CN107988128B - Genetic engineering bacterium for producing D-1,2, 4-butanetriol and application thereof - Google Patents
Genetic engineering bacterium for producing D-1,2, 4-butanetriol and application thereof Download PDFInfo
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Abstract
The invention discloses a genetic engineering bacterium for producing D-1,2, 4-butanetriol and application thereof, wherein a 2-ketonic acid decarboxylase gene, a xylose dehydrogenase gene XylB, a xylonic acid dehydratase YjhG and an alcohol dehydrogenase gene YqhD are transferred into a host bacterium BL21 (DE 3) to obtain the genetic engineering bacterium, and the 2-ketonic acid decarboxylase gene is derived from lactococcus lactis (L.)Lactococcus lactis) The α -ketoacid decarboxylase gene KdcA. Producing D-1,2, 4-butanetriol through fermentation culture. The alpha-keto acid decarboxylase gene KdcA provided by the invention can effectively improve the ability of D-xylose to synthesize D-1,2, 4-butanetriol, and finally can reach 6.82 g/L.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a genetic engineering bacterium for producing D-1,2, 4-butanetriol and application thereof.
Background
D-1,2, 4-butanetriol (BT for short) is a four-carbon polyol with properties similar to those of glycerol, and is widely applied to the fields of medicines, pesticides, cosmetics, papermaking, high polymer materials, tobacco, military industry and the like. BT and its related derivatives are important substrates in the synthesis of many natural products, and are also synthetic precursors for many chiral compounds: in military, because the BT nitro compound has low impact sensitivity, good thermal stability, low toxicity and good hygroscopicity, and is mixed with other energetic plasticizers for use, the low-temperature mechanical property of the fire-fighting agent taking the nitrocellulose as the base can be obviously improved. Therefore, BT is often used as a synthetic precursor of 1,2, 4-butanetriol trinitrate (BTTN), which can replace nitroglycerin as a propellant and an energetic plasticizer, and has better physicochemical properties. In the aspect of medicine, BT can be used as a cationic liposome and a synthetic precursor of various medicines, and BT can also be used for preparing a cholesterol-lowering medicine Movinolin, an anticancer medicine compatin, a dermatological medicine hydroxyeicosatetraenoic acid (12-HETE) and an AIDS medicine 3-hydroxy-tetrahydrofuran. In addition, BT can be used as cigarette additive for eliminating the poison of nitro compound to human body and reducing the harm of tar component, as cross-linking agent for high-molecular material to increase its strength and hardness, and as anti-drying agent for high-grade ink, surface treating agent for high-grade clothes, ceramic processing aid, special-purpose package, storage and transportation, etc. BT is capable of inhibiting microorganisms and is an integral part of antimicrobial agents, such as preservatives.
In 2003, Niu et al first proposed a biosynthesis method of BT. By using in Escherichia coli (Escherichia coli) In the method, a heterogenous metabolic pathway is constructed, and D-xylose or L-arabinose is taken as a substrate and is catalyzed by four steps of enzymes. The whole process uses pseudomonas fragiPseudomonas fragi And Escherichia coliEscherichia coli Two microorganisms act as catalysts. Later, researchers have included four-step enzymes of the BT transformation pathwayE. coli In the expression ofE. coli The biotransformation of the whole BT can be completed, and the transformation process is greatly simplified. However, it has been reported that recombination is usedE. coli The catalytic efficiency for producing BT is low, so that the yield of BT cannot meet the requirement of industrial production. If the conversion efficiency of BT is further improved, the synthesis path of BT must be systematically optimized, the catalytic rate-limiting step is found, and the expression quantity of enzyme catalyzing each step is accurately adjusted to realize the maximum conversion flux.
BT is a polyhydric alcohol with important application prospect, but as a non-natural compound, the biotransformation pathway still faces the problem of low pathway efficiency, so that BT is very important for optimizing the synthesis pathway of BT.
Disclosure of Invention
Aiming at the problem of low efficiency of the existing BT biotransformation pathway, 2-keto acid decarboxylase in the BT synthesis pathway is screened so as to further improve the yield of BT.
The technical scheme adopted by the invention is as follows:
a genetically engineered bacterium for producing D-1,2, 4-butanetriol is prepared from 2-ketoneThe acid decarboxylase gene, the xylose dehydrogenase gene XylB, the xylonic acid dehydratase YjhG and the alcohol dehydrogenase gene YqhD are transferred into host bacteria BL21 (DE 3) to obtain the genetically engineered bacteria. The invention examines the influence of 2-keto acid decarboxylase genes from different sources on the synthesis yield of BT, such as: benzoyl formate decarboxylase (pseudomonas putida,Pseudomonas putida) AY143338.1, alpha-ketoisovalerate decarboxylase (lactococcus lactis)KF147，Lactococcus lactis subsp. lactis (strain KF147) GenBank: ADA65057.1, alpha-keto acid decarboxylase (lactococcus lactis,Lactococcus lactis) GenBank: AAS49166.1, phenylpyruvate decarboxylase (saccharomyces cerevisiae,Saccharomyces cerevisiae) KZV12623.1, GenBank, the yield of BT is the highest when the 2-keto acid decarboxylase gene adopts alpha-keto acid decarboxylase from lactococcus lactis, which is far more than that produced by the current biological method BT.
The host bacterium is escherichia coliBL21(DE3)。
The xylonic acid dehydratase YjhG Gene ID: 946829; the xylose dehydrogenase XylB, Gene ID: 7329904; the alcohol dehydrogenase YqhD, GenBank: ADK 47404.1; the benzoylformate decarboxylase MdlC, GenBank: AY 143338.1; the alpha-ketoisovalerate decarboxylase KivD, GenBank: ADA 65057.1; the alpha-ketoacid decarboxylase KdcA, GenBank: AAS 49166.1; the phenylpyruvic acid decarboxylase Aro10, GenBank KZV 12623.1.
Derived fromLactococcus lactisThe alpha-ketoacid decarboxylase KdcA gene sequence is shown as SEQ.No. 1; derived fromLactococcus lactis subsp. lactis (strain KF147)The KivD sequence of the alpha-ketoisovalerate decarboxylase gene is shown in SEQ.No. 2; derived fromPseudomonas putidaThe sequence of the benzoylformate decarboxylase MdlC is shown in SEQ.No. 3; the sequence of the phenylpyruvate decarboxylase gene Aro10 is shown in SEQ.4
The construction method of the genetic engineering bacteria for producing the D-1,2, 4-butanetriol comprises the following steps:
1) constructing and cloning alpha-keto acid decarboxylase gene KdcA, xylose dehydrogenase gene XylB, D-xylonic acid dehydratase YjhG and alcohol dehydrogenase gene YqhD;
2) the KdcA gene being inserted into the plasmid pTRC99aNcoI andSac between I sites, a plasmid pTRC-KdcA was obtained, and the XylB gene was inserted into the plasmid pTRC99aNcoI andBambetween the HI sites, pTRC99a-XylB was obtained. Then, a XylB fragment having a TRC promoter was obtained by PCR, and the fragment was inserted into the plasmid pTRC-KdcASac I andBambetween the HI sites, plasmid pTRC-KdcA-TRC-xylB was obtained.
3) Inserting the gene segments YjhG and YqhD into pCWJ plasmid respectively to obtain recombinant plasmids pCWJ-YjhG and pCWJ-YqhD; and (3) amplifying a YqhD fragment with a TRC promoter by using the pCWJ-YqhD plasmid as a template, and inserting the fragment into the plasmid pCWJ-YjhG to obtain the plasmid pCWJ-YjhG-TRC-YqhD.
4) The plasmid pTRC-KdcA-TRC-xylB and the plasmid pCWJ-YjhG-TRC-YqhD are jointly transferred into cells of a host bacterium BL21 (DE 3) to obtain the genetic engineering bacterium for producing the D-1,2, 4-butanetriol.
The genetic engineering bacteria are applied to fermentation production of D-1,2, 4-butanetriol. Inoculating the genetically engineered bacteria into a fermentation culture medium, adding IPTG (isopropyl-beta-thiogalactoside) to induce fermentation to obtain a product D-1,2, 4-butanetriol.
The fermentation medium comprises 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl, 25g/L of D-xylose, 100 mg/L of ampicillin and 68 mg/L of chloramphenicol.
The fermentation temperature in the fermentation step is controlled to be 30-37 ℃, and the fermentation time is 60-72 h.
The IPTG induction concentration is 0.1-1.25 mmol/L, and preferably 1 mmol/L.
The invention claims a new medicine from lactococcus lactis (lactococcus lactis)Lactococcus lactis) The use of the alpha-keto acid decarboxylase in the production of D-1,2, 4-butanetriol. Respectively constructing, cloning and expressing different 2-keto acid decarboxylase genes (MdlC, KivD, KdcA and Aro 10), transferring the constructed genes into host bacteria BL21 (DE 3) cells to obtain genetically engineered bacteria, culturing the genetically engineered bacteria and inoculating the genetically engineered bacteria into a fermentation culture medium to produce BT.
The invention first identifies the restrictive steps of the overall catalytic process, Cao et al utilize recombinationE. coli The xylonic acid produced by converting xylose can reach about 88 percent of theoretical yield,valdehusea et al reported the use of recombinationE. coli Conversion of xylose to BT yields 82.28% of xylose converted to xylonic acid, but only 10.25% of xylose was ultimately converted to BT, suggesting that the latter three enzyme catalysis may be the rate limiting step of the overall pathway. Since the decarboxylation reaction in the third step is a non-natural catalytic reaction and is also an irreversible reaction, it is presumed that this step is most likely a limiting step in the overall reaction, and therefore, the present inventors have examined benzoylformate decarboxylase: (a)Pseudomonas putida) GenBank AY143338.1, alpha-ketoisovalerate decarboxylase (A)Lactococcus lactis subsp. lactis (strain KF147) ADA65057.1, alpha-keto acid decarboxylase (C) ((C))Lactococcus lactis) GenBank AAS49166.1, phenylpyruvic acid decarboxylase (C)Saccharomyces cerevisiae) KZV12623.1 in GenBank; a new round of optimization is performed on the synthesis route to further improve the yield of BT.
Advantageous effects
The invention optimizes the path for producing D-1,2, 4-butanetriol by D-xylose by screening different 2-keto acid decarboxylases, adopts the alpha-keto acid decarboxylases from lactococcus lactis to construct the genetic engineering bacteria, and obviously improves the yield of D-1,2, 4-butanetriol, and finally reaches 6.82 g/L.
Drawings
FIG. 1 is a graph of the effect of lactococcus lactis KdcA on BT production by fermentation;
FIG. 2 is a graph showing the effect of different 2-keto acid decarboxylases on the fermentative production of BT;
FIG. 3 is a graph of the effect of IPTG concentration on BT produced by fermentation.
Detailed Description
The following examples are presented to enable one of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way. In the present invention, the recombinant expression vector may be introduced into a host cell by methods well known in the art, including: calcium chloride heat shock method, electrical transformation method, PEG mediated method, gene gun method and so on.
The practice of the present invention will employ conventional techniques in the fields of chemistry, molecular biology, and the like, which are within the capabilities of persons skilled in the art. In addition, unless otherwise indicated, nucleic acids are written from left to right in the 5 'to 3' direction and amino acid sequences are written from left to right in the amino terminus to carboxy terminus direction herein.
The invention is further illustrated below with reference to laboratory specific experimental data. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, such as the molecular cloning guidelines or the conditions recommended by the manufacturer.
Example 1 construction of genetically engineered bacteria BL21-02, BL21-08, BL21-09, BL21-10
1) Constructing, cloning and expressing 2-keto acid decarboxylase genes MdlC, KivD, KdcA and Aro 10; xylose dehydrogenase gene XylB, D-xylonate dehydratase YjhG and alcohol dehydrogenase gene YqhD; d-xylonic acid dehydratase YjhG (A)Escherichia coli) Gene ID 946829; the xylose dehydrogenase Gene XylB, Gene ID: 7329904; the alcohol dehydrogenase gene YqhD, GenBank: ADK 47404.1; the alpha-keto acid decarboxylase (a)Lactococcus lactis), GenBank: AAS49166.1；
2) The 2-keto acid decarboxylase genes MdlC, KivD, KdcA, and Aro10 are inserted into the plasmid pTRC99a (TransGen), respectivelyNcoI andSac the plasmid pTRC-MdlC, pTRC-KivD, pTRC-KdcA, pTRC-Aro10 was obtained between the I sites, and the XylB gene was inserted into the plasmid pTRC99aNcoI andBambetween the HI sites, pTRC99a-XylB was obtained. Then, XylB fragment containing TRC promoter was obtained by PCR, and the fragments were inserted into plasmids pTRC-MdlC, pTRC-KivD, pTRC-KdcA and pTRC-Aro10Sac I andBamplasmids pTRC-MdlC-TRC-xylB, pTRC-KivD-TRC-xylB, pTRC-KdcA-TRC-xylB and pTRC-Aro10-TRC-xylB were obtained between the HI sites.
3) The gene fragments YjhG and YqhD are respectively inserted into the pCWJ plasmid to obtain recombinant plasmids pCWJ-YjhG and pCWJ-YqhD. And (3) amplifying a YqhD fragment with a TRC promoter by using the pCWJ-YqhD plasmid as a template, and inserting the fragment into the plasmid pCWJ-YjhG to obtain the plasmid pCWJ-YjhG-TRC-YqhD.
4) The plasmids pTRC-MdlC-TRC-XylB, pTRC-KivD-TRC-XylB, pTRC-KdCA-TRC-XylB and pTRC-Aro10-TRC-XylB are respectively co-transferred with the plasmid pCWJ-YjhG-TRC-YqhD into the host bacterium BL21 (DE 3) to obtain genetically engineered bacteria BL21-02, BL21-08, BL21-09 and BL 21-10.
The construction method adopts 2-keto acid decarboxylase genes from different sources to construct different genetic engineering bacteria, wherein the 2-keto acid decarboxylase is benzoylformate decarboxylase (A), (B), (C), (Pseudomonas putida) MdlC, GenBank AY143338.1, alpha-ketoisovalerate decarboxylase: (Lactococcus lactis subsp. lactis (strain KF147)) KivD, GenBank AIS03677.1, alpha-keto acid decarboxylase: (A)Lactococcus lactis) GenBank: AJG88423.1, phenylpyruvic acid decarboxylase: (Saccharomyces cerevisiae),GenBank: KZV12623.1。
EXAMPLE 2 fermentation of genetically engineered bacteria to produce D-1,2, 4-butanetriol
Plate culture:
the glycerol cryopreserved cells stored at-80 ℃ were taken out, streaking was performed on a plate medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, ampicillin 100 mg/L, chloramphenicol 68 mg/L), and the plate was then cultured in a 37 ℃ incubator for 12-16 hours.
Seed liquid culture:
5 mL of seed culture medium (10 g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl, 100 mg/L of ampicillin and 68 mg/L of chloramphenicol) is added into a 50 mL centrifuge tube, a small amount of glycerol frozen bacteria is picked up for inoculation, and the culture is carried out for 10-12 h under the conditions of 200 rpm and 37 ℃.
And (3) shake flask fermentation culture:
50 mL of fermentation medium (10 g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl, 25g/L of D-xylose, 100 mg/L of ampicillin and 68 mg/L of chloramphenicol) is filled into a 500 mL shake flask, the inoculation amount is 1 percent, the rotating speed is 200 rpm, the culture temperature is 37 ℃, IPTG with the final concentration of 1mmol/L is added for induction at the beginning of fermentation, and 10g/LCaCO is added3Fermenting for 60-72h as buffer. Sampling 1-2mL every 12h, centrifuging, taking supernatant, and performing liquid phase detection.
Results of fermentation were compared (FIG. 1, FIG. 2) and derived fromLactococcus lactisAlpha-ketoacid decarboxylase KdcA gene of (1)The engineering bacteria have higher catalytic efficiency, can improve the BT synthesizing capacity of the recombinant strains, and the yield of D-1,2, 4-butanetriol reaches 6.82g/L, and the molar yield is 38.59%. Ratio is derived fromPseudomonas putidaThe benzoylformate decarboxylase MdlC and the derivatives derived fromLactococcus lactis subsp. lactis (strain KF147)The alpha-ketoisovalerate decarboxylase KivD is respectively improved by 102 percent and 58.97 percent.
Example 3 Effect of IPTG concentration on BT production by fermentation of genetically engineered bacterium BL21-09
IPTG is isopropylthio-beta-D-galactose, is a very effective inducer of lactose operon, and can be combined with a repressor to promote the expression of genes. IPTG is used as an inducer and has the function of combining with a repressor and playing a role of derepression. Thus, the theoretically optimal concentration is obtained when the amount of IPTG added is sufficient to block all repressor sites. However, if the concentration of IPTG is too high, IPTG will act as a toxic substance and affect the propagation and growth of cells. According to the invention, BL21-09 is taken as an experimental strain, the influence of IPTG with different concentrations on BT synthesis is examined, the set concentration range is 0.1-1.25 mmol/L, and the result is shown in figure 3. When the inducer concentration is below 1mmol/L, BT yield increases with increasing concentration of IPTG. When the concentration reaches 1mmol/L, the yield of BT is highest, and when the concentration exceeds 1mmol/L and reaches 1.25mmol/L, the yield of products is reduced, which indicates that the inducer has some inhibiting effect on the strain.
The high performance liquid chromatography detection method of the D-1,2, 4-butanetriol comprises the following steps:
the detection conditions of the D-1,2, 4-butanetriol are as follows: agilent Technologies1290 high performance liquid chromatography; Bio-Rad Aminex HPX-87H IonExclusion Column (300 mm. times.7.8 mm) organic acid Column; the mobile phase is 5mmol/L H2SO 4; the flow rate is 0.6 mL/min, the column temperature is 55 ℃, the sample injection amount is 20 mu L,
a difference detector.
Example 4 production of D-1,2, 4-butanetriol by fermentation of genetically engineered bacterium BL21-09
The glycerol cryopreserved cells stored at-80 ℃ were taken out, streaking was performed on a plate medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, ampicillin 100 mg/L, chloramphenicol 68 mg/L), and the plate was then cultured in a 37 ℃ incubator for 12-16 hours.
5 mL of seed culture medium (10 g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl, 100 mg/L of ampicillin and 68 mg/L of chloramphenicol) is added into a 50 mL centrifuge tube, a small amount of glycerol frozen bacteria is picked up for inoculation, and the culture is carried out for 10-12 h under the conditions of 200 rpm and 37 ℃.
50 mL of fermentation medium (10 g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl, 25g/L of D-xylose, 100 mg/L of ampicillin and 68 mg/L of chloramphenicol) is filled into a 500 mL shake flask, the inoculation amount is 1 percent, the rotating speed is 200 rpm, the culture temperature is 37 ℃, IPTG with the final concentration of 1mmol/L is added for induction at the beginning of fermentation, and 10g/LCaCO is added3As a buffer, the yield of the D-1,2, 4-butanetriol reaches 6.82g/L after fermentation for 60 hours, and the molar yield is 38.59%.
Sequence listing
<110> Nanjing university of industry
<120> genetic engineering bacterium for producing D-1,2, 4-butanetriol and application thereof
<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
<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 (6)
1. A gene engineering bacterium for producing D-1,2, 4-butanetriol, which is obtained by transferring a 2-ketonic acid decarboxylase gene, a xylose dehydrogenase gene XylB, a xylonic acid dehydratase gene YjhG and an alcohol dehydrogenase gene YqhD into escherichia coli BL21 (DE 3), and is characterized in that the 2-ketonic acid decarboxylase gene is derived from lactococcus lactis (L.)Lactococcus lactis) The sequence of the alpha-ketoacid decarboxylase gene KdcA is shown as SEQ.No. 1; the alcohol dehydrogenase gene YqhD is GenBank ADK 47404.1; the xylose dehydrogenase Gene XylB is Gene ID 7329904; the xylonic acid dehydratase Gene YjhG is Gene ID: 946829.
2. The method for constructing the genetically engineered bacterium producing D-1,2, 4-butanetriol as claimed in claim 1, characterized by comprising the steps of:
1) constructing clone expression alpha-ketoacid decarboxylase gene KdcA, xylose dehydrogenase gene XylB, D-xylonic acid dehydratase gene YjhG and alcohol dehydrogenase gene YqhD;
2) the KdcA gene being inserted into the plasmid pTRC99aNcoI andSac between I sites, a plasmid pTRC-KdcA was obtained, and the XylB gene was inserted into the plasmid pTRC99aNcoI andBambetween the HI sites, thus obtaining pTRC99 a-XylB; then, a XylB fragment having a TRC promoter was obtained by PCR, and the fragment was inserted into the plasmid pTRC-KdcASac I andBamobtaining a plasmid pTRC-KdcA-TRC-XylB between the Hl sites;
3) inserting the gene segments YjhG and YqhD into pCWJ plasmid respectively to obtain recombinant plasmids pCWJ-YjhG and pCWJ-YqhD, amplifying a YqhD segment with TRC promoter by using the pCWJ-YqhD plasmid as a template, inserting the segment into the plasmid pCWJ-YjhG to obtain plasmid pCWJ-YjhG-TRC-YqhD;
4) the plasmid pTRC-KdcA-TRC-XylB and the plasmid pCWJ-YjhG-TRC-YqhD are jointly transferred into the cells of a host bacterium BL21 (DE 3) to obtain the genetically engineered bacterium BL 21-09.
3. The use of the genetically engineered bacterium of claim 1 in the fermentative production of D-1,2, 4-butanetriol.
4. The application of claim 3, wherein the genetically engineered bacteria are inoculated into a fermentation medium, IPTG is added to induce fermentation to obtain the product D-1,2, 4-butanetriol.
5. The use according to claim 4, wherein the fermentation medium is peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, D-xylose 25g/L, ampicillin 100 mg/L, chloramphenicol 68 mg/L.
6. The use according to claim 4, wherein the fermentation temperature is 30-37 ℃ and the fermentation time is 60-72 h.
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| CN101512004A (en) * | 2006-07-19 | 2009-08-19 | 密歇根州州立大学托管委员会 | Microbial synthesis of D-1,2,4-butanetriol |
| CN104450798A (en) * | 2014-11-24 | 2015-03-25 | 中国科学院青岛生物能源与过程研究所 | Method of generating 1,2,4-butantriol by in vitro enzyme reaction and application thereof |
| CN106148429A (en) * | 2016-08-25 | 2016-11-23 | 南京工业大学 | Method for producing D-1,2, 4-butanetriol by biotransformation of cellulose hydrolysate |
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| CN101512004A (en) * | 2006-07-19 | 2009-08-19 | 密歇根州州立大学托管委员会 | Microbial synthesis of D-1,2,4-butanetriol |
| CN104450798A (en) * | 2014-11-24 | 2015-03-25 | 中国科学院青岛生物能源与过程研究所 | Method of generating 1,2,4-butantriol by in vitro enzyme reaction and application thereof |
| CN106148429A (en) * | 2016-08-25 | 2016-11-23 | 南京工业大学 | Method for producing D-1,2, 4-butanetriol by biotransformation of cellulose hydrolysate |
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