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CN108410875B - A kind of method for improving the yield of 1,2,4-butanetriol in recombinant Escherichia coli - Google Patents

A kind of method for improving the yield of 1,2,4-butanetriol in recombinant Escherichia coli Download PDF

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CN108410875B
CN108410875B CN201810219618.5A CN201810219618A CN108410875B CN 108410875 B CN108410875 B CN 108410875B CN 201810219618 A CN201810219618 A CN 201810219618A CN 108410875 B CN108410875 B CN 108410875B
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诸葛斌
景培源
曹曦
陆信曜
宗红
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Abstract

The invention discloses a method for improving the yield of 1,2, 4-butanetriol in recombinant escherichia coli, belonging to the field of genetic engineering. The invention relates to a method for improving the yield of 1,2, 4-butanetriol by using recombinant escherichia coli E.coli W3110 delta yagE E delta yjhH delta yiaE delta ycdW (pEtac-mdLC-tac-xdh) (ATCC 27325) as an initial strain, and by constructing a recombinant expression vector containing an antisense RNA fragment of xylose isomerase XylA, inhibiting the expression level of xylose isomerase in a xylose branching pathway and enhancing the carbon flow to a metabolic end product. Finally, the plasmid pEtac-mdLC-tac-xdh-lac-asRNA3 improves the yield of the recombinant Escherichia coli 1,2, 4-butanetriol by 44 percent.

Description

Method for improving yield of 1,2, 4-butanetriol in recombinant escherichia coli
Technical Field
The invention relates to a method for improving the yield of 1,2, 4-butanetriol in recombinant escherichia coli, in particular to a method for improving the yield of 1,2, 4-butanetriol in recombinant escherichia coli by inhibiting the expression level of xylose isomerase in a branch metabolic pathway through antisense RNA, belonging to the field of genetic engineering.
Background
1,2, 4-Butanetriol (BT) is a four-carbon polyhydric alcohol, is colorless, odorless and tasteless, has the property similar to that of glycerol, and is mainly applied to the fields of military industry, medicines, cosmetics, chemical engineering and the like. At present, 1,2, 4-butanetriol is mainly produced by a chemical method, but the chemical method has the defects of harsh reaction conditions, expensive catalyst, easy environmental pollution, more byproducts, difficult separation and purification and the like, so in recent years, the focus of research is gradually shifted to a biological synthesis method.
To date, 1,2, 4-butanetriol has not been found in organisms, and the biological synthesis of butanetriol is a synthetic pathway constructed by introducing foreign proteins. The synthesis routes which have been successfully constructed and much studied in escherichia coli are as follows: starting from xylose, xylonic acid is generated under the action of xylose dehydrogenase, and the xylonic acid generates 1,2, 4-butanetriol under the action of xylonic acid dehydratase, benzoylformate decarboxylase and alcohol dehydrogenase. In addition, competitive branched metabolic pathways are knocked out to enhance carbon flow to the metabolic end product butanetriol, but knocking out cell growth related genes such as xylose isomerase gene xylA can improve the unit cell yield, and can also seriously affect cell growth to prevent butanetriol accumulation. Thus, balancing the carbon flow for cell growth and product synthesis contributes to further increasing the yield of 1,2, 4-butanetriol.
Antisense RNA (asRNA) refers to RNA molecules complementary to mRNA, and also includes RNA molecules complementary to other RNAs. Since ribosomes are unable to translate double-stranded RNA, antisense RNA specifically binds complementary to mRNA, i.e., inhibits translation of the mRNA. Control of mRNA translation by antisense RNA is a means of regulation of prokaryotic gene expression, originally found in e.coli enterobactin-producing Col E1 plasmid, and many experiments have demonstrated that antisense RNA is also present in eukaryotes. In recent years, by artificially synthesizing a gene of antisense RNA and introducing it into cells to transcribe it into antisense RNA, the expression of a specific gene can be conditionally inhibited and the function of the gene can be partially blocked.
Disclosure of Invention
The invention aims to partially inhibit the expression level of xylose isomerase by an antisense RNA technology, optimize the distribution of xylose flow for thallus growth and BT synthesis and further improve the BT yield. The invention designs 4 antisense RNAs with different target gene binding sites and different lengths according to the antisense RNA design principle, and the four antisense RNAs have different degrees of inhibition on the transcription level of a target gene, wherein the introduction of the asRNA3 and the asRNA4 obviously improves the yield of the 1,2, 4-butanetriol of the recombinant escherichia coli.
The present invention first provides antisense RNA for increasing the production of 1,2, 4-butanetriol in recombinant E.coli: asRNA1, asRNA2, asRNA3, asRNA 4.
The nucleotide sequence encoding the antisense RNA is as follows:
asRNA1:
CGGCATTACCTGATTATGGAGTTCAATATGCAAGCCTATTTTGACCAGCTCGATCGCGTTCGTTATGAAGGCTCAAAATCCTCAAACCCGTTAGCATTCCGTCACTACAATCCCGACGAACTGGTGTTGGGTA
asRNA2:
CGGCATTACCTGATTATGGAGTTCAATATGCAAGCCTATTTTGACCAGCTCGATCGCGTTCGTTATGAAGGCTCAAAATCCTCAAACCCGTTAGCATTCCGTCACTACAATCCCGACGAACTGGTGTTGGGTAAGCGTATGGAAGAGCACTTGCGTTTTGCCGCCTGCTACTGGCACACCTTCTGCTGGAACGGGGCGGATATGTTTGGTGTGGGGGCGTTTAATCGTCCGTGGCAGCAGCCTGGTGAGGCACTGGCGTTGGCGAAGCGTAAAGCAGATGTCGCATTTGAGT
asRNA3:
ATGCAAGCCTATTTTGACCAGCTCGATCGCGTTCGTTATGAAGGCTCAAAATCCTCAAACCCGTTAGCATTCCGTCACTACAATCCCGACGAACTGGTGTTGGGTAAGCGTATGGAAGAGCACTTGCGTTTTGCCGCCTGCTACTGGCACACCTTCTGCTGGAACGGGGCGGATATGTTTGGTGTGGGGGCGTTTAATCGTCCGTGGCAGCAGCCTGGTGAGGCACTGGCGTTGGCGAAGCGTAAAGCAGATGTCGCATTTGAGT
asRNA4:
TTTTCCACAAGTTACATGTGCCATTTTATTGCTTCCACGATGTGGATGTTTCCCCTGAGGGCGCGTCGTTAAAAGAGTACATCAATAATTTTGCGCAAATGGTTGATGTCCTGGCAGGCAAGCAAGAAGAGAGCGGCGTGAAGCTGCTGTGGGGAACGGCCAACTGCTTTACAAACCCTCGCTACGGCGCGGGTGCGGCGACGAACCCAGATCCTGAAGTCTTCAGCTGGGCGGCAACGCAAGTTGTTACAGCGATGGAAGCAA
the invention also provides a recombinant expression vector carrying the antisense RNA and improving the yield of 1,2, 4-butanetriol in recombinant escherichia coli, which takes pEtac-mdLC-tac-xdh as a starting plasmid to construct an expression vector containing the 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-asRNA 4.
The invention also provides recombinant escherichia coli with improved 1,2, 4-butanetriol yield, which is obtained by transferring the constructed recombinant expression vectors into escherichia coli E.coli W3110 delta yagE delta yjhH delta yiaE delta ycdW serving as a starting bacterium respectively.
The construction method of the recombinant escherichia coli with the improved 1,2, 4-butanetriol yield comprises the following steps:
step 1, cloning a corresponding xylA gene segment by using an antisense RNA primer pair as1-F/as1-R, as2-F/as2-R, as3-F/as3-R, as4-F/as4-R by taking an E.coli W3110 genome as a template;
step 2, connecting the cloned fragment with the hairpin structure by utilizing enzyme digestion and connecting molecule operation technologies, and reversely inserting the antisense RNA fragment carrying the hairpin structure into a lac promoter of an expression vector pEtac-mdLC-tac-xdh to obtain a series of antisense RNA recombinant expression vectors;
and 3, introducing the recombinant expression vector into an E.coli W3110 delta yagE delta yjhH delta yiaE delta ycdW competent cell to obtain the 1,2, 4-butanetriol-producing recombinant escherichia coli capable of inhibiting the expression of xylose isomerase.
The invention has the beneficial effects that:
(1) the transcription levels of xylose isomerase gene xylA of the four recombinant escherichia coli carrying antisense RNA expression vectors constructed by the invention are respectively inhibited by 72%, 57%, 32% and 23%.
(2) The yields of the recombinant Escherichia coli E.coli W3110 delta yagE delta yjhH delta yaE delta ycdW (pEtac-mdLC-tac-xdh-lac-asRNA3) and E.coli W3110 delta yagE delta yjhH delta yaE delta ycdW (pEtac-mdLC-tac-xdh-lac-asRNA4) butanetriol carrying the antisense RNA expression vector constructed by the invention are respectively improved by 44% and 20% and reach 5.71g/L and 4.75 g/L.
Drawings
FIG. 1 is a schematic diagram of the metabolic synthesis of butanetriol.
FIG. 2 antisense RNA fragment binding region.
FIG. 3 flow chart of antisense RNA expression vector construction.
FIG. 4 antisense expression recombinant strain xylose isomerase gene xylA transcript level analysis. Coli MXW 004: w3110. delta. yagE. delta. yjhH. delta. yiaE. delta. ycdW (pEtac-mdLC-tac-xdh); coli as1MXW 004: w3110 Δ yagE Δ yjhH Δ yiaE Δ ycdW (pEtac-mdLC-tac-xdh-lac-asRNA 1); coli as2MXW 004: coli W3110 Δ yagE Δ yjhH Δ yiaE Δ ycdW (pEtac-mdLC-tac-xdh-lac-asRNA 2); coli as3MXW 004: coli W3110 Δ yagE Δ yjhH Δ yiaE Δ ycdW (pEtac-mdLC-tac-xdh-lac-asRNA 3); coli as4MXW 004: coli W3110 Δ yagE Δ yjhH Δ yiaE Δ ycdW (pEtac-mdLC-tac-xdh-lac-asRNA4)
FIG. 5 analysis of fermentation performance of antisense expression recombinant strain. Coli MXW 004: w3110. delta. yagE. delta. yjhH. delta. yiaE. delta. ycdW (pEtac-mdLC-tac-xdh); coli MXW 005: w3110 Δ yagE Δ yjhH Δ yiaE Δ ycdW Δ xylA (pEtac-mdLC-tac-xdh); coli as1MXW 004: w3110 Δ yagE Δ yjhH Δ yiaE Δ ycdW (pEtac-mdLC-tac-xdh-lac-asRNA 1); coli as2MXW 004: coli W3110 Δ yagE Δ yjhH Δ yiaE Δ ycdW (pEtac-mdLC-tac-xdh-lac-asRNA 2); coli as3MXW 004: coli W3110 Δ yagE Δ yjhH Δ yiaE Δ ycdW (pEtac-mdLC-tac-xdh-lac-asRNA 3); coli as4MXW 004: coli W3110 Δ yagE Δ yjhH Δ yiaE Δ ycdW (pEtac-mdLC-tac-xdh-lac-asRNA4)
Detailed Description
The starting strain E.coli W3110 delta yagE delta yjhH delta yaE delta ycdW takes E.coli W3100 as a host, yagE, yjhH, yiaE and ycdW genes are knocked out, and a specific construction method is disclosed in the following documents: the knock-out of a byproduct pathway in the synthesis of the recombinant escherichia coli D-1,2, 4-butanetriol and the enhanced expression of key enzymes [ D ]. Jiangnan university, 2017 ].
The plasmid pEtac-mdLC-tac-Xdh uses tac promoter to regulate the expression of Xdh and MdlC, and the specific construction method is shown in the literature: metabolic engineering of Escherichia coli to synthesize D-1,2, 4-butanetriol [ J ]. Microbiol bulletin, 2014,41(10) pEtac-mdLC-tac-xylB.
The xylA-encoding Gene is shown as NCBI-Gene ID: 948141.
EXAMPLE 1 construction of recombinant E.coli carrying antisense RNA expression vector
(1) Step 1, cloning a corresponding xylA gene segment by using E.coli W3110 genome as a template and an antisense RNA primer pair as1-F/as1-R, as2-F/as2-R, as3-F/as3-R, as4-F/as 4-R.
The primer sequence is as follows:
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) and 2, connecting the cloned fragment with the hairpin structure by utilizing enzyme digestion and connecting molecule operation technologies, and reversely inserting the antisense RNA fragment carrying the hairpin structure into a lac promoter of an expression vector pEtac-mdLC-tac-xdh to obtain a series of antisense RNA recombinant expression vectors.
The pMD19-T-asRNA was constructed by inserting the desired xylA fragment, which was amplified by PCR using the corresponding primers, in the reverse direction into the plasmid pMD19-T (mut) BamHI-HindIII cleavage site, wherein pMD19-T (mut) was constructed by inserting a hairpin structure (Nakashima N, Tamura T. conditional gene cloning of multiple genes with antisense RNAs and generation of a tissue strain of Escherichia coli [ J ] and SpeI, Sac II, BamH I and HindIII into the commercial plasmid pMD19-T (simple). Then, a fragment of the asRNA carrying the hairpin structure was obtained by double digestion with Spe I and SacII and ligated to pEtac (mut) SpeI-SacII site to construct pEtac-asRNA, wherein pEtac (mut) was constructed by introducing speI and SacII cleavage sites into the plasmid pEtac (Shenmu, Wangxiang, Tangxuening, etc.. the secretory expression of the hyperthermophilic alpha-amylase gene of the archaebacterium Pyrococcus furiosus in Escherichia coli [ J ]. China, 2003,22(1): 12-14.). Finally, plasmids pEtac-asRNA and pEtac-mdLC-tac-xdh were double-digested with restriction enzymes BlpI and XbaI and ligated to form expression vector pEtac-mdLC-tac-xdh-lac-asRNA.
Plasmids pEtac-mdLC-tac-xdh-lac-asRNA1, pEtac-mdLC-tac-xdh-lac-asRNA2, pEtac-mdLC-tac-xdh-lac-asRNA3 and pEtac-mdLC-tac-xdh-lac-asRNA4, which were constructed stepwise according to the above-described procedure, contained different xylA gene fragments. Wherein the asRNA1 carries xylA gene-27-105 bp sequence, the asRNA2 carries xylA gene-27-265 bp sequence, the asRNA3 carries xylA gene 1-265bp sequence, the asRNA4 carries xylA gene 266-530bp sequence, and the asRNA gene is regulated and expressed by a promoter lac.
(3) And 3, introducing the strain into E.coli W3110 delta yagE delta yjhH delta yiaE delta ycdW competent cells by an electrical transformation method to obtain the 1,2, 4-butanetriol producing recombinant escherichia coli capable of inhibiting the expression of xylose isomerase.
Coli W3110. DELTA. yagE. DELTA. yjhH. DELTA. yaE. DELTA. ycdW cells were cultured to OD600When the concentration is equal to 0.4-0.6, taking out from the shaking table, carrying out ice bath for 30min, and then centrifuging. With 0.1mol/L CaCl2The corresponding antisense RNA expression vector constructed in the transformation step (2) is washed, heat-shocked at 42 ℃ and spread on a Kan resistant plate after being cultured for 1h, and the correct transformant is picked up after the growth of the vector, thereby obtaining recombinant large intestine stems E.coli W3110 delta yagE delta yjhH delta yayiaE delta ycdW (pEtac-mdC-tac-xdh-lac-asRNA 1), E.coli W3110 delta yagE delta yjhH delta yjayiaE delta ycdW (pEtac-mdC-tac-xdh-lac-asRNA 2), E.coli W3110 delta yagE delta yjhH delta yiaE delta ycdW (pEtac-mdC-tac-xdh-lac-asRNA 3) E.coli W3110 delta yagE delta yhH delta yjayyyyyyyyyiaE delta-asRNA 389-5-ctadlRNA.
EXAMPLE 2 Synthesis of D-1,2, 4-butanetriol Using recombinant antisense expression Strain
Single colonies of the different recombinant bacteria constructed in example 1 were picked from the plates, cultured overnight, and inoculated in 50mL of a fermentation medium (1.5-fold concentration of LB medium, 30g/L xylose, 10g/L CaCO) at an inoculum size of 1% as a seed solution3) In (1), culturing to OD600When the concentration is 0.6, adding IPTG with the final concentration of 0.5mmol/L, culturing at 37 ℃ and 200rpm, and taking a logarithmic phase growth bacterial liquid to measure the relative transcription level of mRNA; and (5) taking 48h of fermentation liquor to measure the yield of BT.
(1) Extraction of cellular RNA and quantitative PCR procedure: 10mL of logarithmic phase cell culture was collected by centrifugation. Total RNA of the cells was extracted with a total RNA extraction Kit from Tiangen Biochemical technology Ltd, and the resulting purified RNA was treated with DNase I, and further purified to remove genomic DNA which may be present by the RNeasy Mini Kit from Takara. Then, cDNA was synthesized by reverse transcription of script II Q RT supermix (Vazyme Nanjing, China) using the extracted RNA as a template. The transcription inhibition level of xylA is obtained by measuring the mRNA level of the strain by real-time quantitative PCR by using cDNA as a template and qxylA-F (ATGCAGATGGTGGTTGAGCA) qxylA-R (GTCGCGGCATCGTAATCATTA) as a primer. Each real-time quantitative PCR assay was performed by One Step SYBR Prime Script RT-PCR Kit II from Takara using a Roche Light Cycler480 real-time fluorescent quantitative PCR instrument from Germany. In addition, quantitative PCR values were calibrated by analyzing the expression level of intracellular 16S rRNA using X16S-F (CAGAAGAAGCACCGGCTAAC)/X16S-R (GGGATTTCACATCCGACTTG) as primers. The results showed that the E.coli W3110. delta. yagE. delta. yjhH. delta. yaE. delta. ycdW (pEtac-mdlC-tac-xdh-lac-asRNA1), E.coli W3110. delta. yagE. delta. yjhH. delta. yaE. delta. ycdW (pEtac-mdlC-tac-xdh-lac-asRNA2), E.coli W3110. delta. yagE. delta. yjhH. delta. yyaE. delta. ycdW (pEtac-mdlC-tac-xdh-lac-asRNA3) E.coli W0. delta. yagE. delta. yjhE. delta. ycdW (pEtac-mdlC-tac-xdh-lac-asRNA4) strains were reduced compared to the control strain E.coli W3110. delta. yagE. delta. yjhH. delta. yjyyaE. delta. yycdW (pEtac-tad-tac-6778%, 57-lac-asRNA 4), respectively, the level was reduced by 72%, and by pEtac-mdlC-mC-14%.
(2) And (3) detecting the yield of BT: firstly, cells are removed by centrifuging the fermentation liquor at 12000rpm, and the supernatant is filtered by a 0.22 mu m mixed cellulose ester microporous membrane to be used for HPLC detection. Detection conditions are as follows: agilent 1260, RID detector, Bio-Rad AminexHPX-87column (300 mm. times.7.8 mm), column temperature 60 deg.C, mobile phase 5mmol/L H2SO4The flow rate was 0.6 mL/min. The result shows that the yield of the recombinant Escherichia coli 1,2, 4-butanetriol containing the plasmid pEtac-mdLC-tac-xdh-lac-asRNA3 is optimally 5.71g/L and is improved by 44 percent.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> a method for increasing the yield of 1,2, 4-butanetriol in recombinant Escherichia coli
<160> 16
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<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 (5)

1.一种生产1,2,4-丁三醇的重组大肠杆菌的构建方法，其特征在于，包括以下步骤：1. a construction method of the recombinant Escherichia coli of production 1,2,4-butanetriol, is characterized in that, comprises the following steps: 步骤1:以E.coli W3110基因组为模板，利用核苷酸序列如SEQ ID NO.9-10所示的反义RNA引物对克隆相应的xylA基因片段，获得核苷酸序列如SEQ ID NO.3所示的反义RNA片段asRNA3；Step 1: take E.coli W3110 genome as template, utilize the antisense RNA primer pair shown in nucleotide sequence as shown in SEQ ID NO.9-10 to clone corresponding xylA gene fragment, obtain nucleotide sequence as shown in SEQ ID NO. The antisense RNA fragment asRNA3 shown in 3; 步骤2:利用酶切、连接分子操作技术将克隆片段与发夹结构连接，然后将携带发夹结构的反义RNA片段反向插入到表达载体pEtac-mdlC-tac-xdh的lac启动子后，获得反义RNA重组表达载体pEtac-mdlC-tac-xdh-lac-asRNA3；Step 2: utilize enzyme cleavage, link molecular manipulation technology to connect the cloned fragment with the hairpin structure, then insert the antisense RNA fragment carrying the hairpin structure into the lac promoter of the expression vector pEtac-mdlC-tac-xdh in reverse, Obtain the antisense RNA recombinant expression vector pEtac-mdlC-tac-xdh-lac-asRNA3; 步骤3:将重组表达载体导入E.coli W3110ΔyagEΔyjhHΔyiaEΔycdW感受态细胞中，获得能够抑制木糖异构酶表达的产1,2,4-丁三醇重组大肠杆菌。Step 3: The recombinant expression vector was introduced into E.coli W3110Δ yagE Δ yjhH Δ yiaE Δ ycdW competent cells to obtain 1,2,4-butanetriol-producing recombinant Escherichia coli that can inhibit the expression of xylose isomerase. 2.权利要求1构建得到的生产1 ,2 ,4-丁三醇的重组大肠杆菌，其特征在于，是利用核苷酸序列如SEQ ID NO.3所示的反义RNA asRNA3部分抑制木糖异构酶基因xylA的表达；所述重组大肠杆菌是以E.coli W3110ΔyagEΔyjhHΔyiaEΔycdW为出发菌株，将携带核苷酸序列如SEQ ID NO.3所示的反义RNA asRNA3的重组表达载体转入大肠杆菌；所述重组表达载体是以pEtac-mdlC-tac-xdh为出发质粒，构建的用于表达反义RNA的表达载体：pEtac-mdlC-tac-xdh-lac-asRNA3。2. the recombinant Escherichia coli of the production 1,2,4-butanetriol that claim 1 constructs is characterized in that, utilizes the antisense RNA asRNA3 shown in nucleotide sequence as SEQ ID NO.3 to partially suppress xylose Expression of isomerase gene xylA; the recombinant Escherichia coli is based on E.coli W3110ΔyagEΔyjhHΔyiaEΔycdW as the starting strain, and the recombinant expression vector carrying the antisense RNA asRNA3 whose nucleotide sequence is shown in SEQ ID NO.3 is transferred into Escherichia coli The recombinant expression vector is an expression vector for expressing antisense RNA constructed from pEtac-mdlC-tac-xdh as a starting plasmid: pEtac-mdlC-tac-xdh-lac-asRNA3. 3.应用权利要求2所述的重组大肠杆菌生产1,2,4-丁三醇的方法。3. The method for producing 1,2,4-butanetriol using the recombinant Escherichia coli of claim 2. 4.根据权利要求3所述的方法，其特征在于，以木糖为碳源。4. The method according to claim 3, wherein xylose is used as the carbon source. 5.根据权利要求3所述的方法，其特征在于，将种子培养液接种于发酵培养基中，所述发酵培养基含1.5倍浓度的LB培养基、30 g/L木糖、10 g/L CaCO3；待培养至OD600接近0 .6时加入IPTG诱导培养。5. method according to claim 3, is characterized in that, seed culture liquid is inoculated in fermentation medium, and described fermentation medium contains LB substratum, 30 g/L xylose, 10 g/L of 1.5 times of concentration. L CaCO 3 ; IPTG was added to induce the culture when the OD 600 was close to 0.6.
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