following steps：
1）Build clonal expression 2-ketoacid decarboxylase gene KdcA, xylose dehydrogenase gene XylB, D- xylonate dehydratase YjhG
With alcohol dehydrogenase gene YqhD；
2）KdcA genes are inserted into plasmid pTRC99a'sNcoI HeSac Between I site, plasmid pTRC-KdcA, XylB are obtained
Gene is inserted into plasmid pTRC99a'sNcoI HeBamBetween I sites of H, so as to obtain pTRC99a-XylB.Then PCR is passed through
The XylB fragments with TRC promoters are obtained, by fragment insertion plasmid pTRC-KdcA'sSac I HeBamBetween I sites of H,
Obtain plasmid pTRC-KdcA-TRC-XylB.
3）Genetic fragment YjhG, YqhD is inserted into pCWJ plasmids respectively, to recombinant plasmid pCWJ-YjhG and pCWJ-
YqhD；Go out the YqhD fragments with TRC promoters by template amplification of pCWJ-YqhD plasmids, by fragment insertion plasmid pCWJ-
YjhG, obtains plasmid pCWJ-YjhG-TRC-YqhD.
4）Plasmid pTRC-KdcA-TRC-XylB and plasmid pCWJ-YjhG-TRC-YqhD are transferred to host strain BL21 jointly
（DE3）The intracellular genetic engineering bacterium that must produce D-1,2,4- butantriols.
Application of the genetic engineering bacterium in fermenting and producing D-1,2,4- butantriols.Genetic engineering bacterium is seeded to hair
In ferment culture medium, add IPTG induction fermentations and obtain product D-1,2,4- butantriols.
The fermentation medium is:Peptone 10g/L, dusty yeast 5g/L, NaCl 10 g/L, D- xyloses 25g/L
, ammonia benzyl mycin 100 mg/L, 68 mg/L of chloramphenicol.
It is 30-37 DEG C that the fermentation step, which controls fermentation temperature, fermentation time 60-72h.
The IPTG induced concentrations are 0.1 ~ 1.25mmol/L, are preferably 1mmol/L.
Claimed one kind derives from Lactococcus lactis（Lactococcus lactis）2-ketoacid decarboxylase
Application in D-1,2,4- butantriol production processes.The different 2- keto-acid decarboxylase enzyme genes of clonal expression are built respectively（MdlC、
KivD, KdcA and Aro10）, and the gene built is transferred to host strain BL21（DE3）It is intracellular to obtain genetic engineering bacterium, culture
Genetic engineering bacterium is simultaneously seeded in fermentation medium and produces BT.
The present invention identifies the Steps of whole catalytic process, Cao etc. using restructuring firstE. coli Convert xylose
Production xylonic can reach nearly 88%, Valdehuesa of theoretical yield etc. and report with restructuringE. coli Convert xylose life
When producing BT, the xylose for having 82.28% is xylonic, but only 10.25% xylose is eventually converted into BT, this shows
Three step enzymatics are probably the rate-limiting step of whole approach afterwards.Since the 3rd step decarboxylic reaction is non-native catalytic reaction, at the same time
Decarboxylic reaction is irreversible reaction again, can speculate that step reaction is particularly likely that the Steps entirely reacted, therefore, this hair
It is bright investigated benzoylformate decarboxylase (Pseudomonas putida), GenBank:AY143338.1, α-ketoisovaleric acid
Decarboxylase (Lactococcus lactis subsp. lactis (strain KF147), GenBank: ADA65057.1、
2-ketoacid decarboxylase (Lactococcus lactis), GenBank:AAS49166.1, phenylpyruvate decarboxylase
(Saccharomyces cerevisiae), GenBank: KZV12623.1；The optimization of a new round is carried out to route of synthesis, with
Further improve the yield of BT.
Beneficial effect
The present invention optimizes D- xylose productions D-1, the path of 2,4- butantriols, is adopted by screening different 2- keto acid decarboxylases
Genetic engineering bacterium, D-1 are built with the 2-ketoacid decarboxylase from Lactococcus lactis, the yield of 2,4- butantriols significantly improves,
Final reachable 6.82g/L.
Brief description of the drawings
Fig. 1 is influences of the Lactococcus lactis KdcA to fermenting and producing BT；
Fig. 2 is influence of the different 2- keto acid decarboxylases to fermenting and producing BT；
Fig. 3 is influence of the IPTG concentration to fermenting and producing BT.
Embodiment
The following examples can make those skilled in the art that the present invention be more fully understood, but not limit this in any way
Invention.In the present invention, recombinant expression carrier can be imported in host cell with method well known in the art, these methods include：
Calcium chloride heat shock method, electrotransformation, PEG mediated methods, particle bombardment etc..
The implementation of the present invention is by using fields such as the chemistry within the limit of power of those skilled in the art, molecular biology
Conventional art.In addition, unless otherwise indicated, herein, nucleic acid is write from left to right with 5 ' to 3 ' direction, amino acid sequence
Row are then write from left to right with the direction of aminoterminal to c-terminus.
With reference to the specific experimental data in laboratory, the present invention is further explained.It is to be understood that these embodiments are only used for
Illustrate the present invention rather than limit the scope of the invention.The experimental method of actual conditions is not specified in the following example, usually
Operated according to normal condition, such as《Molecular Cloning:A Laboratory guide》, or the condition proposed by manufacturer.
Embodiment 1 builds genetic engineering bacterium BL21-02, BL21-08, BL21-09, BL21-10
1）Build clonal expression 2- keto acid decarboxylase gene Ms dlC, KivD, KdcA, Aro10；Xylose dehydrogenase gene XylB, D-
Xylonate dehydratase YjhG and alcohol dehydrogenase gene YqhD；D- xylonate dehydratases YjhG (Escherichia coli),Gene
ID: 946829；Xylose dehydrogenase gene XylB, the Gene ID: 7329904；The alcohol dehydrogenase gene YqhD,
GenBank: ADK47404.1；The 2-ketoacid decarboxylase (Lactococcus lactis), GenBank:
AAS49166.1；
2）2- keto acid decarboxylase gene Ms dlC, KivD, KdcA, Aro10 are respectively inserted plasmid pTRC99a（TransGen）'sNcoI HeSac Between I site, plasmid pTRC-MdlC, pTRC-KivD, pTRC-KdcA, pTRC-Aro10, XylB genes are obtained
It is inserted into plasmid pTRC99a'sNcoI HeBamBetween I sites of H, so as to obtain pTRC99a-XylB.Then obtained by PCR
XylB fragments with TRC promoters, by the fragment be inserted into respectively plasmid pTRC-MdlC, pTRC-KivD, pTRC-KdcA,
PTRC-Aro10'sSac I HeBamBetween I sites of H, plasmid pTRC-MdlC-TRC-XylB, pTRC-KivD-TRC- are obtained
XylB、pTRC-KdcA-TRC-XylB、pTRC-Aro10-TRC-XylB。
3）Genetic fragment YjhG, YqhD is inserted into pCWJ plasmids respectively, obtains recombinant plasmid pCWJ-YjhG and pCWJ-
YqhD.Go out the YqhD fragments with TRC promoters by template amplification of pCWJ-YqhD plasmids, by fragment insertion plasmid pCWJ-
YjhG, obtains plasmid pCWJ-YjhG-TRC-YqhD.
4）By plasmid pTRC-MdlC-TRC-XylB, pTRC-KivD-TRC-XylB, pTRC-KdcA-TRC-XylB,
PTRC-Aro10-TRC-XylB is transferred to host strain BL21 jointly with plasmid pCWJ-YjhG-TRC-YqhD respectively（DE3）Into the cell
Obtain genetic engineering bacterium BL21-02, BL21-08, BL21-09, BL21-10.
The 2- keto-acid decarboxylase enzyme genes of separate sources are used in the construction method, structure obtains different genetic engineerings
Bacterium, wherein, 2- keto acid decarboxylases be respectively benzoylformate decarboxylase (Pseudomonas putida) MdlC, GenBank:
AY143338.1, α-ketoisovaleric acid decarboxylase (Lactococcus lactis subsp. lactis (strain KF147))
KivD, GenBank:AIS03677.1,2-ketoacid decarboxylase (Lactococcus lactis), GenBank:
AJG88423.1, phenylpyruvate decarboxylase (Saccharomyces cerevisiae),GenBank: KZV12623.1。
2 engineering bacteria fermentation of embodiment produces D-1,2,4- butantriols
Tablet culture：
The glycerine for being stored in -80 DEG C is frozen bacterium to take out, in plating medium（Peptone 10g/L, dusty yeast 5g/L, NaCl
10 g/L, ammonia benzyl mycin 100 mg/L, 68 mg/L of chloramphenicol）Upper carry out scribing operation, is then placed in 37 DEG C of trainings by tablet
Support case culture 12-16h.
Seed liquor culture：
5 mL seed culture mediums are added into 50 mL centrifuge tubes（10 g/L of peptone 10g/L, dusty yeast 5g/L, NaCl, ammonia
100 mg/L of benzyl mycin, 68 mg/L of chloramphenicol）, a small amount of glycerine of picking freezes bacterium and is inoculated with, in 200 rpm, 37 DEG C of bars
10 ~ 12 h are cultivated under part.
Shake flask fermentation culture：
Load 50 mL fermentation mediums in 500 mL shaking flasks（10 g/L of peptone 10g/L, dusty yeast 5g/L, NaCl,
D- xylose 25g/L, ammonia benzyl mycin 100 mg/L, 68 mg/L of chloramphenicol）, inoculum concentration is 1 %, and rotating speed is 200 rpm, training
It is 37 DEG C to support temperature, and the IPTG inductions of final concentration of 1mmol/L are added when fermenting initial, and adds 10g/LCaCO3As slow
Punching, ferment 60 ~ 72h.1-2mL is sampled every 12h, centrifuging and taking supernatant, carries out liquid phase detection.
Compared by fermentation results（Fig. 1, Fig. 2）, derive fromLactococcus lactis2-ketoacid decarboxylase KdcA
Genetic engineering bacterium have the catalytic efficiency of higher, it is possible to increase recombinant bacterial strain synthesizes the ability of BT, D-1,2,4- butantriol yield
Reach 6.82g/L, molar yield 38.59%.Than fromPseudomonas putidaBenzoylformate decarboxylase MdlC
With fromLactococcus lactis subsp. lactis (strain KF147)α-ketoisovaleric acid decarboxylase KivD
It has been respectively increased 102%, 58.97%.
3 IPTG concentration of embodiment, which ferments genetic engineering bacterium BL21-09, produces the influence of BT
IPTG is isopropylthio-β-D- galactolipins, is a kind of derivant of highly effective lactose operon, can be with repressor
With reference to the expression for promoting gene.Using IPTG as derivant, its effect is combined with repressor, plays depressor effect.Therefore work as
It is theoretic optium concentration when the IPTG amounts added are enough to close all repressor sites.But IPTG excessive concentrations
When, it will as toxicant, influence the breeding and growth of cell.The present invention has been investigated not using BL21-09 as experimental strain
The influence synthesized with the IPTG of concentration to BT, the concentration range of setting is 0.1 ~ 1.25mmol/L, and the results are shown in Figure 3.When luring
When leading agent concentration and being less than 1mmol/L, BT yield is increased with the concentration increase of IPTG.When concentration reaches 1mmol/L, BT
Yield highest, when concentration reaches 1.25mmol/L more than 1mmol/L, Product yields reduce, illustrate that derivant is to bacterial strain at this time
Generate some inhibitory action.
D-1, the high-efficiency liquid chromatography method for detecting of 2,4- butantriols：
D-1, the testing conditions of 2,4- butantriols are：Agilent Technologies1290 high performance liquid chromatography；Bio-Rad
Aminex HPX-87H IonExclusion Column (300 mm × 7.8 mm) organic acid column；Mobile phase is 5
mmol/L H2SO4；0.6 mL/min of flow velocity, 55 DEG C of column temperature, 20 μ L of sample size,
Composition distribution.
4 genetic engineering bacterium BL21-09 fermenting and producing D-1,2,4- butantriols of embodiment
The glycerine for being stored in -80 DEG C is frozen bacterium to take out, in plating medium（Peptone 10g/L, dusty yeast 5g/L, NaCl
10 g/L, ammonia benzyl mycin 100 mg/L, 68 mg/L of chloramphenicol）Upper carry out scribing operation, is then placed in 37 DEG C of trainings by tablet
Support case culture 12-16h.
5 mL seed culture mediums are added into 50 mL centrifuge tubes（10 g/ of peptone 10g/L, dusty yeast 5g/L, NaCl
L, ammonia benzyl mycin 100 mg/L, 68 mg/L of chloramphenicol）, a small amount of glycerine of picking freezes bacterium and is inoculated with, in 200 rpm, 37
10 ~ 12 h are cultivated under the conditions of DEG C.
Load 50 mL fermentation mediums in 500 mL shaking flasks（10 g/ of peptone 10g/L, dusty yeast 5g/L, NaCl
L, D- xylose 25g/L, ammonia benzyl mycin 100 mg/L, 68 mg/L of chloramphenicol）, inoculum concentration is 1 %, and rotating speed is 200 rpm,
Cultivation temperature is 37 DEG C, and the IPTG inductions of final concentration of 1mmol/L are added when fermenting initial, and adds 10g/LCaCO3As slow
Punching, ferment 60h, D-1,2, and the yield of 4- butantriols reaches 6.82g/L, molar yield 38.59%.
Sequence table
<110>Nanjing University of Technology
<120>A kind of genetic engineering bacterium of production D-1,2,4- butantriols and its application
<141> 2017-11-27
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1644
<212> DNA
<213>Lactococcus lactis (Lactococcus lactis)
<400> 1
atgtataccg tgggcgatta tctgctggat cgcctgcatg aactgggcat cgaagaaatt 60
tttggcgtgc cgggcgatta taatctgcag ttcctggatc agattattag ccgcgaagac 120
atgaaatgga ttggcaatgc gaatgaactg aatgcgagct atatggcgga tggctatgcg 180
cgcaccaaaa aagcagcggc gtttctgact acctttggcg tgggtgaact gagcgcgatt 240
aatggtctgg cgggcagcta tgcagaaaat ctgccggtgg tggaaattgt tggtagcccg 300
accagcaaag tgcagaatga tggcaaattt gtgcatcata ccctggcgga tggcgatttt 360
aaacacttca tgaagatgca tgaaccggtt accgcggcgc gtactttatt gaccgcggaa 420
aatgcgacct atgaaattga tcgcgtgctg agccagttgc tgaaagaacg caaaccggtg 480
tatattaatc tgccggtgga tgttgcggca gcgaaagcgg aaaaaccagc gctgagcctg 540
gaaaaagaaa gcagcaccac caataccacc gaacaggtga ttctgagcaa aattgaggag 600
agcctgaaaa atgcgcagaa accggtggtt attgcgggcc atgaagtgat tagctttggc 660
ctggaaaaaa ccgtgaccca gtttgtgagc gaaaccaaac tgccgattac caccctgaat 720
tttggcaaaa gcgcggtgga tgaaagcctg ccgagctttc tgggcattta taatggcaag 780
ctgagcgaaa ttagcctgaa gaactttgtg gaaagcgcgg attttattct gatgctgggc 840
gtgaaactga ccgatagcag caccggcgcg tttacccatc atctggacga gaataagatg 900
atcagcctga atattgacga gggcatcatc tttaacaagg tggtggagga ttttgatttt 960
cgcgcggtgg ttagcagtct gagcgaactg aaaggcattg aatatgaggg ccagtatatc 1020
gataaacagt acgaggagtt tattccgagc agcgcgccat taagccagga tcgtttgtgg 1080
caagcggtgg aaagcctgac ccagagcaat gaaaccattg tggcggaaca gggcaccagt 1140
ttttttggcg cgagcaccat ttttctgaaa agcaacagcc gctttattgg ccaaccactg 1200
tggggcagca ttggctatac ctttccagcg gcgttgggta gccagattgc ggataaagaa 1260
agccgccatc tgctgtttat tggcgatggc agcttgcaac tgaccgtgca agaactgggc 1320
ctgagcattc gcgaaaaact gaatccgatc tgcttcatca tcaacaacga tggctatacc 1380
gtggaacgcg aaattcatgg cccgacccag agctataatg atatcccgat gtggaattat 1440
agcaaactgc cggaaacctt tggcgcgacc gaagatcgtg tggtgagcaa aattgtgcgc 1500
accgagaatg aatttgtgag cgtgatgaaa gaagcgcagg cggatgtgaa tcgcatgtat 1560
tggattgaac tggtgctgga aaaagaagat gcgccgaaac tgctgaaaaa aatgggcaaa 1620
ctgtttgcgg aacagaacaa gtga 1644
<210> 2
<211> 1647
<212> DNA
<213>Lactococcus lactis (Lactococcus lactis)
<400> 2
atgtataccg tgggcgatta tctgctggat cgcctgcatg aactgggcat cgaagaaatt 60
tttggcgtgc cgggcgatta taatctgcag ttcctggatc agattattag ccgcaaggat 120
atgaaatggg tgggcaatgc gaatgaactg aatgcgagct atatggcgga tggctatgcg 180
cgcaccaaaa aagcggcggc gtttctgact acctttggcg ttggtgaact gagcgcggtt 240
aatggtctgg cgggcagcta tgcagaaaat ctgccggtgg tggaaattgt tggtagcccg 300
accagcaaag tgcagaatga aggcaaattt gtgcatcata ccctggcgga tggcgatttt 360
aagcacttca tgaagatgca tgaaccggtt accgcggcgc gtaccttatt gaccgcggaa 420
aatgcgaccg tggaaattga tcgcgtgctg agcgcgttac tgaaagaacg caagccggtg 480
tatattaatc tgccggtgga tgttgcggca gcgaaagcgg aaaaaccaag cctgccgctg 540
aaaaaagaaa atccgaccag caataccagc gatcaggaaa tcctgaataa aatccaggag 600
agcctgaaaa atgcgaaaaa gccgattgtg attaccggcc acgaaattat tagcttcggc 660
ctggaaaata ccgtgaccca gtttattagc aaaaccaagc tgccgattac caccctgaat 720
tttggcaaaa gcagcgtgga tgaaaccctg ccgagctttc tgggcattta taatggcaaa 780
ctgagcgaac cgaacctgaa agaatttgtg gaaagcgcgg attttattct gatgctgggc 840
gtgaaactga ccgatagcag cactggcgcg tttacccatc atctgaacga gaacaagatg 900
atcagcctga atatcgacga aggcaagatt tttaacgaaa gcatccagaa ctttgacttt 960
gaaagcctga ttagcagcct gctggatctg agcggcattg aatataaggg caagtacatc 1020
gataagaagc aggaggattt tgtgccgagc aatgcgctgt taagccagga tcgtctgtgg 1080
caggcggttg aaaatctgac ccagagcaat gaaaccattg tggcggaaca gggcaccagc 1140
ttttttggcg cgagcagcat ttttctgaaa ccgaagagcc attttattgg ccagccgctg 1200
tggggtagca ttggctatac ctttccagca gcgctgggca gtcaaattgc ggataaagaa 1260
agccgccatc tgctgtttat tggcgatggc agcctgcaac tgaccgtgca agaactgggt 1320
ctggcgattc gcgaaaaaat taacccgatc tgcttcatca tcaacaatga tggctatacc 1380
gtggaacgcg aaattcatgg cccgaatcag agctataatg atatcccgat gtggaattat 1440
agcaaactgc cggaaagctt tggcgcgacc gaagaacgtg tggtgagcaa aattgtgcgc 1500
accgagaatg aatttgtgag cgtgatgaaa gaagcgcagg ccgatccgaa tcgcatgtat 1560
tggattgaac tggtgctggc gaaagaagat gcgccgaaag tgctgaaaaa aatgggcaaa 1620
ctgtttgcgg aacagaacaa gagctaa 1647
<210> 3
<211> 1587
<212> DNA
<213>Pseudomonas putida (Pseudomonas putida)
<400> 3
atggcttctg ttcacggtac cacctacgaa ctgctgcgtc gtcagggtat cgacaccgtt 60
ttcggtaacc cgggttctaa cgaactgccg ttcctgaaag acttcccgga agacttccgt 120
tacatcctgg ctctgcagga agcttgcgtt gttggtatcg ctgacggtta cgctcaggct 180
tctcgtaaac cggctttcat caacctgcac tctgctgctg gtaccggtaa cgctatgggt 240
gctctgtcta acgcttggaa ctctcactct ccgctgatcg ttaccgctgg tcagcagacc 300
cgtgctatga tcggtgttga agctctgctg accaacgttg acgctgctaa cctgccgcgt 360
ccgctggtta aatggtctta cgaaccggct tctgctgctg aagttccgca cgctatgtct 420
cgtgctatcc acatggcttc tatggctccg cagggtccgg tttacctgtc tgttccgtac 480
gacgactggg acaaagacgc tgacccgcag tctcaccacc tgttcgaccg tcacgtttct 540
tcttctgttc gtctgaacga ccaggacctg gacatcctgg ttaaagctct gaactctgct 600
tctaacccgg ctatcgttct gggtccggac gttgacgctg ctaacgctaa cgctgactgc 660
gttatgctgg ctgaacgtct gaaagctccg gtttgggttg ctccgtctgc tccgcgttgc 720
ccgttcccga cccgtcaccc gtgcttccgt ggtctgatgc cggctggtat cgctgctatc 780
tctcagctgc tggaaggtca cgacgttgtt ctggttatcg gtgctccggt tttccgttac 840
caccagtacg acccgggtca gtacctgaaa ccgggtaccc gtctgatctc tgttacctgc 900
gacccgctgg aagctgctcg tgctccgatg ggtgacgcta tcgttgctga catcggtgct 960
atggcttctg ctctggctaa cctggttgaa gaatcttctc gtcagctgcc gaccgctgct 1020
ccggaaccgg ctaaagttga ccaggacgct ggtcgtctgc acccggaaac cgttttcgac 1080
accctgaacg acatggctcc ggaaaacgct atctacctga acgaatctac ctctaccacc 1140
gctcagatgt ggcagcgtct gaacatgcgt aacccgggtt cttactactt ctgcgctgct 1200
ggtggtctgg gtttcgctct gccggctgct atcggtgttc agctggctga accggaacgt 1260
caggttatcg ctgttatcgg tgacggttct gctaactact ctatctctgc tctgtggacc 1320
gctgctcagt acaacatccc gaccatcttc gttatcatga acaacggtac ctacggtgct 1380
ctgcgttggt tcgctggtgt tctggaagct gaaaacgttc cgggtctgga cgttccgggt 1440
atcgacttcc gtgctctggc taaaggttac ggtgttcagg ctctgaaagc tgacaacctg 1500
gaacagctga aaggttctct gcaggaagct ctgtctgcta aaggtccggt tctgatcgaa 1560
gtttctaccg tttctccggt taaataa 1587
<210> 4
<211> 1908
<212> DNA
<213>Saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 4
atggcaccgg tgaccattga aaaattcgtg aaccaggaag aacgccatct ggtgagcaat 60
cgcagcgcga ccattccatt tggcgagtac atttttaaac gcctgctgag cattgatacc 120
aaaagcgtgt ttggcgtgcc gggcgatttt aatctgagcc tgctggaata tctgtatagc 180
ccgagcgttg aaagcgcggg cttacgttgg gttggcacct gcaatgaact gaatgcggcg 240
tatgcggcgg atggttatag ccgctacagc aataaaattg gctgcctgat taccacctat 300
ggcgtgggtg aactgagtgc gctgaatggt attgcgggca gctttgcgga aaatgtgaag 360
gtgctgcata ttgtgggcgt ggcgaaaagc attgatagcc gcagcagcaa ttttagcgat 420
cgcaatctgc atcatctggt gccgcagctg catgatagca attttaaagg cccgaaccat 480
aaagtgtatc acgacatggt gaaagatcgc gtggcatgca gtgtggcgta tctggaagat 540
attgaaaccg cgtgcgatca ggtggataat gtgatccgcg acatttacaa gtatagcaag 600
ccgggctata tttttgtgcc ggcggatttt gcggatatga gcgtgacctg cgataatctg 660
gtgaatgtgc cgcgcattag ccagcaggat tgcattgtgt atccgagcga aaatcagctg 720
agcgacatca ttaataagat caccagctgg atctatagca gcaaaacccc ggcgattctg 780
ggtgatgtgc tgaccgatcg ctatggcgtg agcaattttc tgaacaagct gatttgcaaa 840
accggcatct ggaattttag caccgtgatg ggcaaaagcg tgattgatga aagcaatccg 900
acctatatgg gccagtataa tggcaaagaa ggcctgaaac aggtgtatga gcattttgaa 960
ctgtgcgatc tggtgctgca ttttggcgtg gatatcaacg aaatcaacaa cggccattac 1020
accttcacct ataaaccgaa cgcgaagatt attcagttcc acccgaatta tattcgcctg 1080
gtggataccc gtcagggcaa tgaacagatg ttcaagggca ttaattttgc gccgatcctg 1140
aaagaactgt acaagcgcat tgatgtgagc aaactgagcc tgcagtatga tagcaatgtg 1200
acccagtata ccaatgaaac catgcgcctg gaagatccga ccaatggcca gagcagcatt 1260
attacccagg tgcatctgca gaaaaccatg ccgaaatttc tgaatccggg cgatgtggtg 1320
gtttgtgaaa ccggcagctt tcagtttagc gtgcgcgatt ttgcgtttcc gagccagctg 1380
aaatatatta gccagggctt ctttctgagc attggcatgg cattgccagc agcgttaggt 1440
gttggcattg cgatgcagga tcatagcaac gcgcatatta atggcggcaa cgtgaaggaa 1500
gattataaac cgcgcctgat tctgtttgaa ggcgatggtg cggcgcaaat gaccattcag 1560
gaactgagca ccattctgaa atgcaatatt ccgctggaag tgatcatttg gaacaacaac 1620
ggctatacca ttgaacgcgc gattatgggt ccaacccgca gctataatga tgtgatgagc 1680
tggaaatgga ccaaactgtt tgaagcgttc ggcgattttg atggcaaata caccaatagc 1740
accctgattc agtgcccgag caaactggcg ctgaaactgg aagaactgaa aaacagcaat 1800
aaacgcagcg gcattgaact gctggaagtg aaactgggcg aactggattt tccggaacag 1860
ctgaaatgca tggttgaagc ggcagcgctg aaacgcaata agaagtga 1908
Claims (8)
1. one kind production D-1, the genetic engineering bacterium of 2,4- butantriols, the genetic engineering bacterium is by 2- keto-acid decarboxylases enzyme gene, wood
Glucocorticoid dehydrogenase gene XylB, xylonate dehydratase YjhG and alcohol dehydrogenase gene YqhD are transferred to host strain BL21（DE3）Obtain base
Because of engineering bacteria, it is characterised in that the 2- keto-acid decarboxylases enzyme gene is from Lactococcus lactis（Lactococcus lactis）2-ketoacid decarboxylase gene KdcA.
2. a kind of production D-1 according to claim 1, the genetic engineering bacterium of 2,4- butantriols, it is characterised in that the α -one
Pyruvate decarboxylase gene KdcA sequences are as shown in SEQ.No.1.
3. a kind of production D-1 according to claim 1, the genetic engineering bacterium of 2,4- butantriols, it is characterised in that the host
Bacterium is Escherichia coliBL21(DE3)。
4. the production D-1 described in claim 1 or 2, the construction method of the genetic engineering bacterium of 2,4- butantriols, it is characterised in that bag
Include following steps：
1）Build clonal expression 2-ketoacid decarboxylase gene KdcA, xylose dehydrogenase gene XylB, D- xylonate dehydratase YjhG
With alcohol dehydrogenase gene YqhD；
2）KdcA genes are inserted into plasmid pTRC99a'sNcoI HeSac Between I site, plasmid pTRC-KdcA, XylB are obtained
Gene is inserted into plasmid pTRC99a'sNcoI HeBamBetween I sites of H, so as to obtain pTRC99a-XylB；Then PCR is passed through
The XylB fragments with TRC promoters are obtained, by fragment insertion plasmid pTRC-KdcA'sSac I HeBamBetween I sites of H,
Obtain plasmid pTRC-KdcA-TRC-XylB;
3）Genetic fragment YjhG, YqhD is inserted into pCWJ plasmids respectively, to recombinant plasmid pCWJ-YjhG and pCWJ-YqhD;With
PCWJ-YqhD plasmids go out the YqhD fragments with TRC promoters for template amplification, and fragment is inserted into plasmid pCWJ-YjhG, is obtained
Plasmid pCWJ-YjhG-TRC-YqhD;
4）Plasmid pTRC-KdcA-TRC-XylB and plasmid pCWJ-YjhG-TRC-YqhD are transferred to host strain BL21 jointly（DE3）
It is intracellular to obtain genetic engineering bacterium BL21-09.
5. application of the genetic engineering bacterium in fermenting and producing D-1,2,4- butantriols described in claim 1.
6. application according to claim 5, it is characterised in that genetic engineering bacterium is seeded in fermentation medium, is added
IPTG induction fermentations obtain product D-1,2,4- butantriols.
7. application according to claim 6, it is characterised in that the fermentation medium is:Peptone 10g/L, yeast
Powder 5g/L, NaCl 10 g/L, D- xylose 25g/L, ammonia benzyl mycin 100 mg/L, 68 mg/L of chloramphenicol.
8. application according to claim 6, it is characterised in that fermentation temperature is 30-37 DEG C, fermentation time 60-72h.
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