CN108410875A - A kind of method of 1,2,4- butantriol yield in raising recombination bacillus coli - Google Patents

A kind of method of 1,2,4- butantriol yield in raising recombination bacillus coli Download PDF

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CN108410875A
CN108410875A CN201810219618.5A CN201810219618A CN108410875A CN 108410875 A CN108410875 A CN 108410875A CN 201810219618 A CN201810219618 A CN 201810219618A CN 108410875 A CN108410875 A CN 108410875A
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诸葛斌
景培源
曹曦
陆信曜
宗红
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Jiangnan University
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Abstract

The invention discloses a kind of methods of 1,2,4 butantriol yield in raising recombination bacillus coli, belong to field of genetic engineering.The present invention is using recombination bacillus coli E.coli W3110 Δ yagE Δ yjhH Δ yiaE Δs ycdW (pEtac mdlC tac xdh) (ATCC 27325) as starting strain, by the recombinant expression carrier for building the antisense RNA fragment containing xylose isomerase XylA, inhibit the expression of xylose isomerase in xylose branch approach, reinforce carbon flow and flows to end product of metabolism, to improve 1, the method of 2,4 butantriol yield.Last plasmid pEtac mdlC tac xdh lac asRNA3, make 1,2,4 butantriol output increased of recombination bacillus coli 44%.

Description

A kind of method of 1,2,4- butantriol yield in raising recombination bacillus coli
Technical field
The present invention relates to a kind of method of 1,2,4-butanetriol yield in raising recombination bacillus coli, especially a kind of antisenses RNA inhibits the method that branched metabolic pathway xylose isomerase expression improves recombination bacillus coli 1,2,4-butanetriol yield, Belong to field of genetic engineering.
Background technology
1,2,4-butanetriol (BT) is a kind of four carbon polyols, and colourless, odorless, tasteless, property is similar with glycerine, mainly answers For fields such as military project, medicine, cosmetics, chemical industry.Currently, 1,2,4-butanetriol is mainly produced by chemical method, but chemical method It is harsh that there are reaction conditions, and used catalyst is expensive, easily causes environmental pollution, and by-product is more the disadvantages such as to be unfavorable for isolating and purifying End, therefore in recent years, the focus of research is increasingly turned to biological synthesis process.
So far, 1,2,4-butanetriol is not yet found in organism, bioanalysis synthesis butantriol is by introducing external source The synthesis path of albumen structure.It is built in Escherichia coli successfully at present and studies more route of synthesis and be：Go out from xylose Hair generates xylonic under xylose dehydrogenase effect, and for xylonic through xylonate dehydratase, benzoylformate decarboxylase and alcohol are de- The effect of hydrogen enzyme generates 1,2,4-butanetriol.In addition, competitive branched metabolic pathway is knocked to reinforce flow direction metabolism production eventually The carbon flow of object butantriol, although unit can be improved with the knockout of the relevant gene of cell growth such as xylose isomerase gene xylA Thalline yield, while can also seriously affect the accumulation that cell growth is unfavorable for butantriol.Therefore, balance is used for cell growth and production The carbon flow of object synthesis, contributes to the yield for further increasing 1,2,4-butanetriol.
Antisense RNA (asRNA) refers to the RNA molecule with mRNA complementations, also includes the RNA molecule with other RNA complementations.By The RNA of double-strand cannot be translated in ribosomes, so antisense RNA is combined with the complementary of mRNA specificity, that is, inhibits the mRNA's Translation.The translation that mRNA is controlled by antisense RNA is a kind of mode of prokaryote gene expression regulation and control, is in E.coli earliest Production enterobacteria Col E1 plasmids in find, many is experiments have shown that there is also antisense RNAs in eucaryote.In recent years Carry out the gene by artificial synthesized antisense RNA, and be conducted into Intracellular transcription into antisense RNA, can inhibit certain to conditionity The expression of specific gene, the function of the part blocks gene.
Invention content
The purpose of the present invention is inhibit xylose isomerase expression, optimization to be used for thalline by Antisense RNA Technique part The distribution of the xylose stream of growth and BT synthesis, and then improve BT yield.The present invention devises 4 according to antisense RNA design principle The antisense RNA of different length difference target gene binding site, four antisense RNAs have target gene transcriptional level different degrees of The importing of inhibiting effect, wherein asRNA3 and asRNA4 significantly improves the 1,2,4-butanetriol yield of recombination bacillus coli.
Present invention firstly provides the antisense RNAs for improving 1,2,4- butantriols yield in recombination bacillus coli：asRNA1、 asRNA2、asRNA3、asRNA4。
The nucleotide sequence for encoding the antisense RNA is as follows：
asRNA1:
CGGCATTACCTGATTATGGAGTTCAATATGCAAGCCTATTTTGACCAGCTCGATCGCGTTCGTTATGAA GGCTCAAAATCCTCAAACCCGTTAGCATTCCGTCACTACAATCCCGACGAACTGGTGTTGGGTA
asRNA2:
CGGCATTACCTGATTATGGAGTTCAATATGCAAGCCTATTTTGACCAGCTCGATCGCGTTCGTTATGAA GGCTCAAAATCCTCAAACCCGTTAGCATTCCGTCACTACAATCCCGACGAACTGGTGTTGGGTAAGCGTATGGAAGA GCACTTGCGTTTTGCCGCCTGCTACTGGCACACCTTCTGCTGGAACGGGGCGGATATGTTTGGTGTGGGGGCGTTTA ATCGTCCGTGGCAGCAGCCTGGTGAGGCACTGGCGTTGGCGAAGCGTAAAGCAGATGTCGCATTTGAGT
asRNA3:
ATGCAAGCCTATTTTGACCAGCTCGATCGCGTTCGTTATGAAGGCTCAAAATCCTCAAACCCGTTAGCA TTCCGTCACTACAATCCCGACGAACTGGTGTTGGGTAAGCGTATGGAAGAGCACTTGCGTTTTGCCGCCTGCTACTG GCACACCTTCTGCTGGAACGGGGCGGATATGTTTGGTGTGGGGGCGTTTAATCGTCCGTGGCAGCAGCCTGGTGAGG CACTGGCGTTGGCGAAGCGTAAAGCAGATGTCGCATTTGAGT
asRNA4:
TTTTCCACAAGTTACATGTGCCATTTTATTGCTTCCACGATGTGGATGTTTCCCCTGAGGGCGCGTCGT TAAAAGAGTACATCAATAATTTTGCGCAAATGGTTGATGTCCTGGCAGGCAAGCAAGAAGAGAGCGGCGTGAAGCTG CTGTGGGGAACGGCCAACTGCTTTACAAACCCTCGCTACGGCGCGGGTGCGGCGACGAACCCAGATCCTGAAGTCTT CAGCTGGGCGGCAACGCAAGTTGTTACAGCGATGGAAGCAA
The present invention also provides the recombinations for improving 1,2,4- butantriol yield in recombination bacillus coli for carrying the antisense RNA It is the plasmid that sets out that expression vector, which is with pEtac-mdlC-tac-xdh, builds the expression vector containing antisense RNA：pEtac- mdlC-tac-xdh-lac-asRNA1、pEtac-mdlC-tac-xdh-lac-asRNA2、pEtac-mdlC-tac-xdh-lac- AsRNA3 or pEtac-mdlC-tac-xdh-lac-asRNA4.
It is to carry constructed recombinant expression the present invention also provides the recombination bacillus coli of 1,2,4-butanetriol output increased Body is transferred to out bacterium germination E. coli W3110 Δ yagE Δ yjhH Δ yiaE Δs ycdW respectively.
The construction method of the recombination bacillus coli of the 1,2,4-butanetriol output increased, includes the following steps：
Step 1:Using E.coli W3110 genomes as template, antisense RNA primer pair as1-F/as1-R, as2-F/ is utilized As2-R, as3-F/as3-R, as4-F/as4-R clone corresponding xylA genetic fragments；
Step 2:Cloned sequence is connect with hairpin structure using digestion, connection molecule operating technology, then will carry and send out After the antisense RNA fragment of clamping structure is reversely inserted into the lac promoters of expression vector pEtac-mdlC-tac-xdh, series is obtained Antisense RNA recombinant expression carrier；
Step 3:It is thin that recombinant expression carrier is imported into E.coli W3110 Δ yagE Δ yjhH Δ yiaE Δ ycdW competence In born of the same parents, the production 1,2,4-butanetriol recombination bacillus coli that can inhibit xylose isomerase expression of enzymes is obtained.
Beneficial effects of the present invention：
(1) recombination bacillus coli for four plants of carrying antisense rna expression carriers that the present invention is built, xylose isomerase gene XylA transcriptional levels are respectively by 72%, 57%, 32%, 23% inhibition.
(2) the recombination bacillus coli E.coli W3110 Δ yagE Δs for the carrying antisense rna expression carrier that the present invention is built YjhH Δ yiaE Δs ycdW (pEtac-mdlC-tac-xdh-lac-asRNA3) and E.coli W3110 Δ yagE Δ yjhH Δs YiaE Δs ycdW (pEtac-mdlC-tac-xdh-lac-asRNA4) butantriol yield is respectively increased 44%, 20%, reaches 5.71g/L、4.75g/L。
Description of the drawings
Fig. 1 butantriols are metabolized synthetic schemes.
Fig. 2 antisense RNA fragment calmodulin binding domain CaMs.
Fig. 3 antisense RNA expression vectors build flow chart.
Fig. 4 antisense expression recombinant bacterial strain xylose isomerase gene xylA transcriptional levels are analyzed.Wherein, E.coli MXW004： W3110ΔyagEΔyjhHΔyiaEΔycdW(pEtac-mdlC-tac-xdh)；E.coli as1MXW004：W3110Δ yagEΔyjhHΔyiaEΔycdW(pEtac-mdlC-tac-xdh-lac-asRNA1)；E.coli as2MXW004：E.coli W3110ΔyagEΔyjhHΔyiaEΔycdW(pEtac-mdlC-tac-xdh-lac-asRNA2)；E.coli as3MXW004：E.coli W3110ΔyagEΔyjhHΔyiaEΔycdW(pEtac-mdlC-tac-xdh-lac- asRNA3)；E.coli as4MXW004：E.coli W3110ΔyagEΔyjhHΔyiaEΔycdW(pEtac-mdlC-tac- xdh-lac-asRNA4)
Fig. 5 antisense expression recombinant bacterial strain fermenting properties are analyzed.Wherein, E.coli MXW004：W3110ΔyagEΔyjhH ΔyiaEΔycdW(pEtac-mdlC-tac-xdh)；E.coli MXW005：W3110ΔyagEΔyjhHΔyiaEΔycdW ΔxylA(pEtac-mdlC-tac-xdh)；E.coli as1MXW004：W3110ΔyagEΔyjhHΔyiaEΔycdW (pEtac-mdlC-tac-xdh-lac-asRNA1)；E.coli as2MXW004：E.coli W3110ΔyagEΔyjhHΔ yiaEΔycdW(pEtac-mdlC-tac-xdh-lac-asRNA2)；E.coli as3MXW004：E.coli W3110ΔyagE ΔyjhHΔyiaEΔycdW(pEtac-mdlC-tac-xdh-lac-asRNA3)；E.coli as4MXW004：E.coli W3110ΔyagEΔyjhHΔyiaEΔycdW(pEtac-mdlC-tac-xdh-lac-asRNA4)
Specific implementation mode
Starting strain E.coli W3110 Δ yagE Δ yjhH Δ yiaE Δs ycdW is struck for host with E.coli W3100 Except yagE, yjhH, yiaE, ycdW gene, specific construction method is referring to document：What pretty grain husk recombination bacillus coli D-1,2,4- fourth The knockout of by-product approach and overexpression [D] the Southern Yangtze University of key enzyme in triol synthesis, 2017..
Plasmid pEtac-mdlC-tac-xdh be with tac promoter regulations Xdh, MdlC express, specific construction method referring to Document：Sun Wenlong, Lu Xinyao, Zong Hong wait metabolic engineering Escherichia coli to synthesize D-1,2,4- butantriols [J] microbiologies It is notified to, the construction method of pEtac-mdlC-tac-xylB in 2014,41 (10).
XylA encoding genes such as NCBI-Gene ID:Shown in 948141.
The structure of the recombination bacillus coli of 1 carrying antisense rna expression carrier of embodiment
(1) step 1:Using E.coli W3110 genomes as template, antisense RNA primer pair as1-F/as1-R, as2- is utilized F/as2-R, as3-F/as3-R, as4-F/as4-R clone corresponding xylA genetic fragments.
Primer sequence：
as1-F：CCCAAGCTTTACCCAACACCAGTTCGTCG
as1-R：CGCGGATCCCGGCATTACCTGATTATGGAG
as2-F：CCCAAGCTTACTCAAATGCGACATCTGCTT
as2-R：CGCGGATCCCGGCATTACCTGATTATGGAG
as3-F：CCCAAGCTTACTCAAATGCGACATCTGCT
as3-R：CGCGGATCCATGCAAGCCTATTTTGACCAGC
as4-F：CCCAAGCTTTTGCTTCCATCGCTGTAACAAC
as4-R：CGCGGATCCTTTTCCACAAGTTACATGTGCC
(2) step 2:Cloned sequence is connect with hairpin structure using digestion, connection molecule operating technology, then will be carried After the antisense RNA fragment of hairpin structure is reversely inserted into the lac promoters of expression vector pEtac-mdlC-tac-xdh, it is Row antisense RNA recombinant expression carrier.
The xylA target fragments expanded with corresponding primer PCR are reversely inserted into plasmid pMD19-T (Mut) BamH I- It builds to form pMD19-T-asRNA at III restriction enzyme sites of Hind, wherein the structure of pMD19-T (Mut) is by business plasmid Hairpin structure (Nakashima N, Tamura T.Conditional gene silencing are inserted into pMD19-T (simple) ofmultiple genes with antisense RNAs and generation ofa mutator strain ofEscherichia coli[J].NucleicAcids Research,2009,37(15):) and Spe I, Sac II, e103. BamH I andHind tetra- restriction enzyme sites of III.Then, the asRNA for carrying hairpin structure is obtained with Spe I, II double digestions of Sac Segment is simultaneously connected to II sites I-Sac of pEtac (Mut) Spe, is built into pEtac-asRNA, wherein pEtac (Mut) is by right (Shen Wei, Wang Zhengxiang, Tang Xueming wait archeobacteria Pyrococcusfuriosus high thermophilic alpha-amylase genes to exist to plasmid pEtac Secreting, expressing [J] China brewing in Escherichia coli, 2003,22 (1):12-14.) introduce speI, sacII restriction enzyme site structure It forms.Finally, plasmid pEtac-asRNA and pEtac-mdlC-tac-xdh are carried out with restriction enzyme Blp I and Xba I double Digestion and connect form expression vector pEtac-mdlC-tac-xdh-lac-asRNA.
Gradually structure obtains plasmid pEtac-mdlC-tac-xdh-lac-asRNA1, pEtac-mdlC- according to the method described above tac-xdh-lac-asRNA2、pEtac-mdlC-tac-xdh-lac-asRNA3、pEtac-mdlC-tac-xdh-lac- AsRNA4 contains different xylA genetic fragments.Wherein asRNA1 carries xylA gene -27-105bp sequences, and asRNA2 carries xylA Gene -27-265bp sequences, asRNA3 carry xylA gene 1-265bp sequences, and asRNA4 carries xylA gene 266-530bp sequences Row, asRNA genes are by promoter lac regulating and expressings.
(3) step 3:It is conducted into the ycdW impressions of E.coli W3110 Δ yagE Δ yjhH Δ yiaE Δs by electrotransformation In state cell, the production 1,2,4-butanetriol recombination bacillus coli that can inhibit xylose isomerase expression of enzymes is obtained.
By E.coli W3110 Δs yagE Δs yjhH Δs yiaE Δs ycdW thalline cultures to OD600When equal to 0.4-0.6, from Shaking table takes out, and is centrifuged after ice bath 30min.With the CaCl of 0.1mol/L2Washing thalline, 42 DEG C of thermal shock step of converting (2) build The corresponding antisense RNA expression vector arrived is coated on Kan resistant panels after cultivating 1h, the correct transformant of picking after it grows, Obtain the recombination large intestine bar E.coli W3110 Δ yagE Δ yjhH Δ yiaE Δ ycdW (pEtac-mdlC- containing corresponding plasmid tac-xdh-lac-asRNA1)、E.coli W3110ΔyagEΔyjhHΔyiaEΔycdW(pEtac-mdlC-tac-xdh- lac-asRNA2)、E.coli W3110ΔyagEΔyjhHΔyiaEΔycdW(pEtac-mdlC-tac-xdh-lac- asRNA3)E.coli W3110ΔyagEΔyjhHΔyiaEΔycdW(pEtac-mdlC-tac-xdh-lac-asRNA4)。
The application recombination antisense expression bacterial strain production butantriol of embodiment 2 synthesizes D-1,2,4- butantriols
The single bacterium colony for the different recombinant bacteriums that picking embodiment 1 is built from tablet is used as seed liquor after being incubated overnight, with 1% inoculum concentration is inoculated in 50mL fermentation mediums (the LB culture mediums of 1.5 times of concentration, 30g/L xyloses, 10g/L CaCO3) in, Wait for culture to OD600The IPTG of final concentration of 0.5mmol/L is added when=0.6, is cultivated at 37 DEG C, 200rpm, takes logarithmic growth Phase bacterium solution measures mRNA relative transcript levels；48h zymotic fluids are taken to measure BT yield.
(1) extraction of cell RNA and quantitative PCR operation：10mL logarithmic phase cell culture fluids are collected by centrifugation.Cell it is total RNA is operated by the total RNA extraction reagent box of Tiangeng biochemical technology Co., Ltd, obtained purifying RNA with DNase I into Row processing, is further further purified by the RNeasy Mini Kit of Takara companies and removes genome that may be present DNA.Then hiscript II Q RT supermix (Vazyme Nanjing, China) are utilized by template of the RNA of extraction Reverse transcription synthesizes cDNA.The Transcription inhibition level of xylA is template with qxylA-F (ATGCAGATGGTGGTTGAGCA) using cDNA QxylA-R (GTCGCGGCATCGTAATCATA) is the mRNA level in-site acquisition that primer measures bacterial strain by real-time quantitative PCR.Each Real-time PCR Analysis is grasped by the One Step SYBR Prime Script RT-PCR Kit II of Takara companies Make, the instrument used is Germany's Roche Light Cycler480 real-time fluorescence quantitative PCR instrument.In addition, with X16S-F (CAGAAGAAGCACCGGCTAAC)/X16S-R (GGGATTTCACATCCGACTTG) analyzes intracellular 16S rRNA's for primer Expression calibrates quantitative PCR value.The result shows that E.coli W3110 Δ yagE Δ yjhH Δ yiaE Δs ycdW (pEtac-mdlC-tac-xdh-lac-asRNA1)、E.coli W3110ΔyagEΔyjhHΔyiaEΔycdW(pEtac- mdlC-tac-xdh-lac-asRNA2)、E.coli W3110ΔyagEΔyjhHΔyiaEΔycdW(pEtac-mdlC-tac- xdh-lac-asRNA3)E.coli W3110ΔyagEΔyjhHΔyiaEΔycdW(pEtac-mdlC-tac-xdh-lac- AsRNA4) bacterial strain and control strain E.coli W3110 Δ yagE Δ yjhH Δ yiaE Δs ycdW (pEtac-mdlC-tac-xdh) Decline 72%, 57%, 32%, 23% respectively compared to xylA transcriptional levels.
(2) BT yield detects：Zymotic fluid 12000rpm is centrifuged off cell, 0.22 μm of composite fibre of supernatant first Plain ester micro-pore-film filtration can be used to HPLC detections.Testing conditions：Agilent 1260, RID detector, chromatographic column Bio- Rad AminexHPX-87column (300mm × 7.8mm), 60 DEG C of column temperature, mobile phase are 5mmol/L H2SO4, flow velocity 0.6mL/ min.The results show that the recombination bacillus coli 1,2,4-butanetriol containing plasmid pEtac-mdlC-tac-xdh-lac-asRNA3 Yield is optimal to reach 5.71g/L, improves 44%.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill The people of art can do various change and modification, therefore the protection model of the present invention without departing from the spirit and scope of the present invention Enclosing be subject to what claims were defined.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>A kind of method of 1,2,4- butantriol yield in raising recombination bacillus coli
<160> 16
<170> PatentIn version 3.3
<210> 1
<211> 133
<212> DNA
<213>Artificial sequence
<400> 1
cggcattacc tgattatgga gttcaatatg caagcctatt ttgaccagct cgatcgcgtt 60
cgttatgaag gctcaaaatc ctcaaacccg ttagcattcc gtcactacaa tcccgacgaa 120
ctggtgttgg gta 133
<210> 2
<211> 292
<212> DNA
<213>Artificial sequence
<400> 2
cggcattacc tgattatgga gttcaatatg caagcctatt ttgaccagct cgatcgcgtt 60
cgttatgaag gctcaaaatc ctcaaacccg ttagcattcc gtcactacaa tcccgacgaa 120
ctggtgttgg gtaagcgtat ggaagagcac ttgcgttttg ccgcctgcta ctggcacacc 180
ttctgctgga acggggcgga tatgtttggt gtgggggcgt ttaatcgtcc gtggcagcag 240
cctggtgagg cactggcgtt ggcgaagcgt aaagcagatg tcgcatttga gt 292
<210> 3
<211> 265
<212> DNA
<213>Artificial sequence
<400> 3
atgcaagcct attttgacca gctcgatcgc gttcgttatg aaggctcaaa atcctcaaac 60
ccgttagcat tccgtcacta caatcccgac gaactggtgt tgggtaagcg tatggaagag 120
cacttgcgtt ttgccgcctg ctactggcac accttctgct ggaacggggc ggatatgttt 180
ggtgtggggg cgtttaatcg tccgtggcag cagcctggtg aggcactggc gttggcgaag 240
cgtaaagcag atgtcgcatt tgagt 265
<210> 4
<211> 264
<212> DNA
<213>Artificial sequence
<400> 4
ttttccacaa gttacatgtg ccattttatt gcttccacga tgtggatgtt tcccctgagg 60
gcgcgtcgtt aaaagagtac atcaataatt ttgcgcaaat ggttgatgtc ctggcaggca 120
agcaagaaga gagcggcgtg aagctgctgt ggggaacggc caactgcttt acaaaccctc 180
gctacggcgc gggtgcggcg acgaacccag atcctgaagt cttcagctgg gcggcaacgc 240
aagttgttac agcgatggaa gcaa 264
<210> 5
<211> 29
<212> DNA
<213>Artificial sequence
<400> 5
cccaagcttt acccaacacc agttcgtcg 29
<210> 6
<211> 30
<212> DNA
<213>Artificial sequence
<400> 6
cgcggatccc ggcattacct gattatggag 30
<210> 7
<211> 30
<212> DNA
<213>Artificial sequence
<400> 7
cccaagctta ctcaaatgcg acatctgctt 30
<210> 8
<211> 30
<212> DNA
<213>Artificial sequence
<400> 8
cgcggatccc ggcattacct gattatggag 30
<210> 9
<211> 29
<212> DNA
<213>Artificial sequence
<400> 9
cccaagctta ctcaaatgcg acatctgct 29
<210> 10
<211> 31
<212> DNA
<213>Artificial sequence
<400> 10
cgcggatcca tgcaagccta ttttgaccag c 31
<210> 11
<211> 31
<212> DNA
<213>Artificial sequence
<400> 11
cccaagcttt tgcttccatc gctgtaacaa c 31
<210> 12
<211> 31
<212> DNA
<213>Artificial sequence
<400> 12
cgcggatcct tttccacaag ttacatgtgc c 31
<210> 13
<211> 20
<212> DNA
<213>Artificial sequence
<400> 13
atgcagatgg tggttgagca 20
<210> 14
<211> 20
<212> DNA
<213>Artificial sequence
<400> 14
gtcgcggcat cgtaatcata 20
<210> 15
<211> 20
<212> DNA
<213>Artificial sequence
<400> 15
cagaagaagc accggctaac 20
<210> 16
<211> 20
<212> DNA
<213>Artificial sequence
<400> 16
gggatttcac atccgacttg 20

Claims (10)

1. the antisense RNA for improving 1,2,4-butanetriol yield in recombination bacillus coli, which is characterized in that encode the antisense The nucleotide sequence of RNA is as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.1.
2. carry coding claim 1 described in antisense RNA nucleotide recombinant expression carrier, which is characterized in that be with PEtac-mdlC-tac-xdh is the plasmid that sets out, expression vector of the structure for antisence RNA：pEtac-mdlC-tac- Xdh-lac-asRNA1, pEtac-mdlC-tac-xdh-lac-asRNA2, pEtac-mdlC-tac-xdh-lac-asRNA3 or pEtac-mdlC-tac-xdh-lac-asRNA4。
3. a kind of recombination bacillus coli of 1,2,4-butanetriol output increased, which is characterized in that be using anti-described in claim 1 The adopted parts RNA inhibit the expression of xylose isomerase.
4. a kind of recombination bacillus coli of 1,2,4-butanetriol output increased according to claim 3, which is characterized in that be The recombinant expression carrier for carrying the nucleotide of antisense RNA described in coding claim 1 is transferred to Escherichia coli.
5. a kind of recombination bacillus coli of 1,2,4-butanetriol output increased according to claim 3 or 4, feature exist In being that expression vector described in claim 2 is transferred to Escherichia coli respectively.
6. according to a kind of recombination bacillus coli of any 1,2,4-butanetriol output increased of claim 3~5, feature It is, using E.coli W3110 Δ yagE Δ yjhH Δs yiaE Δs ycdW as starting strain.
7. a kind of construction method of the recombination bacillus coli of 1,2,4-butanetriol output increased described in claim 6, feature It is, includes the following steps：
Step 1:Using E.coli W3110 genomes as template, corresponding xylA genetic fragments are cloned using antisense RNA primer pair；
Step 2:Cloned sequence is connect with hairpin structure using digestion, connection molecule operating technology, then will carry hair clip knot After the antisense RNA fragment of structure is reversely inserted into the lac promoters of expression vector pEtac-mdlC-tac-xdh, serial antisense is obtained RNA recombinant expression carriers；
Step 3:Recombinant expression carrier is imported in E.coli W3110 Δ yagE Δ yjhH Δ yiaE Δ ycdW competent cells, The production 1,2,4- butantriol recombination bacillus colis of xylose isomerase expression of enzymes can be inhibited by obtaining.
8. the method for any recombination bacillus coli production 1,2,4- butantriols of application claim 3~6.
9. according to the method described in claim 8, it is characterized in that, using xylose as carbon source.
10. described according to the method described in claim 9, it is characterized in that, seed culture fluid is inoculated in fermentation medium Fermentation medium contains LB culture mediums, 30g/L xyloses, the 10g/L CaCO of 1.5 times of concentration3；Wait for culture to OD600Add when close to 0.6 Enter IPTG Fiber differentiations.
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* Cited by examiner, † Cited by third party
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CN115094016A (en) * 2022-06-30 2022-09-23 山东大学 Recombinant escherichia coli with glucose-6-phosphate isomerase gene knocked out and application of recombinant escherichia coli in production of 1,2,4-butanetriol

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ALLISON HOYNES-O’ CONNOR等: "Development of design rules for reliable antisense RNA behavior in E. coli", 《ACS SYNTHETIC BIOLOGY》 *
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115094016A (en) * 2022-06-30 2022-09-23 山东大学 Recombinant escherichia coli with glucose-6-phosphate isomerase gene knocked out and application of recombinant escherichia coli in production of 1,2,4-butanetriol
CN115094016B (en) * 2022-06-30 2024-02-23 山东大学 Recombinant escherichia coli knocked out glucose-6-phosphate isomerase gene and application thereof in production of 1,2,4-butanetriol

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