CN111334459B - A kind of construction method and application of Klebsiella engineering bacteria improving 1,3-propanediol production - Google Patents

Abstract

The invention belongs to the technical field of environmental microorganisms, and in particular relates to a construction method and application of an engineering bacterium for improving the production of 1,3-propanediol synthesized by Klebsiella. The invention utilizes biotechnology to inactivate the activities of Klebsiella diol dehydratase, lactate dehydrogenase, alcohol dehydrogenase and other enzymes. When the constructed engineered bacteria ferment 1,3-propanediol, the yield and conversion rate of 1,3-propanediol are increased, and the production of by-products lactic acid and ethanol is reduced. The engineering bacterium constructed by the method provided by the invention has stable genetic properties, good 1,3-propanediol synthesis performance, and reduced by-product output, and is a relatively valuable construction method for Klebsiella engineering bacteria.

Description

A kind of construction method and application of Klebsiella engineering bacteria for improving 1,3-propanediol production

technical field

The invention relates to the technical field of environmental microbes, in particular to a construction method and application of Klebsiella engineering bacteria for improving the production of 1,3-propanediol.

Background technique

1,3-propanediol is an important bulk chemical, which is widely used in cosmetics, food, lubricants and pharmaceutical industries, especially as a monomer and terephthalic acid to form polytrimethylene terephthalate (PTT ). PTT has good performance and is widely used in the clothing industry, carpet industry, and engineering thermoplastic industry, thereby driving the market demand for 1,3-propanediol.

The technology of biological synthesis of 1,3-propanediol mainly focuses on two directions, one of which uses glucose as the substrate and the other uses glycerol as the substrate. Among the series of microorganisms that metabolize glycerol to synthesize 1,3-propanediol, Klebsiella (including Klebsiella pneumoniae and Klebsiella oxytoca) is a facultative anorexia Klebsiella bacteria have the advantages of simple fermentation operation, high synthesis amount and conversion rate of 1,3-propanediol, etc. Therefore, Klebsiella is the most promising strain for industrial production of 1,3-propanediol.

The pathway of Klebsiella metabolizing glycerol to 1,3-propanediol includes oxidation branch and reduction branch. In the oxidation branch, glycerol is catalyzed by glycerol dehydrogenase and dihydroxyacetone kinase to generate dihydroxyacetone phosphate, and then enters the glycolysis pathway to generate ethanol, lactic acid, succinic acid, acetic acid, 2,3-butanediol and other by-products product. The formation of by-products reduces the conversion rate of the substrate and is unfavorable for the extraction of 1,3-propanediol products in the subsequent process. In the reduction branch, glycerol synthesizes 3-hydroxypropanal under the action of glycerol dehydratase (DhaB), and then is catalyzed by 1,3-propanediol oxidoreductase to generate 1,3-propanediol.

Contents of the invention

In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a Klebsiella engineered bacterium that increases the production of 1,3-propanediol and its construction method and application, so as to solve the problems in the prior art.

To achieve the above object and other related objects, the first aspect of the present invention provides a Klebsiella engineered bacterium, which is a Klebsiella engineered bacterium in which diol dehydratase-related genes have been knocked out.

Preferably, the diol dehydratase-related genes are selected from diol dehydratase genes and/or diol dehydratase activator genes.

More preferably, the diol dehydratase gene is selected from one or more genes among pduC, pduD and pduE; the diol dehydratase activator gene is selected from one or more genes among pduG and pduH.

Preferably, the lactate dehydrogenase gene and/or the alcohol dehydrogenase gene in the Klebsiella engineering bacteria are also knocked out.

More preferably, the lactate dehydrogenase gene is ldhA gene.

More preferably, the alcohol dehydrogenase gene is adhE gene.

The K.pΔpduΔldhΔadh engineering bacteria K.pΔpduΔldhΔadh fermented 30 hours of 1,3-propanediol yield and conversion rate respectively increased by 25.8% and 10.3% compared with the unmodified Klebsiella; K.oΔpduΔldhΔadh fermented for 30 hours 1 , the yield and conversion rate of 3-propanediol were respectively increased by 22.11% and 7.97% compared with the unmodified Klebsiella.

The second aspect of the present invention provides a method for constructing Klebsiella engineering bacteria, the method comprising the following steps: knocking out diol dehydratase-related genes.

Preferably, the gene knockout related to diol dehydratase is selected from diol dehydratase gene and/or diol dehydratase activator gene.

Preferably, the method further includes knocking out the lactate dehydrogenase gene and/or the alcohol dehydrogenase gene.

The third aspect of the present invention provides an application of Klebsiella engineering bacteria in the fermentative production of 1,3-propanediol.

The method that Klebsiella engineering bacteria of the present invention produces 1,3-propanediol comprises the steps:

1) Klebsiella strains are inoculated into the seed culture medium to expand cultivation;

2) Inoculate the Klebsiella engineering bacteria in step 1) into the fermentation medium for fermentation.

Further, the fermentation medium is a fermentation medium containing glycerol,

Furthermore, the initial concentration of glycerol in the fermentation medium is 20-50g/L.

Further, the glycerin concentration is controlled at 10-30g/L during the fermentation process.

Further, the pH value is controlled at 6.5-7.5 during the fermentation process.

Further, the fermentation temperature is controlled at 30-40°C.

Further, stirring is carried out during the fermentation process, and the stirring speed is controlled at 100-300 rpm.

Further, during the fermentation process, the fermenter is ventilated, and the ventilating volume is controlled at 1-4L/min.

Furthermore, the gas introduced is air.

As mentioned above, the construction method and application of Klebsiella engineering bacteria for improving the production of 1,3-propanediol of the present invention have the following beneficial effects: the genetic performance of the constructed engineering bacteria is stable, and the output and conversion of synthetic 1,3-propanediol The yield is obviously increased, and the production of by-products lactic acid and ethanol is reduced. It is a relatively promising engineering bacterium. The construction method of Klebsiella engineering bacteria provided by the present invention is relatively valuable in application.

Description of drawings

Figure 1 shows that the primers pdutest-s and pdutest-a in Example 1 of the present invention verify the knockout of the gene encoding diol dehydratase.

Figure 2 shows that the primers pduGHtest-s and pduGHtest-a in Example 2 of the present invention verify the knockout of the gene encoding the diol dehydratase activating factor.

Figure 3 shows that the primers ldhtest-s and ldhtest-a in Example 3 of the present invention verify the knockout of the gene encoding lactate dehydrogenase.

Figure 4 shows that the primers adhtest-s and adhtest-a in Example 4 of the present invention verify the knockout of the gene encoding alcohol dehydrogenase.

Detailed ways

The first aspect of the present invention provides a Klebsiella engineering bacterium, wherein the Klebsiella engineering bacterium is a Klebsiella engineering bacterium in which diol dehydratase-related genes have been knocked out.

The Klebsiella engineering bacterium is the Klebsiella engineering bacterium in which the diol dehydratase-related gene in the wild type Klebsiella pneumoniae or the wild type Klebsiella acidogens has been knocked out.

The wild-type Klebsiella pneumoniae can be selected from: Klebsiella pneumoniae CGMCC 1.6366.

The wild-type Klebsiella acidogens may be selected from: Klebsiella acidogens M5a1.

Further, the diol dehydratase-related genes are selected from diol dehydratase genes and/or diol dehydratase activator genes.

Further, the diol dehydratase gene is selected from one or more of pduC, pduD, pduE;

Further, the knockout of the diol dehydratase gene can be knocked out in the following ways:

1) Knockout pduC;

2) knock out pduD;

3) knock out pduE;

4) knock out pduC and pduD;

5) Knockout of pduC and pduE;

6) Knockout of pduD and pduE;

7) Knockout pduC, pduD, pduE.

Further, the diol dehydratase activator gene is selected from one or more of pduG and pduH;

Further, when knocking out diol dehydratase-related genes, you can choose to knock out in the following ways:

1) Knockout pduC, pduD, pduE, pduG, pduH;

2) knock out pduG, pduH;

3) knock out any gene in pduC, pduD and pduE and any gene in pduG, pduH;

4) Knocking out any two genes in pduC, pduD and pduE and any gene in pduG and pduH;

5) knock out any two genes and pduG, pduH in pduC, pduD and pduE;

6) Knock out the three genes pduC, pduD, pduE and any gene in pduG, pduH.

Further, the diol dehydratase refers to an enzyme that catalyzes the dehydration of glycerol to generate 3-hydroxypropanal, which is jointly encoded by three genes, and the gene names are respectively: pduC, pduD, and pduE.

The gene reading frames of pduC, pduD, and pduE can be queried through existing databases (such as NCBI), or can be obtained through sequencing.

For example, it can be seen through sequencing that in Klebsiella pneumoniae CGMCC 1.6366, the gene reading frame of pduC is shown in SEQ ID NO.1, the gene reading frame of pduD is shown in SEQ ID NO.2, and the gene reading frame of pduE is shown in SEQ ID NO.2. Shown in ID NO.3.

Further, the diol dehydratase activating factor refers to an enzyme that activates diol dehydratase activity, which is jointly encoded by two genes, and the gene names are: pduG and pduH respectively.

The gene reading frames of pduG and pduH can be queried through existing databases (such as NCBI), or can be obtained through sequencing.

For example, according to sequencing, in Klebsiella pneumoniae CGMCC 1.6366, the gene reading frame of pduG is shown in SEQ ID NO.4, and the gene reading frame of pduH is shown in SEQ ID NO.5.

Further, in the Klebsiella engineering bacteria, the lactate dehydrogenase gene and/or the alcohol dehydrogenase gene are also knocked out. After the lactate dehydrogenase gene and/or the alcohol dehydrogenase gene are further knocked out, the 1,3-propanediol production of the Klebsiella engineered bacteria is further improved.

Furthermore, the lactate dehydrogenase refers to an enzyme that catalyzes the reduction of pyruvate into lactate, and its gene name is ldhA.

The gene reading frame of ldhA can be queried through existing databases (such as NCBI), or obtained through sequencing.

For example, it can be known through sequencing that the gene reading frame of the lactate dehydrogenase gene in Klebsiella pneumoniae CGMCC 1.6366 is shown in SEQ ID NO.6.

Further, the alcohol dehydrogenase refers to an enzyme that catalyzes the generation of ethanol from acetaldehyde, and its gene name is adhE.

The gene reading frame of adhE can be queried through existing databases (such as NCBI), and can also be obtained through sequencing.

For example, it can be known from sequencing that the gene reading frame of the alcohol dehydrogenase gene in Klebsiella pneumoniae CGMCC 1.6366 is shown in SEQ ID NO.7.

The 1,3-propanediol yield and conversion rate of the Klebsiella engineered bacteria K.pΔpduΔldhΔadh fermented for 30 hours are at least 13.825.8% and 5.3710.3% respectively higher than those of the unmodified original Klebsiella bacteria. ; Klebsiella engineered bacteria K.oΔpduΔldhΔadh fermented 1,3-propanediol yield and conversion rate for 30 hours were increased by 22.11% and 7.97% respectively compared with unmodified Klebsiella bacteria;

The output and conversion rate of the Klebsiella engineering bacteria 1,3-propanediol are detected according to the following method:

1) Inoculate the constructed Klebsiella strain and the initial bacterial strain into a 250mL Erlenmeyer flask containing 50mL of seed medium, the inoculation amount is 1% of the volume of the seed medium, and culture on a shaker at 37°C and 200rpm for 12h.

2) Inoculate 50mL of Klebsiella engineering bacteria from step 1) into a fermenter containing 3L of fermentation medium, at 37°C, with an air volume of 2L/min, and a rotation speed of 200rpm, to carry out continuous fermentation to synthesize 1,3-propanediol, and ferment During the process, the pH value was adjusted to 6.8 with 30% NaOH solution.

3) Fermentation process gets a certain amount of fermented liquid at regular intervals, with high performance liquid chromatography (selects the Aminex HPX-87H chromatographic column of Bio-Rad Company, and the detector is the RID-20A type differential detector of Japan Shimadzu Company) , mobile phase 0.005mol/L H ₂ SO ₄ , flow rate 0.8mL/min, column oven temperature 65°C, injection volume generally 20μL) to measure the consumption of substrate glycerol in the fermentation broth, if the glycerol in the fermentor is consumed , fed with a glycerol aqueous solution with a concentration of 75% (g/g). After 30 hours of fermentation, the consumption of substrate glycerol and the production of product 1,3-propanediol were detected by high performance liquid chromatography.

4) Calculate the conversion rate according to formula ①, and calculate the conversion rate and the increase rate of 1,3-propanediol output according to formula ②.

Concentration of 1,3-propanediol (g/L)*fermentation broth volume (L)/[initial glycerol concentration of fermentation medium (g/L)*medium volume in the fermentation broth of conversion rate (%)=30h (L)+concentration of feeding material (g/L)*consumed volume of feeding material (L)－concentration of residual glycerol in the fermentation broth after 30 hours of fermentation (g/L)*volume of fermentation broth (L)]*

100%①

Improvement rate (%) = [c (engineering) - c (initial)] / c (initial) × 100%②

Among them, c (engineering) refers to the conversion rate of Klebsiella engineering bacteria or the concentration (g/L) of 1,3-propanediol produced, and c (initial) refers to the initial Klebsiella, that is, the unmodified The transformation rate of the strain or the concentration of 1,3-propanediol produced.

The second aspect of the present invention provides a method for constructing Klebsiella engineering bacteria, the method comprising the following steps: knocking out genes related to diol dehydratase.

Further, the diol dehydratase-related genes are selected from diol dehydratase genes and/or diol dehydratase activator genes.

Further, the method also includes knocking out the lactate dehydrogenase gene and/or the alcohol dehydrogenase gene.

Further, the method also includes one or more of the following:

1) The diol dehydratase gene is selected from one or more genes in pduC, pduD, pduE;

2) The diol dehydratase activator gene is selected from one or more genes in pduG and pduH;

3) The lactate dehydrogenase gene is the ldhA gene;

4) The lactate dehydrogenase gene is the adhE gene;

5) The method of homologous recombination is used to knock out the gene;

The third aspect of the present invention provides an application of Klebsiella engineering bacteria in the fermentative production of 1,3-propanediol.

Klebsiella engineering bacterium of the present invention produces the method for 1,3-propanediol, comprises the steps:

1) Inoculate Klebsiella engineering bacteria into the seed medium for cultivation.

2) Inoculate the Klebsiella engineering bacteria in step 1) into the fermentation medium for cultivation.

Further, in step 1), Klebsiella engineering bacteria are inoculated according to 0.5%-2% of the volume of the seed medium.

Further, in step 1), the culture conditions of Klebsiella engineering bacteria are 30°C-40°C, 100-300rpm culture for 10-20h.

Further, the seed medium components are: peptone 10g/L, yeast extract 5g/L, sodium chloride 5g/L.

Further, all the Klebsiella engineering bacteria in step 1) are inoculated into the fermenter containing the fermentation medium for cultivation.

Further, the amount of the fermentation medium is 50%-80% of the capacity of the fermenter.

Further, the fermentation medium is a fermentation medium containing glycerol. The composition of the fermentation medium is 0.69 g/L of dipotassium hydrogen phosphate, 0.25 g/L of potassium dihydrogen phosphate, 1.5 g/L of yeast powder, 4.0 g/L of ammonium sulfate, and 0.2 g/L of magnesium sulfate.

Furthermore, the initial concentration of glycerol in the fermentation medium is 20-50g/L.

Further, the glycerin concentration is controlled at 10-30g/L during the fermentation process.

Further, the pH value is controlled at 6.5-7.5 during the fermentation process.

Further, the fermentation temperature is controlled at 30-40°C.

Further, stirring is carried out during the fermentation process, and the stirring speed is controlled at 100-300 rpm.

Further, during the fermentation process, the fermenter is ventilated, and the ventilating volume is controlled at 1-4L/min.

Furthermore, the gas introduced is air.

Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes, and various modifications or changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention.

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are to describe specific specific embodiments, It is not intended to limit the protection scope of the present invention; in the description and claims of the present invention, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural forms.

When the examples give numerical ranges, it should be understood that unless otherwise specified in the present invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to those skilled in the art's grasp of the prior art and the description of the present invention, the methods, equipment, and materials described in the embodiments of the present invention can also be used Any methods, apparatus and materials of the prior art similar or equivalent to the practice of the present invention.

Embodiment 1: Construction of Klebsiella engineering bacteria with diol dehydratase inactivation

For the operation principle of knocking out the coding gene of diol dehydratase in this part and materials such as plasmids and bacterial strains used, see (Wei et.al.Red recombinase assisted gene replacement in Klebsiella pneumoniaeJournal of Industrial Microbiology&Biotechnology 2012), the specific steps are as follows:

1) Construction of a homologous recombination fragment to knock out the gene encoding diol dehydratase

According to the gene sequence of Klebsiella pneumoniae CGMCC 1.6366 encoding diol dehydratase, the upstream and downstream primers were designed as follows:

Upstream primer pdu-s1: CAACGTGGAAGTCGTGGCGTATAGCT (SEQ ID NO.8)

Downstream primer pdu-a1: GCGTTGAGGTGGAATACGGTGGC (SEQ ID NO.9)

Using the designed primers pdu-s1 and pdu-a1, using the genomic DNA of Klebsiella pneumoniae CGMCC 1.6366 as a template, PCR amplification was used to obtain partial genes encoding diol dehydratase (including pduC, pduD, pduE), It was connected to pMD-19T simple plasmid (commercial product, Treasure Bioengineering Dalian Co., Ltd.) by TA cloning method, and the obtained recombinant plasmid was named pMD19T-pdu, and was verified by sequencing by Shanghai Sangon Biotechnology Co., Ltd.

The successfully constructed plasmid pMD19T-pdu was transformed into the strain DH5α-pIJ790 (obtained from the NTCC Type Culture Collection) to obtain the double plasmid-carrying strain DH5α-pIJ790-pMD19T-pdu.

According to the gene sequence of the plasmid pIJ778 (obtained from the NTCC Type Culture Collection) carrying the streptomycin resistance gene cassette, the upstream and downstream primers were designed:

Upstream primer pdu-FRT-s1:

CAGGCGCACGTCACCAACGTCAAAGATAACCCGGTACAGATTCCGGGGATCCGTCGACC (SEQ ID NO. 10)

Downstream primer pdu-FRT-a1:

AATCGTCGCCTTTGAGTTTTTTACGCTCGACGTACAGCGTTGTAGGCTGGAGCTGCTTC (SEQ ID NO. 11)

Using the designed primers pdu-FRT-s1 and pdu-FRT-a1, using the plasmid pIJ778 as a template, the fragment of the streptomycin resistance gene cassette was obtained by PCR amplification.

Make the competent cells of the strain DH5α-pIJ790-pMD19T-pdu, and then electrotransform DH5α-pIJ790-pMD19T-pdu competent cells with the fragment of the streptomycin resistance gene cassette obtained by PCR and recovered after cleaning, and recover at 37°C for 1 hour The streptomycin-resistant plate was centrifuged (the concentration of streptomycin used was 50 mg/L), and the positive strain carrying pMD19T-Δpdu778 was screened. After colonies grew on the plate, they were verified by primers pdu-s1 and test778 (test778: AGAATCTCGCTCTCTCCAGGGGAAG) (SEQ ID NO.12). If recombination does not occur, the gene fragment cannot be amplified. If the recombination is successful, a gene fragment of about 1.0 kb is amplified. The strains with successful recombination verified by PCR were inoculated in streptomycin-resistant LB test tube culture medium, and the plasmid was extracted, which was named pMD19T-Δpdu778.

Using the constructed pMD19T-Δpdu778 as a template, use primers pdu-s1 and pdu-a1 under the action of high-fidelity enzyme KOD to amplify the homologous recombination fragment that knocked out the pdu gene, and the obtained fragment is abbreviated as "A". The size of the "A" fragment is 2700bp, with homology arms of about 500bp at both ends, and a streptomycin resistance gene in the middle.

2) Homologous recombination knockout of the gene encoding Klebsiella diol dehydratase

A. Knockout of the gene encoding Klebsiella pneumoniae diol dehydratase

Klebsiella pneumoniae CGMCC 1.6366 strain (this strain is also called TUAC01, AC01), CGMCC1.6366 strain has been disclosed in the literature (Journal of Industrial Microbiology & Biotechnology. 2012 39:1219-1226). This strain is a strain used to produce 1,3-propanediol. The bacterium was isolated from soil, and the isolation process and description of its properties can be found in (World Journal of Microbiology Biotechnology 2008, 24:1731-1740).

Competent cells of Klebsiella pneumoniae CGMCC 1.6366 were made, and the plasmid pDK6-red was transformed (the reference for the construction process of the plasmid was Wei Dong, Wang Min, Shi Jiping, Hao Jian. Red recombinase assisted gene replacement in Klebsiella pneumoniae. Journal of Industrial Microbiology & Biotechnology. 201239:1219–1226), resulting in strain K.p/red.

Make electroporation competent cells of strain K.p/red, add IPTG to induce the expression of Red recombinase when culturing competent cells, and add EDTA to improve the electroporation transformation efficiency of Klebsiella. K.p/red competent cells were transformed by electroporation with homologous recombination fragment "A". After recovery, they were all spread on streptomycin-resistant plates and cultured overnight at 37°C. After a single colony grows on the plate, use primers pdutest-s and pdutest-a to verify [pdutest-s: CGGCGATGTTTACGGCAATGAAG (SEQ ID NO.13), pdutest-a: CTGGGCCAGCAGCTCAAGGTTAC) (SEQ ID NO.14) ]. If the recombination is successful, a band with a size of 3.0 kb can be amplified. If the recombination is not successful, the size of the amplified fragment is 4.0 kb. The results of PCR verification are shown in Figure 1. Verify that the correct PCR product was sequenced by Shanghai Sangon Biotechnology Co., Ltd. to further determine whether the gene encoding diol dehydratase was successfully knocked out. The sequencing results verified that the correct strain was subcultured to eliminate the plasmid pDK6-red, and the finally obtained engineering strain was recorded as K.pΔpdu778.

B. Knockout of the gene encoding Klebsiella acidogens diol dehydratase

Klebsiella acidogenic bacteria M5a1 is a widely used strain of Klebsiella acidogenic bacteria, which has been considered as Klebsiella pneumoniae for a long time, so it is also called Klebsiella pneumonia in some literatures Bacillus M5a1.

Competent cells of Klebsiella acidogens M5a1 were made, and the plasmid pDK6-red was transformed to obtain strain K.o/red.

Make electroporation competent cells of the strain K.o/red, and add IPTG to induce the expression of Red recombinase when culturing the competent cells, and add EDTA to improve the electroporation transformation efficiency of Klebsiella. K.o/red competent cells were transformed by electric shock with homologous recombination fragment "A". After recovery, they were all spread on streptomycin-resistant plates and cultured overnight at 37°C. After a single colony grows on the plate, use the primers pdutest-s and pdutest-a to verify it. If the recombination is successful, a band with a size of 3.0kb can be amplified. If the recombination is not successful, it will be amplified. The size of the fragment is 4.0 kb. PCR verification The correct PCR product was sequenced by Shanghai Sangon Biotechnology Co., Ltd. to further determine whether the gene encoding diol dehydratase was successfully knocked out. Sequencing results verified that the correct strain was subcultured to eliminate the plasmid pDK6-red, and the finally obtained engineering strain was recorded as K.oΔpdu778.

3) Elimination of resistance marker genes

A. Eliminate the resistance gene carried by the engineered bacteria K.pΔpdu778

Making competent cells of Klebsiella pneumoniae engineering bacteria K.pΔpdu778, transforming plasmid pDK6-flp (references for the construction process of plasmids Wei Dong, Wang Min, Shi Jiping, Hao Jian. Red recombinase assisted gene replacement in Klebsiella pneumoniae.Journal of Industrial Microbiology & Biotechnology.2012 39:1219-1226), to obtain bacterial strain K.pΔpdu778/flp.

Subculture K.pΔpdu778/flp, and add IPTG to induce plasmid pDK6-flp to express FLP recombinase to eliminate the resistance marker. After subculture, it was diluted and spread on the non-antibiotic LB plate, and a certain number of colony markers was selected and inoculated on the streptomycin resistance plate in turn. Single colonies that do not grow on streptomycin-resistant plates were further verified with primers pdutest-s and pdutest-a. If the carried resistance gene fragment is eliminated successfully, the amplified size is 1.1 kb; if not eliminated, the amplified size is 3.0 kb. The PCR product with a size of 1.1 kb was verified by sequencing by Shanghai Sangon Biotechnology Co., Ltd. Sequencing verified that the correct bacteria were subcultured to eliminate the plasmid pDK6-flp, and finally obtained the engineering bacteria K.pΔpdu.

B. Eliminate the resistance gene carried by the engineered bacteria K.oΔpdu778

Competent cells of Klebsiella acidogens engineering strain K.oΔpdu778 were produced, and plasmid pDK6-flp was transformed to obtain bacterial strain K.oΔpdu778/flp.

Subculture K.oΔpdu778/flp, and add IPTG to induce plasmid pDK6-flp to express FLP recombinase to eliminate the resistance marker. After subculture, it was diluted and spread on the non-antibiotic LB plate, and a certain number of colony markers was selected and inoculated on the streptomycin resistance plate in turn. Single colonies that do not grow on streptomycin-resistant plates were further verified with primers pdutest-s and pdutest-a. If the carried resistance gene fragment is eliminated successfully, the amplified size is 1.1 kb; if not eliminated, the amplified size is 3.0 kb. The PCR product with a size of 1.1 kb was verified by sequencing by Shanghai Sangon Biotechnology Co., Ltd. Sequencing verified that the correct bacteria were subcultured to eliminate the plasmid pDK6-flp, and finally obtained the engineering bacteria K.oΔpdu.

Embodiment 2: Construction of Klebsiella engineering bacteria with inactivated diol dehydratase activating factor

The technical scheme for knocking out the gene encoding the diol dehydratase activating factor in this part is consistent with the method for knocking out the diol dehydratase in Example 1, and the specific steps are as follows:

1) Construction of a homologous recombination fragment to knock out the gene encoding the diol dehydratase activator

According to the gene sequence of Klebsiella pneumoniae CGMCC 1.6366 encoding diol dehydratase activator, the upstream and downstream primers were designed as follows:

Upstream primer pduGH-s1: GCTGCCGATTGTTGACGAAGTGC (SEQ ID NO.15)

Downstream primer pduGH-a1: CAGGATTTCACTTCGCCTACGAC (SEQ ID NO.16)

Utilizing the designed primers pduGH-s1 and pduGH-a1, using the genomic DNA of Klebsiella pneumoniae CGMCC 1.6366 as a template, a partial gene (including pduG, pduH) encoding diol dehydratase activating factor was obtained through PCR amplification, It was connected to the pMD-19T simple plasmid by the TA cloning method, and the resulting recombinant plasmid was named pMD19T-pduGH, and was verified by sequencing by Shanghai Sangon Biotechnology Co., Ltd.

The successfully constructed plasmid pMD19T-pduGH was transformed into strain DH5α-pIJ790 to obtain double-plasmid-carrying strain DH5α-pIJ790-pMD19T-pduGH.

According to the gene sequence of the plasmid pIJ773 (NTCC Type Culture Collection Center) carrying the apramycin resistance gene cassette, design upstream and downstream primers:

Upstream primer pduGH-FRT-s1:

GGACCTGCTGGCCGTCGATACCTCGGTGCCGGTGAGCGGATTCCGGGGATCCGTCGACC (SEQ ID NO. 17)

Downstream primer pduGH-FRT-a1:

GGAGCAGGAAAGGAATGCCTTCCTCTTCGATACCCAGCTGTAGGCTGGAGCTGCTTC (SEQ ID NO. 18)

Using the designed primers pduGH-FRT-s1 and pduGH-FRT-a1, and using the plasmid pIJ773 as a template, the fragment of the apramycin resistance gene cassette was obtained by PCR amplification.

Make the competent cells of the strain DH5α-pIJ790-pMD19T-pduGH, then electrotransform DH5α-pIJ790-pMD19T-pduGH competent cells with the fragment of the apramycin resistance gene cassette obtained by PCR and recovered after cleaning, and recover at 37°C for 1 hour Afterwards, the apramycin-resistant plate was coated by centrifugation (the concentration of apramycin used was 50 mg/L), and the positive strain carrying pMD19T-ΔpduGH773 was screened. After colonies grow on the plate, use primers pduGH-s1 and test773 to verify (test773: GCAAATACGGCATCAGTTACC) (SEQ ID NO.19). If recombination does not occur, the gene fragment cannot be amplified. If the recombination is successful, a gene fragment of about 1.0 kb is amplified. The strains with successful recombination verified by PCR were inoculated in apramycin-resistant LB test tube culture medium, and the plasmid was extracted, which was named pMD19T-ΔpduGH778.

Using the constructed pMD19T-ΔpduGH773 as a template, use primers pduGH-s1 and pduGH-a1 under the action of high-fidelity enzyme KOD to amplify the homologous recombination fragment of knockout pduGH gene, and the obtained fragment is abbreviated as "B". The size of the "B" fragment is 2700bp, and the two ends carry homology arms of about 500bp respectively, and the apramycin resistance gene is in the middle.

2) Homologous recombination to knock out the gene encoding Klebsiella diol dehydratase activator

A. Knockout of the gene encoding Klebsiella pneumoniae diol dehydratase activator

Competent cells of Klebsiella pneumoniae CGMCC 1.6366 were produced, and plasmid pDK6-red was transformed to obtain strain K.p/red.

Make electroporation competent cells of strain K.p/red, add IPTG to induce the expression of Red recombinase when culturing competent cells, and add EDTA to improve the electroporation transformation efficiency of Klebsiella. The homologous recombination fragment "B" was used to transform K.p/red competent cells by electric shock, and after recovery, they were all spread on apramycin-resistant plates and cultured overnight at 37°C. After a single colony grows on the plate, use primers pduGHtest-s and pduGHtest-a to verify [pduGHtest-s: TGCGTAACGTGTTCGGTATTCA (SEQ ID NO.20), pduGHtest-a: GCGGTGCTCCTTATTCGCCATCA) (SEQ ID NO.21)] . If the recombination is successful, a band with a size of 3.0 kb can be amplified. If the recombination is not successful, the size of the amplified fragment is 2.5 kb. The result of PCR verification is shown in Figure 2. Verify that the correct PCR product was sequenced by Shanghai Sangon Biotechnology Co., Ltd. to further determine whether the gene encoding the diol dehydratase activating factor was successfully knocked out. The sequencing results verified that the correct strain was subcultured to eliminate the plasmid pDK6-red, and the finally obtained engineering strain was recorded as K.pΔpduGH773.

B. Knockout of the gene encoding Klebsiella acidogens diol dehydratase activator

Competent cells of Klebsiella acidogens M5a1 were made, and the plasmid pDK6-red was transformed to obtain strain K.o/red.

Make electroporation competent cells of the strain K.o/red, and add IPTG to induce the expression of Red recombinase when culturing the competent cells, and add EDTA to improve the electroporation transformation efficiency of Klebsiella. K.o/red competent cells were transformed by electroporation with homologous recombination fragment "B". After recovery, they were all spread on apramycin-resistant plates and cultured overnight at 37°C. After a single colony grows on the plate, use the primers pduGHtest-s and pduGHtest-a to verify the same. If the recombination is successful, a band with a size of 3.0kb can be amplified. If the recombination is not successful, it will be amplified. The fragment size is 2.5kb. PCR verification The correct PCR product was sequenced by Shanghai Sangon Biotechnology Co., Ltd. to further determine whether the gene encoding the diol dehydratase activating factor was successfully knocked out. Sequencing results verified that the correct strain was subcultured to eliminate the plasmid pDK6-red, and the finally obtained engineering strain was recorded as K.oΔpduGH773.

3) Elimination of resistance marker genes

A. Eliminate the resistance gene carried by the engineered bacteria K.pΔpduGH773

Competent cells of Klebsiella pneumoniae engineering strain K.pΔpduGH773 were produced, and plasmid pDK6-flp was transformed to obtain bacterial strain K.pΔpduGH773/flp.

K.pΔpduGH773/flp was subcultured, and IPTG was added to induce the expression of FLP recombinase in the plasmid pDK6-flp to eliminate the resistance marker. After subculture, it was diluted and spread on the non-antibiotic LB plate, and a certain number of colony markers was selected and inoculated on the apramycin resistance plate in turn. The single colony that does not grow on the apramycin-resistant plate was further verified with primers pduGHtest-s and pduGHtest-a. If the carried resistance gene fragment is eliminated successfully, the amplified size is 1.1 kb; if not eliminated, the amplified size is 3.0 kb. The PCR product with a size of 1.1 kb was verified by sequencing by Shanghai Sangon Biotechnology Co., Ltd. The correct bacteria verified by sequencing were subcultured to eliminate the plasmid pDK6-flp, and finally obtained the engineering bacteria K.pΔpduGH.

B. Eliminate the resistance gene carried by the engineered bacteria K.oΔpduGH773

Competent cells of Klebsiella acidogens engineering strain K.oΔpduGH773 were produced, and plasmid pDK6-flp was transformed to obtain bacterial strain K.oΔpduGH773/flp.

K.oΔpduGH773/flp was subcultured, and IPTG was added to induce the expression of FLP recombinase in the plasmid pDK6-flp to eliminate the resistance marker. After subculture, it was diluted and spread on the non-antibiotic LB plate, and a certain number of colony markers was selected and inoculated on the streptomycin resistance plate in turn. The single colony that does not grow on the apramycin-resistant plate was further verified with primers pduGHtest-s and pduGHtest-a. If the carried resistance gene fragment is eliminated successfully, the amplified size is 1.1 kb; if not eliminated, the amplified size is 3.0 kb. The PCR product with a size of 1.1 kb was verified by sequencing by Shanghai Sangon Biotechnology Co., Ltd. Sequencing verified that the correct bacteria were subcultured to eliminate the plasmid pDK6-flp, and finally obtained the engineering bacteria K.oΔpduGH.

Embodiment 3: Construction of Klebsiella engineering bacteria with lactate dehydrogenase inactivation

The technical scheme for knocking out lactate dehydrogenase in this part is consistent with the method for knocking out diol dehydratase in Example 1, and the specific steps are as follows:

1) Construction of homologous recombination fragments to knock out the gene encoding lactate dehydrogenase

Design upstream and downstream primers according to the gene sequence encoding lactate dehydrogenase of Klebsiella pneumoniae CGMCC 1.6366

Upstream primer ldh-s1: AGAGCGCACAGGACCACTATCCA (SEQ ID NO.22)

Downstream primer ldh-a1: TCGGCGAGCTTATAGACCAGCGT (SEQ ID NO.23)

Using the designed primers ldh-s1 and ldh-a1, using Klebsiella pneumoniae CGMCC 1.6366 genomic DNA as a template, the gene encoding lactate dehydrogenase was obtained by PCR amplification, and connected to pMD-19T simple by TA cloning method On the plasmid, the obtained recombinant plasmid was named pMD19T-ldh, and was verified by sequencing by Shanghai Sangon Biotechnology Co., Ltd.

The successfully constructed plasmid pMD19T-ldh was transformed into strain DH5α-pIJ790 to obtain double-plasmid-carrying strain DH5α-pIJ790-pMD19T-ldh.

According to the gene sequence of the plasmid pIJ773 carrying the apramycin resistance gene cassette, design upstream and downstream primers and upstream primer ldh-FRT-s1:

CCAGCTGCCTAGGGGCCTTACTACGTATGGCGAAGCGTTGGCACCCGCCAAAACCGCCA (SEQ ID NO. 24)

Downstream primer ldh-FRT-a1:

CTTCGTCGAGGTCGGATGTCAAGTGGCCGGTAGTCCGCAAGGAGTGGCGGCTCCGCGAC (SEQ ID NO. 25)

Using the designed primers ldh-FRT-s1 and ldh-FRT-a1, using the plasmid pIJ773 as a template, the fragment of the apramycin resistance gene cassette was obtained by PCR amplification.

Make competent cells of the strain DH5α-pIJ790-pMD19T-ldh, and then electrotransform DH5α-pIJ790-pMD19T-ldh competent cells with the fragment of the apramycin resistance gene cassette obtained by PCR and recovered after cleaning, and recover at 37°C for 1 hour After centrifugation, the apramycin-resistant plate was coated, and the positive strain carrying pMD19T-Δldh773 was screened. After colonies grow on the plate, they are verified with primers ldh-s1 and test773. If recombination does not occur, the gene fragment cannot be amplified. If the recombination is successful, a gene fragment of about 1.3 kb is amplified. The strains with successful recombination verified by PCR were inoculated in apramycin-resistant LB test tube culture medium, and the plasmid was extracted, which was named pMD19T-Δldh773.

Using the constructed plasmid pMD19T-Δldh773 as a template, primers ldh-s1 and ldh-a1 were used under the action of high-fidelity enzyme KOD to amplify the homologous recombination fragment that knocked out the ldh gene, and the obtained fragment was abbreviated as "C". The size of the "C" fragment is 2810bp, the two ends carry homology arms of about 800bp and 700bp respectively, and the apramycin resistance gene is in the middle.

2) Homologous recombination knockout of the gene encoding lactate dehydrogenase in Klebsiella engineering bacteria

A. Knockout the gene encoding K.pΔpdu lactate dehydrogenase

Competent cells of K.pΔpdu were made, and plasmid pDK6-red was transformed to obtain strain K.pΔpdu/red.

Make electroporation competent cells of strain K.pΔpdu/red, add IPTG to induce the expression of Red recombinase when culturing competent cells, and add EDTA to improve the electroporation transformation efficiency of Klebsiella. K.pΔpdu/red competent cells were transformed by electroporation with homologous recombination fragment "C". After recovery, they were all spread on apramycin-resistant plates and cultured overnight at 37°C. After a single colony grows on the plate, use primers ldhtest-s and ldhtest-a to verify [ldhtest-s: CCTGTTAAAGCATAGTTGCCAGCCGGAC (SEQ ID NO.26), ldhtest-a: TCGTCAGCTCGATGGTTCGGCGATTGG (SEQ ID NO.27)] . If the recombination is successful, a band with a size of 3.1 kb can be amplified. If the recombination is not successful, the size of the amplified fragment is 2.4 kb. The result of PCR verification is shown in Figure 3. Verify that the correct PCR product was sequenced by Shanghai Sangon Biotechnology Co., Ltd. to further determine whether the gene encoding lactate dehydrogenase was successfully knocked out. The sequencing results verified that the correct strain was subcultured to eliminate the plasmid pDK6-red, and the finally obtained engineering strain was recorded as K.pΔpduΔldh773.

B. Knockout the gene encoding K.oΔpdu diol dehydratase

Competent cells of K.oΔpdu were made, and plasmid pDK6-red was transformed to obtain strain K.pΔpdu/red.

Make electroporation competent cells of strain K.oΔpdu/red, add IPTG to induce the expression of Red recombinase when culturing competent cells, and add EDTA to improve the electroporation transformation efficiency of Klebsiella. K.oΔpdu/red competent cells were transformed by electroporation with homologous recombination fragment "C". After recovery, they were all spread on apramycin-resistant plates and cultured overnight at 37°C. After a single colony grows on the plate, use primers ldhtest-s and ldhtest-a to verify. If the recombination is successful, a band with a size of 3.1 kb can be amplified, and if the recombination is not successful, the amplified fragment has a size of 2.4 kb. Verify that the correct PCR product was sequenced by Shanghai Sangon Biotechnology Co., Ltd. to further determine whether the gene encoding lactate dehydrogenase was successfully knocked out. Sequencing results verified that the correct strain was subcultured to eliminate the plasmid pDK6-red, and the finally obtained engineering strain was recorded as K.oΔpduΔldh773.

3) Elimination of resistance marker genes

A. Eliminate the resistance gene carried by the engineered bacteria K.pΔpduΔldh773

Competent cells of Klebsiella pneumoniae engineering strain K.pΔpduΔldh773 were produced, and plasmid pDK6-flp was transformed to obtain bacterial strain K.pΔpduΔldh773/flp.

Subculture K.pΔpduΔldh773/flp, and add IPTG to induce the expression of FLP recombinase in plasmid pDK6-flp to eliminate the resistance marker. After subculture, it was diluted and spread on the non-antibiotic LB plate, and a certain number of colony markers was selected and inoculated on the apramycin resistance plate in turn. The single colonies that do not grow on the apramycin-resistant plate were further verified with primers ldhtest-s and ldhtest-a. If the carried resistance gene fragment is eliminated successfully, the amplified size is 1.5kb; if not eliminated, the amplified size is 3.1kb. The PCR product with a size of 1.5kb was verified by sequencing by Shanghai Sangon Biotechnology Co., Ltd. Sequencing verified that the correct bacteria were subcultured to eliminate the plasmid pDK6-flp, and finally obtained the engineering bacteria K.pΔpduΔldh.

B. Eliminate the resistance gene carried by the engineered bacteria K.oΔpduΔldh773

The competent cells of K.oΔpduΔldh773 were produced, and the plasmid pDK6-flp was transformed to obtain strain K.oΔpduΔldh773/flp.

K.oΔpduΔldh773/flp was subcultured, and IPTG was added to induce the expression of FLP recombinase in the plasmid pDK6-flp to eliminate the resistance marker. After subculture, it was diluted and spread on the non-antibiotic LB plate, and a certain number of colony markers was selected and inoculated on the apramycin resistance plate in turn. The single colonies that do not grow on the apramycin-resistant plate were further verified with primers ldhtest-s and ldhtest-a. If the carried resistance gene fragment is eliminated successfully, the amplified size is 1.5kb; if not eliminated, the amplified size is 3.1kb. The PCR product with a size of 1.5kb was verified by sequencing by Shanghai Sangon Biotechnology Co., Ltd. Sequencing verified that the correct bacteria were subcultured to eliminate the plasmid pDK6-flp, and finally obtained the engineering bacteria K.oΔpduΔldh.

Embodiment 4: Construction of Klebsiella engineering bacteria with alcohol dehydrogenase inactivation

The technical scheme for knocking out alcohol dehydrogenase in this part is consistent with the method for knocking out diol dehydratase in Example 1, and the specific steps are as follows:

1) Construction of homologous recombination fragments to knock out the gene encoding alcohol dehydrogenase

According to the gene sequence of Klebsiella pneumoniae CGMCC 1.6366 encoding alcohol dehydrogenase, design upstream and downstream primers:

Upstream primer adh-s1: GCAGCATCATCAAAATTGGCGGTTGAC (SEQ ID NO.28)

Downstream primer adh-a1: GTGCTTTGCTATGGCTTGCGGACAGAC (SEQ ID NO.29)

Using the designed primers adh-s1 and adh-a1, Klebsiella pneumoniae CGMCC 1.6366 genomic DNA was used as a template, and the gene encoding alcohol dehydrogenase was obtained by PCR amplification, which was connected to pMD-19T simple by TA cloning method On the plasmid, the obtained recombinant plasmid was named pMD19T-adh, and was verified by sequencing by Shanghai Sangon Biotechnology Co., Ltd.

The successfully constructed plasmid pMD19T-adh was transformed into strain DH5α-pIJ790 to obtain double-plasmid-carrying strain DH5α-pIJ790-pMD19T-adh.

According to the gene sequence of the plasmid pIJ778 carrying the streptomycin resistance gene cassette, design upstream and downstream primers

Upstream primer adh-FRT-s1:

CAGTTTCACTCAAGAACAAGTCGACAAAATTCCGGGGATCCGTCGACC (SEQ ID NO. 30)

Downstream primer adh-FRT-a1:

CGACCGTAGTAGGTATCCAGCAGGATCTGTAGGCTGGAGCTGCTTC (SEQ ID NO. 31)

Using the designed primers adh-FRT-s1 and adh-FRT-a1, using the plasmid pIJ778 as a template, the fragment of the streptomycin resistance gene cassette was obtained by PCR amplification.

Make the competent cells of the strain DH5α-pIJ790-pMD19T-adh, and then electrotransform DH5α-pIJ790-pMD19T-adh competent cells with the fragment of the streptomycin resistance gene cassette obtained by PCR and recovered after cleaning, and recover at 37°C for 1 hour The streptomycin-resistant plates were centrifuged and positive strains carrying pMD19T-Δadh778 were screened. After colonies grow on the plate, they are verified with primers adh-s1 and test778. If recombination does not occur, the gene fragment cannot be amplified. If the recombination is successful, a gene fragment of about 1.3 kb is amplified. The strains with successful recombination verified by PCR were inoculated in streptomycin-resistant LB test tube culture medium, and the plasmid was extracted, which was named pMD19T-Δadh778.

Using the constructed plasmid pMD19T-Δadh778 as a template, using primers adh-s1 and adh-a1 under the action of high-fidelity enzyme KOD, amplify the homologous recombination fragment that knocked out the adh gene, and the obtained fragment is abbreviated as "D". The size of the "D" fragment is 2.5kb, the two ends carry homology arms of about 600bp and 500bp respectively, and the streptomycin resistance gene is in the middle.

2) Homologous recombination to knock out the gene encoding alcohol dehydrogenase of Klebsiella engineering bacteria

A. Knockout the gene encoding K.pΔpduΔldh alcohol dehydrogenase

Competent cells of K.pΔpduΔldh were made, and plasmid pDK6-red was transformed to obtain strain K.pΔpduΔldh/red.

The electroporation competent cells of the strain K.pΔpduΔldh/red were made, and IPTG was added to induce the expression of Red recombinase during culture, and EDTA was added to improve the electroporation transformation efficiency of Klebsiella. K.pΔpduΔldh/red competent cells were transformed by electroporation with the homologous recombination fragment "D". After recovery, they were all spread on streptomycin-resistant plates and cultured overnight at 37°C. After a single colony grows on the plate, use primers adhtest-s and adhtest-a to verify [adhtest-s: GTTAACCAGGGCAAATAAGCCGATG (SEQ ID NO.32), adhtest-a: CGATTCACTGCGTGCTGGTGGATGA (SEQ ID NO.33)]. If the recombination is successful, a band with a size of 2.9 kb can be amplified. If the recombination is not successful, the size of the amplified fragment is 1.8 kb. The result of PCR verification is shown in Figure 4. Verify that the correct PCR product was sequenced by Shanghai Sangon Biotechnology Co., Ltd. to further determine whether the gene encoding alcohol dehydrogenase was successfully knocked out. The sequencing results verified that the correct strain was subcultured to eliminate the plasmid pDK6-red, and the finally obtained engineering strain was recorded as K.pΔpduΔldhΔadh778.

B. Knockout the gene encoding K.oΔpduΔldh alcohol dehydrogenase

Competent cells of K.oΔpduΔldh were made, and the plasmid pDK6-red was transformed to obtain strain K.oΔpduΔldh/red.

The electroporation competent cells of the strain K.oΔpduΔldh/red were made, and IPTG was added to induce the expression of Red recombinase during culture, and EDTA was added to improve the electroporation transformation efficiency of Klebsiella. K.oΔpduΔldh/red competent cells were transformed by electroporation with the homologous recombination fragment "D". After recovery, they were all spread on streptomycin-resistant plates and cultured overnight at 37°C. After a single colony grows on the plate, use primers adhtest-s and adhtest-a to verify. If the recombination is successful, a band with a size of 2.9 kb can be amplified, and if the recombination is not successful, the amplified fragment has a size of 1.8 kb. Verify that the correct PCR product was sequenced by Shanghai Sangon Biotechnology Co., Ltd. to further determine whether the gene encoding alcohol dehydrogenase was successfully knocked out. The sequencing results verified that the correct strain was subcultured to eliminate the plasmid pDK6-red, and the finally obtained engineering strain was recorded as K.oΔpduΔldhΔadh778.

3) Elimination of resistance marker genes

A. Eliminate the resistance gene carried by the engineered bacteria K.pΔpduΔldhΔadh778

Competent cells of Klebsiella pneumoniae engineering strain K.pΔpduΔldhΔadh778 were produced, and plasmid pDK6-flp was transformed to obtain strain K.pΔpduΔldhΔadh778/flp.

K.pΔpduΔldhΔadh778/flp was subcultured, and IPTG was added to induce the expression of FLP recombinase in the plasmid pDK6-flp to eliminate the resistance marker. After subculture, it was diluted and spread on the non-antibiotic LB plate, and a certain number of colony markers was selected and inoculated on the streptomycin resistance plate in turn. Single colonies that do not grow on streptomycin-resistant plates were further verified with primers adh-s and adh-a. If the carried resistance gene fragment is eliminated successfully, the amplified size is 1.4kb; if not eliminated, the amplified size is 2.9kb. The PCR product with a size of 1.4kb was verified by sequencing by Shanghai Sangon Biotechnology Co., Ltd. The correct bacteria verified by sequencing were subcultured to eliminate the plasmid pDK6-flp, and finally obtained the engineering bacteria K.pΔpduΔldhΔadh.

B. Eliminate the resistance gene carried by the engineered bacteria K.oΔpduΔldhΔadh778

Competent cells of Klebsiella pneumoniae engineering strain K.oΔpduΔldhΔadh778 were produced, and plasmid pDK6-flp was transformed to obtain strain K.oΔpduΔldhΔadh778/flp.

K.oΔpduΔldhΔadh778/flp was subcultured, and IPTG was added to induce the expression of FLP recombinase in the plasmid pDK6-flp to eliminate the resistance marker. After subculture, it was diluted and spread on the non-antibiotic LB plate, and a certain number of colony markers was selected and inoculated on the streptomycin resistance plate in turn. Single colonies that do not grow on streptomycin-resistant plates were further verified with primers adh-s and adh-a. If the carried resistance gene fragment is eliminated successfully, the amplified size is 1.4kb; if not eliminated, the amplified size is 2.9kb. The PCR product with a size of 1.4kb was verified by sequencing by Shanghai Sangon Biotechnology Co., Ltd. Sequencing verified that the correct bacteria were subcultured to eliminate the plasmid pDK6-flp, and finally obtained the engineering bacteria K.oΔpduΔldhΔadh.

Embodiment 5: Klebsiella and the engineered bacteria of construction carry out fermentation experiment

The engineering bacteria obtained in the starting strain Klebsiella pneumoniae CGMCC 1.6366 (abbreviated as K.p), Klebsiella acidogens M5a1 (abbreviated as K.o) and embodiments 1-4: K.pΔpdu, K. pΔpduGH, K.pΔpduΔldh, K.pΔpduΔldhΔadh, K.oΔpdu, K.oΔpduGH, K.oΔpduΔldh, K.oΔpduΔldhΔadh were continuously fed-fed in a 5L fermenter to investigate the performance of the strains in synthesizing 1,3-propanediol. Three batches of fermentation were repeated for each strain.

The above bacterial strains were respectively inoculated into a 250mL Erlenmeyer flask with 50mL seed medium, inoculated at a ratio of 1% (volume), and cultivated at 37° C. on a shaker at 200 rpm for 12 hours. After the cultivation, the seeds obtained were inoculated into the fermenter.

The components of the seed medium are: peptone 10g/L, yeast extract 5g/L, sodium chloride 5g/L.

The cultured seeds were inoculated into a 5L fermenter for 1,3-propanediol fermentation. Configure 3L of fermentation medium in the fermenter, and inoculate all 50mL of seed liquid. The temperature of the fermenter was controlled at 37° C., the air flow rate was 2 L/min, and the rotation speed was 200 rpm. During the fermentation process, 30% NaOH solution was used to adjust the pH value to 6.8. During the fermentation process, a certain amount of fermentation broth was taken at regular intervals for analysis. Spectrophotometer was used to measure the growth of bacteria, and high performance liquid chromatography was used to measure the consumption of substrate glycerol and the production of 1,3-propanediol, by-product lactic acid and ethanol in the fermentation broth. At the same time, after the glycerin in the fermenter was consumed, feeding was carried out with a sterilized glycerin aqueous solution with a concentration of 75% (g/g). It is not easy to feed too quickly, and it needs to be controlled in conjunction with the residual amount of glycerin in the tank. Generally, the concentration of glycerin in the fermentation broth is controlled at the initial stage of feeding to 10g/L. After 30 hours of fermentation, the results of 1,3-propanediol yield, lactic acid, ethanol and conversion rate are shown in Table 1.

Fermentation medium composition: dipotassium hydrogen phosphate 0.69g/L, potassium dihydrogen phosphate 0.25g/L, yeast powder 1.5g/L, ammonium sulfate 4.0g/L, magnesium sulfate 0.2g/L, glycerol 30.0g/L.

Table 1 Fermentation results of Klebsiella and engineering bacteria

Conversion rate (%)=concentration of 1,3-propanediol in the fermented liquid of fermentation 30h g/L*fermented liquid volume L/(concentration of the initial glycerol of fermentation medium g/L*medium volume L+concentration of feed g/L*consumed volume of feeding material L－concentration of residual glycerol in the fermentation broth after 30 hours of fermentation g/L*volume of fermentation broth)*100

It can be seen from Table 1:

Compared with the initial strain K.p, the 1,3-propanediol production of K.pΔpdu knockout diol dehydratase engineered strain reached 63.65g/L after 30h fermentation, which was 14.19% higher than K.p's synthetic 1,3-propanediol production; The output of by-product lactic acid is 15.84g/L, which is 45.99% lower than that of K.p synthetic lactic acid; the output of by-product ethanol is 3.09g/L, which is 56.11% lower than that of K.p synthetic ethanol; K.pΔpdu metabolizes glycerol synthesis The conversion rate of 1,3-propanediol was 46.16%, which was 6.9% higher than that of K.p; the fermentation results of K.pΔpdu showed that knocking out the gene encoding diol dehydratase in Klebsiella pneumoniae could significantly improve the synthesis of 1,3-Propanediol performance while reducing by-product lactic acid and ethanol formation.

Also compared with the initial strain K.p, the 1,3-propanediol production of the engineered strain K.pΔpduGH, which knocked out the diol dehydratase activating factor, reached 58.82 g/L after 30 hours of fermentation, which was higher than that of K.p. 5.52%; the output of by-product lactic acid is 24.56g/L, which is 16.26% lower than the output of K.p synthetic lactic acid; the output of by-product ethanol is 5.61g/L, which is 20.31% lower than the output of K.p synthetic ethanol; K.pΔpduGH The conversion rate of metabolizing glycerol to 1,3-propanediol was 42.13%, which was 2.87% higher than that of K.p; the fermentation results of K.pΔpduGH indicated that the encoding of diol dehydratase activating factor was knocked out in Klebsiella pneumoniae The gene can also improve the performance of the strain to synthesize 1,3-propanediol, but the yield of the strain to synthesize 1,3-propanediol is lower than that of K.pΔpdu.

On the engineered bacteria K.pΔpdu, the gene encoding lactate dehydrogenase was further knocked out, and the obtained engineered bacteria K.pΔpduΔldh fermented for 30 hours, and the production of 1,3-propanediol reached 68.07g/L, which was more synthetic than K.p The output of the by-product lactic acid is no longer synthesized; the output of the by-product ethanol is 3.18g/L, which is 54.82% lower than that of K.p synthetic ethanol; the conversion rate of K.pΔpduΔldh metabolism of glycerol to 1,3-propanediol It is 48.14%, which is 8.88% higher than the conversion rate of K.p. The fermentation results of K.pΔpduΔldh indicate that further knockout of the Klebsiella pneumoniae lactate dehydrogenase coding gene can continue to improve the performance of the strain in metabolizing glycerol to synthesize 1,3-propanediol, and the synthesis of by-product lactic acid is completely eliminated due to the knockout of the ldh gene block.

On the engineered bacteria K.pΔpduΔldh, the gene encoding alcohol dehydrogenase was further knocked out, and the obtained engineered bacteria K.pΔpduΔldhΔadh fermented for 30 hours, and the production of 1,3-propanediol reached 70.12g/L, which was more synthetic than K.p The yield increased by 25.80%; the by-products lactic acid and ethanol were no longer synthesized; the conversion rate of K.pΔpduΔldhΔadh to metabolize glycerol to 1,3-propanediol was 49.56%, which was 10.3% higher than that of K.p. The fermentation results of K.pΔpduΔldhΔadh indicate that further knockout of the Klebsiella pneumoniae alcohol dehydrogenase gene can continue to improve the performance of the strain in metabolizing glycerol to synthesize 1,3-propanediol, and the synthesis of by-product ethanol is completely eliminated due to the knockout of the adh gene block.

Compared with the initial strain K.o, the 1,3-propanediol production reached 54.76g/L after 30 hours of fermentation with the engineered strain K.oΔpdu which knocked out the diol dehydratase, which was 13.80% higher than the amount of 1,3-propanediol synthesized by K.o The output of by-product lactic acid is 14.12g/L, which is 47.35% lower than that of K.o's synthetic lactic acid; the output of by-product ethanol is 4.1g/L, which is 46.05% lower than that of K.o's synthetic ethanol; K.oΔpdu metabolizes glycerol The conversion rate of synthetic 1,3-propanediol is 40.6%, which is 5.37% higher than that of K.p; the fermentation result of K.oΔpdu shows that knocking out the diol dehydratase gene in Klebsiella acidogens can significantly improve the strain Performance in synthesizing 1,3-propanediol while reducing by-product lactic acid and ethanol formation.

Also compared with the original strain K.o, the 1,3-propanediol production reached 50.21g/L after 30h fermentation of the engineered strain K.oΔpduGH which knocked out the diol dehydratase activating factor, which was higher than the amount of 1,3-propanediol synthesized by K.o The output of by-product lactic acid is 22.32g/L, which is 16.77% lower than the output of K.o synthetic lactic acid; the output of by-product ethanol is 6.41g/L, which is 15.66% lower than the output of K.o synthetic ethanol; K. The conversion rate of oΔpdu to metabolize glycerol to 1,3-propanediol was 37.22%, which was 1.99% higher than that of K.p; the fermentation results of K.oΔpdu indicated that the diol dehydratase activating factor was knocked out in Klebsiella acidogens The gene can significantly improve the performance of the strain to synthesize 1,3-propanediol, and at the same time reduce the production of by-products lactic acid and ethanol. The gene encoding lactate dehydrogenase was further knocked out on the engineering bacterium K.oΔpdu. After 30 hours of fermentation, the yield of 1,3-propanediol reached 58.11g/L, which was higher than that of K.o. The output increased by 20.76%; the by-product lactic acid was no longer synthesized; the output of by-product ethanol was 4.6g/L, which was 39.47% lower than that of K.p synthetic ethanol; It is 42.1%, which is 6.87% higher than the conversion rate of K.o. The fermentation results of K.oΔpduΔldh also indicate that further knockout of the gene encoding lactate dehydrogenase can continue to improve the performance of Klebsiella acidogens to metabolize glycerol to synthesize 1,3-propanediol, and the synthesis of by-product lactic acid is also due to knockout of the ldh gene is completely blocked.

On the engineered bacteria K.oΔpduΔldh, the gene encoding alcohol dehydrogenase was further knocked out, and the obtained engineered bacteria K.oΔpduΔldhΔadh fermented for 30 hours, and the yield of 1,3-propanediol reached 58.76g/L, which was more synthetic than K.o The yield increased by 22.11%; the by-products lactic acid and ethanol were no longer synthesized; the conversion rate of K.oΔpduΔldhΔadh to metabolize glycerol to 1,3-propanediol was 43.2%, which was 7.97% higher than that of K.o. The fermentation results of K.oΔpduΔldhΔadh indicate that further knockout of the alcohol dehydrogenase coding gene of Klebsiella acidogens can continue to improve the performance of the strain in metabolizing glycerol to synthesize 1,3-propanediol, and the synthesis of by-product ethanol is also due to knockout of the ahd gene is completely blocked.

The above examples are intended to illustrate the disclosed embodiments of the present invention, and should not be construed as limiting the present invention. In addition, various modifications set forth herein, as well as changes in the method and composition of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in connection with various specific preferred embodiments of the invention, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as mentioned above which are obvious to those skilled in the art to obtain the invention should be included in the scope of the present invention.

sequence listing

<110> Shanghai Advanced Research Institute, Chinese Academy of Sciences

<120> A construction method and application of Klebsiella engineering bacteria for improving 1,3-propanediol production

<160> 33

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1665

<212>DNA

<213> Klebsiella pneumoniae

<400> 1

atgagatcga aaagatttga agcactggcg aaacgccctg tgaatcagga cggcttcgtt 60

aaggagtgga tcgaagaagg ctttatcgcg atggaaagcc cgaacgatcc gaaaccgtcg 120

ataaaaatcg ttaacggcgc ggtgaccgag ctggacggga aaccggtgag cgattttgac 180

ctgatcgacc actttatcgc ccgctacggt atcaacctga atcgcgccga agaagtgatg 240

gcgatggatt cggttaagct ggccaacatg ctgtgcgatc cgaacgttaa acgcagcgaa 300

atcgtcccgc tgaccaccgc gatgacgccg gcaaaaattg tcgaagtggt ttcgcatatg 360

aacgtcgttg agatgatgat ggcgatgcag aaaatgcgcg cccgccgcac cccatcccag 420

caggcgcacg tcaccaacgt caaagataac ccggtacaga ttgccgccga cgccgccgaa 480

ggcgcatggc gcggatttga cgaacaggaa accaccgttg cggtagcgcg ctacgcgccg 540

ttcaacgcca tcgcgctgct ggtcggctcg caggtaggcc gtccgggcgt actgactcag 600

tgctcgctgg aagaagccac cgagctgaag ctcggcatgc tgggccacac ctgctacgcc 660

gaaaccatct ccgtctacgg taccgaaccg gtctttaccg acggcgacga cacgccgtgg 720

tcgaagggtt tcttagcctc ctcctacgcc tctcgcggtc tgaaaatgcg cttcacctcc 780

ggctccggct ccgaagtgca gatgggctac gccgaaggca aatccatgct ttacctggaa 840

gcgcgctgca tctacatcac caaagccgcg ggcgtacagg gcctgcaaaa cggttccgtt 900

agctgcatcg gcgtgccgtc tgcggtgcct tccggcattc gcgcggtgct ggcggaaaac 960

ctgatctgtt cgtcgctgga tctggagtgc gcctccagta acgaccagac cttcacccac 1020

tccgatatgc gtcgtaccgc gcgcctgctg atgcagtttc tgccggggac cgactttatc 1080

tcttccggtt attccgcggt gccgaactac gacaacatgt tcgccggttc caacgaagat 1140

gccgaagact ttgacgacta caacgtcatc cagcgcgacc tgaaggtgga cggcggtctg 1200

cgtccggttc gcgaagaaga tgttatcgcc atccgtaata aggctgcccg cgcgctgcag 1260

gccgtttttg ccggaatggg gctgccgccg attaccgatg aagaagttga agccgcgacc 1320

tacgcccacg gttcgaaaga tatgccggag cgcaacatcg tcgaagacat caagttcgcc 1380

caggaaatca tcaataaaaa ccgcaacggc ctggaagtag tgaaagcgct ggcgcagggc 1440

gggttcaccg acgttgccca ggacatgctc aacatccaga aagccaagct gaccggagac 1500

tatctgcata cctccgcaat tatcgtcggc gatgggcagg tgctgtcagc cgtcaacgac 1560

gttaacgact atgccggtcc ggcaacgggc tatcgcctgc agggcgaacg ctgggaagag 1620

attaaaaaca tccctggcgc tcttgatccc aacgagattg attaa 1665

<210> 2

<211> 675

<212>DNA

<213> Klebsiella pneumoniae

<400> 2

atggaaatta atgaaaaatt gctgcgccag ataattgaag aagtgctcag cgagatgaag 60

ggcagcgata aacccgtctc gtttactgcg ccagcggcct ccgcggcgcc ccagcccacg 120

ccgcccgccg gcgacggctt cctgacggaa gtgggcgaag cgcgtcaggg aacccagcag 180

gacgaagtga ttatcgccgt cggcccggct ttcggcctgg cgcagaccgt caatatcgtc 240

ggcataccgc ataagagcat tttgcgcgaa gtcattgccg gtattgaaga agaaggcatt 300

aaggcgcgcg tgattcgctg ctttaaatcc tccgacgtgg ccttcgtcgc cgttgaaggc 360

aatcgcctga gcggttccgg catctctatc ggcatccagt caaaaggcac cacggtgatt 420

caccagcagg ggctgccgcc gctctctaac ctggagctgt tcccccaggc gccgctgctg 480

accctggaaa cctatcgcca gattggtaaa aacgccgccc gctatgcgaa acgcgaatcg 540

ccgcagccgg tcccgacgct gaacgaccag atggcgcggc caaaatacca ggcgaaatcg 600

gccattttgc acattaaaga gaccaagtac gtggtgacgg gcaaaaaccc gcaggaactg 660

cgcgtggcgc tttga 675

<210> 3

<211> 522

<212>DNA

<213> Klebsiella pneumoniae

<400> 3

atgaataccg acgcaattga atcaatggtg cgcgacgtat taagccgcat gaacagcctg 60

cagggcgagg cgccgacggc ggctccggcg gcagacggcg cgtcccgtag cgcaaaggtc 120

agcgactacc cgctggcgaa caaacacccg gaatgggtga aaaccgccac caataaaacg 180

ctggacgact ttacgctgga aaacgtgctg agcaacaaag ttaccgccca ggatatgcgt 240

attaccccgg aaacgctgcg cttacaggct tctatcgcca gggacgcggg ccgcgaccgg 300

ctggcgatga acttcgaacg cgccgccgaa ctgaccgcgg taccggacga tcgcattctt 360

gaaatctaca acgccctccg cccctatcgc tcgacgaaag aggagctgct ggcgatcgcc 420

gacgatctcg aaagccgcta tcaggcgaag atttgcgccg ctttcgttcg cgaagcggca 480

acgctgtacg tcgagcgtaa aaaactcaaa ggcgacgatt aa 522

<210> 4

<211> 1833

<212>DNA

<213> Klebsiella pneumoniae

<400> 4

atgcgatata tagctggcat tgatatcggc aactcatcga cggaagtcgc cctggcgact 60

ctgaatgagg ctggcacgct gacaatcacc cacagcgcgc tggcggaaac caccgggatc 120

aaaggcacgt tgcgtaacgt gttcggtatt caggaggcgc tcgccctcgt cgccagaggc 180

gccgggatcg ctgtcagcga tatttcgctc atccgcatta atgaagctac gccggtgatt 240

ggcgatgtgg cgatggaaac cattaccgag accatcatca ccgaatcgac catgatcggc 300

cataacccga aaacgcccgg cggcgcgggg cttggcgtgg gcatcaccat tacgccgcag 360

gagctgttaa cccgcccggc ggacgcgccc tatatcctgg tggtgtcgtc ggcgttcgat 420

tttgccgata tcgccagcgt gattaacgct tccctgcgcg ccggatatca gattaccggc 480

gtcattttgc aacgtgacga tggcgtactg gtcagcaacc ggctggaaaa accgctgccg 540

attgttgacg aagtgctgta catcgaccgc attcctctgg gaatgctggc ggcaattgag 600

gtcgccgtcc cggggaaggt catcgaaacg ctctctaacc cctacggcat cgccaccgta 660

ttccacctca acgccgagga gacgaaaaac atcgtcccga tggctcgcgc gctgattggc 720

aaccgttccg ccgtggtggt caaaacgcca tccggtgacg tcaaagcgcg cgcgataccc 780

gccggtaacc ttgagctgct ggcccagggc cggagcgtac gcgtagacgt tgccgctggc 840

gccgaagcca tcatgaaagc ggtcgacggc tgcggcaagc tcgataacgt caccggcgag 900

tcggggacca atatcggcgg catgctggaa cacgtgcgcc agaccatggc cgagctgacc 960

aacaagccca gcagcgaaat attcattcag gacctgctgg ccgtcgatac ctcggtgccg 1020

gtgagcgtca ccggcggtct tgccggggag ttctcgctgg agcaggccgt gggcatcgcc 1080

tcaatggtga aatcggatcg tttgcagatg gcgatgattg cccgcgaaat cgagcagaag 1140

ctcaatatcg acgtgcagat cggcggcgcc gaggccgaag ccgccattct gggcgcgctg 1200

accacgccgg gtaccacccg accgctggcg atcctcgacc tcggcgcagg ctccaccgat 1260

gcctccatca tcaaccccaa aggcgacatc atcgccactc acctcgccgg agctggcgat 1320

atggtgacga tgattattgc ccgcgagctg gggctggaag accgctatct ggcggaagag 1380

atcaagaagt atccgctggc caaggtggaa agtctgttcc atttacgcca cgaggacggc 1440

agcgtgcagt tcttctccac gccgctgccc cccgccgtat tcgcccgcgt ctgcgtggtg 1500

aaaccggacg aactggtgcc gctacccggc gacttagcgc tggaaaaagt acgcgccatt 1560

cgccgcagcg ccaaagagcgggtctttgtcaccaacgccctgcgcgcact gcgtcaggtc 1620

agccccaccg gcaacattcg cgatattccg ttcgtggtgc tggtcggcgg ttcgtcgctg 1680

gatttcgagg tcccgcagct ggtcaccgat gcgctggcgc actaccgcct ggttgccggg 1740

cggggaaata ttcgcggcag cgagggcccc agaaacgcgg tggccaccgg cctgattctc 1800

tcctggcata aggagttcgc gcatggacag taa 1833

<210> 5

<211> 378

<212>DNA

<213> Klebsiella pneumoniae

<400> 5

atggacagta atcacagcgc cccggctatt gtgatcgccg ccatcgacgg ctgcgacggc 60

ctgtggcgcg acgtgctgct gggtatcgaa gaggaaggca ttcctttcct gctccagcat 120

cacccggccg gggatgtcgt ggacagcgcc tggcaggcgg cgcgcagctc gccgctactg 180

gtgggcatcg cctgcgaccg ccatacgctg gtcgtgcact acaagaattt acccgcatcg 240

gcgccgcttt ttacgctgat gcatcatcag gacagtcagg cccaacgtaa caccggtaat 300

aacgcggcac ggctggtcaa agggatcccg ttccgggatc tgaatagcga agcaacagga 360

gaacagcagt atgaataa 378

<210> 6

<211> 990

<212>DNA

<213> Klebsiella pneumoniae

<400> 6

atgaaaatcg cggtttacag tacgaagcag tacgataaaa agtacctgca gcacgttaat 60

gatacttacg gctttgaact ggaattcttc gacttcctgc tgacagagaa aaccgccaaa 120

accgcccacg gttgcgaagc ggtatgcatc ttcgtcaatg acgacggcag ccgtccggtg 180

ctggaggagc tgaaggccca cggcgtgaag tatatcgccc tgcgctgcgc cgggtttaac 240

aacgtcgacc tcgaggcggc gaaagcgctt ggcctgcgcg tcgtgcgcgt cccggcctac 300

tcgccggaag cggtcgctga acatgcgatc ggcatgatga tgtcgcttaa ccgccgcatc 360

caccgcgcct accagcgtac ccgtgatgcc aatttctccc tcgaaggcct caccggtttc 420

accatgtacg gcaaaaccgc cggggtgatc ggcaccggga aaattggcgt ggcgatgcta 480

cggatcctta aaggcttcgg catgcgtctg ctggcttttg acccgtaccc aagcgccgcc 540

gcgctggagc tgggggtgga atatgttgac ctggccacgc tgtacaagga gtcggacgtg 600

atctccctgc actgcccgct caccgacgaa aactaccatc tgctcaatcg cgaggccttc 660

gatcagatga aggacggggt gatggtgatt aacaccagcc gcggcgcgct gatcgattcc 720

caggcggcca tcgatgccct gaagcaccag aaaatcggcg cgctggggct ggacgtttat 780

gagaacgaac gcgatctgtt cttcgaagac aaatctaacg acgtgatcca ggacgacgtc 840

ttccgtcgcc tctccgcctg ccacaacgtg ctgttcaccg gccatcaggc gttcctcacc 900

gccgaggcgc tgatcagcat ttcggagacc accctgggca atctgcagca ggtcgccaac 960

ggtgaaacct gcccgaacgc tatcgtctga 990

<210> 7

<211> 2676

<212>DNA

<213> Klebsiella pneumoniae

<400> 7

atggctgtta ctaatatcgc tgaactgaac gcgcttgttg agcgtgtcaa gaaagcccag 60

cgtgaatatg ccagtttcac tcaagaacaa gtcgacaaaa tcttccgcgc cgccgcgctg 120

gccgctgcag atgctcgaat ccctctcgcc aaaatggccg ttgccgaatc cggcatgggc 180

atcgttgaag acaaagtgat caaaaaccac ttcgcttccg aatacattta caacgcttat 240

aaagacgaaa aaacctgtgg cgtcctgtca gaagacgaca cctttggtac catcactatc 300

gctgaaccaa tcggcatcat ctgcggtatc gtaccgacca ctaacccgac ttcaacagcc 360

atcttcaaat cgctcatcag cctgaagacg cgtaacgcca tcattttctc tccgcacccg 420

cgtgctaaag aagccaccaa caaagcggct gacatcgtcc tgcaggctgc catcgctgcc 480

ggcgcgccga aagacctgat tggctggatc gatcagccgt ctgttgaact gtctaacgcc 540

ctgatgcacc acccggacat caacctgatc cttgcgaccg gtggtccagg catggtgaaa 600

gcagcataca gctccggtaa acctgctatc ggcgtaggtg ccggtaacac cccggtggtg 660

attgatgaaa cggctgacat caaacgcgcc gttgcttccg ttctgatgtc taaaaccttc 720

gataacggcg ttatctgtgc ttctgagcag tccgttgtgg tggttgattc cgtttatgac 780

gcggttcgcg aacgtttcgc cagccacggc ggctacctgc tgcagggtaa agagctgaaa 840

gccgttcagg acattatcct gaaaaatggc gcgctgaacg ccgctatcgt tggtcagcct 900

gcggcgaaaa tcgctgaact ggcaggcttc accgtaccgg ccacgactaa aattctgatt 960

ggcgaagtca ccgacgttga cgaaagcgag ccgtttgctc acgaaaaact gtctccgacg 1020

ctggcgatgt accgtgcgaa agatttcgaa gacgccgtga ccaaagctga aaaactggtc 1080

gccatgggcg gtatcggtca tacctcttgc ctgtacaccg accaggataa ccagccggct 1140

cgcgtggctt acttcggcca gatgatgaaa actgcgcgta tcctgatcaa caccccggct 1200

tctcagggtg gtatcggtga cctgtacaac ttcaaactcg ctccttccct gactctgggt 1260

tgtggttcct gggtggtaa ctccatctct gaaaacgttg gtccgaaaca cctgatcaac 1320

aagaaaaccg ttgctaagcg agctgaaaac atgttgtggc acaaacttcc gaaatctatc 1380

tacttccgtc gtggctccct gcctatcgca ctggatgaag tgattactga tggtcacaaa 1440

cgcgcgctga tcgtgactga ccgcttcctg ttcaacaacg gctatgccga tcagatcacc 1500

tccgtactga aagcagcggg tgttgaaact gaagtcttct tcgaagttga agctgacccg 1560

acgctgacca tcgtacgtaa aggcgccgac ctggcgaact cctttaaacc agacgtgatc 1620

atcgcgctgg gcggcggttc cccgatggat gcggcaaaaa tcatgtgggt catgtacgaa 1680

catccggaaa cccacttcga agaactggcg ctgcgcttta tggacatccg taaacgtatc 1740

tacaagttcc cgaaaatggg tgttaaagcc aagatggtgg cgatcactac cacctccggt 1800

accggttctg aagttacgcc gttcgccgtt gtgaccgatg atgcgacagg tcagaaatat 1860

ccgctggcgg actacgcgct gaccccggat atggccatcg tcgatgccaa cctggtgatg 1920

gatatgccga aatcgctgtg tgcgttcggt ggtctggatg ccgtgaccca cgccctggaa 1980

gcttacgttt ccgtgctggc ttctgagttc tctgacggcc aggctctgca ggcgctgaaa 2040

ctgctgaaag agtacctgcc ggcttcctac cacgaaggtt ccaagaaccc ggttgcccgc 2100

gagcgcgtac acagtgccgc caccatcgcc ggtatcgcgt ttgctaacgc cttcctcggc 2160

gtgtgtcact ccatggcgca caaactgggt tcccagttcc atattccgca cggtctggcc 2220

aacgccctgc tgatctgcaa cgttatccgc tacaacgcga atgacaaccc gaccaagcag 2280

accgcgttca gccagtacga ccgtccgcag gctcgtcgtc gctacgctga aatcgccgat 2340

cacctgggtc tctccgcacc gggcgaccgt accgcagcga aaatcgagaa gttgctggca 2400

tggctggaaa gcgtgaaagc tgagctgggt attccgaaat ctatccgcga agctggcgtt 2460

caggaagctg acttcctggc ccacgttgat aagctgtctg aagatgcatt cgatgaccag 2520

tgcaccggcg ctaacccgcg ctacccgctg atctccgagc tgaaacagat cctgctggat 2580

acctactacg gtcgcgagtt cgtcgaaggt gaagcagccg cgaaagccga agcagctccg 2640

gtgaaagctg agaaaaaagc gaaaaaatcc gcttaa 2676

<210> 8

<211> 26

<212>DNA

<213> Artificial Sequence

<400> 8

caacgtggaa gtcgtggcgt atagct 26

<210> 9

<211> 23

<212>DNA

<213> Artificial Sequence

<400> 9

gcgttgaggt ggaatacggt ggc 23

<210> 10

<211> 59

<212>DNA

<213> Artificial Sequence

<400> 10

caggcgcacg tcaccaacgt caaagataac ccggtacaga ttccggggat ccgtcgacc 59

<210> 11

<211> 59

<212>DNA

<213> Artificial Sequence

<400> 11

aatcgtcgcc tttgagtttt ttacgctcga cgtacagcgt tgtaggctgg agctgcttc 59

<210> 12

<211> 25

<212>DNA

<213> Artificial Sequence

<400> 12

agaatctcgc tctctccagg ggaag 25

<210> 13

<211> 23

<212>DNA

<213> Artificial Sequence

<400> 13

cggcgatgtt tacggcaatg aag 23

<210> 14

<211> 23

<212>DNA

<213> Artificial Sequence

<400> 14

ctgggccagc agctcaaggt tac 23

<210> 15

<211> 23

<212>DNA

<213> Artificial Sequence

<400> 15

gctgccgatt gttgacgaag tgc 23

<210> 16

<211> 23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

caggatttca cttcgcctac gac 23

<210> 17

<211> 59

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ggacctgctg gccgtcgata cctcggtgcc ggtgagcgga ttccggggat ccgtcgacc 59

<210> 18

<211> 57

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ggagcaggaa aggaatgcct tcctcttcga taccccagctg taggctggag ctgcttc 57

<210> 19

<211> 21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gcaaatacgg catcagttac c 21

<210> 20

<211> 22

<212>DNA

<213> Artificial Sequence

<400> 20

tgcgtaacgt gttcggtatt ca 22

<210> 21

<211> 23

<212>DNA

<213> Artificial Sequence

<400> 21

gcggtgctcc ttattcgcca tca 23

<210> 22

<211> 23

<212>DNA

<213> Artificial Sequence

<400> 22

agagcgcaca ggaccactat cca 23

<210> 23

<211> 23

<212>DNA

<213> Artificial Sequence

<400> 23

tcggcgagct tatagaccag cgt 23

<210> 24

<211> 59

<212>DNA

<213> Artificial Sequence

<400> 24

ccagctgcct aggggcctta ctacgtatgg cgaagcgttg gcacccgcca aaaccgcca 59

<210> 25

<211> 59

<212>DNA

<213> Artificial Sequence

<400> 25

cttcgtcgag gtcggatgtc aagtggccgg tagtccgcaa ggagtggcgg ctccgcgac 59

<210> 26

<211> 28

<212>DNA

<213> Artificial Sequence

<400> 26

cctgttaaag catagttgcc agccggac 28

<210> 27

<211> 27

<212>DNA

<213> Artificial Sequence

<400> 27

tcgtcagctc gatggttcgg cgattgg 27

<210> 28

<211> 27

<212>DNA

<213> Artificial Sequence

<400> 28

gcagcatcat caaaattggc ggttgac 27

<210> 29

<211> 27

<212>DNA

<213> Artificial Sequence

<400> 29

gtgctttgct atggcttgcg gacagac 27

<210> 30

<211> 48

<212>DNA

<213> Artificial Sequence

<400> 30

cagtttcact caagaacaag tcgacaaaat tccggggatc cgtcgacc 48

<210> 31

<211> 46

<212>DNA

<213> Artificial Sequence

<400> 31

cgaccgtagt aggtatccag caggatctgt aggctggagc tgcttc 46

<210> 32

<211> 25

<212>DNA

<213> Artificial Sequence

<400> 32

gttaaccagg gcaaataagc cgatg 25

<210> 33

<211> 25

<212>DNA

<213> Artificial Sequence

<400> 33

cgattcactg cgtgctggtg gatga 25

Claims (7)

1. The Klebsiella engineered bacterium is characterized in that a diol dehydratase gene is knocked out, and the diol dehydratase gene is pduC, pduD and pduE.

2. The Klebsiella engineered bacterium of claim 1, wherein the lactate dehydrogenase gene and/or the alcohol dehydrogenase gene are also deleted.

3. The Klebsiella engineering bacterium according to claim 2, wherein the lactate dehydrogenase gene is an ldhA gene, and the alcohol dehydrogenase gene is an adhE gene.

4. A method for constructing Klebsiella engineering bacteria is characterized by comprising the following steps: knocking out a diol dehydratase gene of a wild type Klebsiella, wherein the diol dehydratase gene is pduC, pduD and pduE.

5. The method of claim 4, further comprising knocking out a lactate dehydrogenase gene and/or an alcohol dehydrogenase gene.

6. The method of claim 5, further comprising one or more of:

1) The lactate dehydrogenase gene is an ldhA gene;

2) The lactate dehydrogenase gene is an adhE gene;

3) Knocking out genes by adopting a homologous recombination method.

7. Use of the engineered Klebsiella as claimed in any of claims 1 to 3 for the production of 1, 3-propanediol.

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