CN111500514A

CN111500514A - Genetically engineered bacterium for co-production of 1, 3-propylene glycol and polyhydroxybutyrate and construction method and application thereof

Info

Publication number: CN111500514A
Application number: CN202010331996.XA
Authority: CN
Inventors: 纪晓俊; 王维鉴; 陶春平; 徐俊; 蒋文彬
Original assignee: Changzhou Xindong Chemical Industry Development Co ltd; Nanjing Tech University
Current assignee: Changzhou Xindong Chemical Industry Development Co ltd; Nanjing Tech University
Priority date: 2020-04-24
Filing date: 2020-04-24
Publication date: 2020-08-07

Abstract

The invention provides a genetic engineering bacterium for co-producing 1, 3-propylene glycol and polyhydroxybutyrate and a construction method and application thereof, belonging to the technical field of genetic engineering. The genetic engineering bacteria are classified and named as Klebsiella pneumoniaeKlebsiella pneumoniae) Q3-PHB with preservation registration number of CCTCC M20191090 the strain of the invention has simple construction method, and adopts fed-batch fermentation method to finally realize the great increase of biomass of the thalli, the yield of 1,3 propanediol reaches 76.21 g/L, the conversion rate of 1, 3-propanediol is 0.56mol/mol, the contents of ethanol and lactic acid are effectively controlled below 1 g/L, and simultaneously, the PHB accumulated by the thalli reaches 42.36 percent of the dry weight of the thalli, thereby not only reducing the production cost, but also greatly increasing the production costThe downstream separation cost is reduced, the comprehensive economic benefit is higher, and the industrial application value is realized.

Description

Genetically engineered bacterium for co-production of 1, 3-propylene glycol and polyhydroxybutyrate and construction method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and relates to a genetic engineering bacterium for co-producing 1, 3-propylene glycol and Polyhydroxybutyrate (PHB), and a construction method and application thereof.

Background

1, 3-propanediol (1,3-PD) is an important platform chemical, and is widely applied to industries such as medicines, cosmetics and high polymer materials after being developed in the last 90 th century. 1, 3-propanediol is mainly used as a monomer to polymerize with terephthalic acid to produce a novel polyester material, polytrimethylene terephthalate (PTT). PTT has many excellent properties such as elongation, flexibility, easy processability, heat resistance and stability. Based on the properties, PTT has extremely high application value in the fields of synthetic fibers and engineering plastics, and further promotes the research and development of the synthetic monomer PDO.

There are some microorganisms that can naturally synthesize 1, 3-propanediol using glycerol in nature, such as Klebsiella pneumoniae (Klebsiella pneumoniae), Klebsiella oxytoca (Klebsiella oxytoca), Clostridium butyricum (Clostridium butyricum), and the like. The Klebsiella pneumoniae has the characteristics of high glycerol metabolism rate, high 1, 3-propylene glycol yield and high tolerance to high-concentration 1, 3-propylene glycol, and is widely applied to the industrial production of the 1, 3-propylene glycol.

However, the fermentation production of 1, 3-propanediol by using Klebsiella pneumoniae is often accompanied by the synthesis of many byproducts, such as 2,3-butanediol, ethanol, lactic acid, succinic acid, acetic acid, and the like. The accumulation of these byproducts not only reduces the yield of glycerol from 1, 3-propanediol, but also causes inhibition of bacterial growth, increasing the cost of raw materials and the cost of separation of downstream products in the fermentation process.

Disclosure of Invention

The invention mainly aims to provide a genetically engineered bacterium for co-producing 1, 3-propylene glycol and polyhydroxybutyrate, which has the advantages of simple construction method, less accumulation of byproducts in the production process, high biomass of the bacterium, capability of realizing high-efficiency accumulation of extracellular 1, 3-propylene glycol and intracellular Polyhydroxybutyrate (PHB), great reduction of production cost and product separation cost and higher industrial application value.

The invention also aims to provide a construction method of the genetic engineering bacteria for co-producing the 1, 3-propanediol and the polyhydroxybutyrate, which is simple and efficient.

The invention also aims to provide application of the genetic engineering strain in preparation of 1, 3-propanediol and polyhydroxybutyrate.

In order to realize the purpose of the invention, the invention adopts the following technical scheme:

the genetically engineered bacterium for co-production of 1, 3-propylene glycol and polyhydroxybutyrate is classified and named as Klebsiella pneumoniae Q3-PHB (Klebsiella pneumoniae Q3-PHB), and the preservation registration number of the genetically engineered bacterium is CCTCC M20191090.

The invention also provides a construction method of the genetic engineering bacteria, which is characterized in that the genetic engineering bacteria are obtained by taking the Klebsiella pneumoniae ATCC 25955 as an original strain, knocking out aldehyde/alcohol dehydrogenase genes, lactic acid dehydrogenase genes and fumaric acid reductase genes of the Klebsiella pneumoniae ATCC 25955, and introducing a PhbCBA gene from a Cupriavidusnecator source.

In a preferred technical scheme, the construction method comprises the following steps:

(1) taking Klebsiella pneumoniae ATCC 25955 as an original strain, and knocking out an aldehyde/alcohol dehydrogenase gene, a lactate dehydrogenase gene and a fumarate reductase gene in sequence to obtain the recombinant Klebsiella pneumoniae of which three genes are deleted simultaneously;

(2) inserting the phbCBA gene into pDK7 to construct recombinant plasmid pDK7-phbCBA;

(3) and (3) introducing the recombinant plasmid pDK7-phbCBA into the recombinant Klebsiella pneumoniae lacking three genes to obtain the genetic engineering bacteria co-producing the 1, 3-propanediol and the polyhydroxybutyrate.

In the invention, the construction method of the recombinant Klebsiella pneumoniae with three genes deleted in the step (1) is as follows: homologous recombination is carried out on the upstream and downstream homologous arms of the Klebsiella pneumoniae aldehyde/alcohol dehydrogenase gene and the Klebsiella pneumoniae ATCC 25955 by taking a suicide plasmid pKR6K as a vector, so as to knock out the aldehyde/alcohol dehydrogenase gene; and then, taking a suicide plasmid pKR6K as a vector to carry out homologous recombination on the upstream and downstream homologous arms of the lactate dehydrogenase gene and the upstream and downstream homologous arms of the fumarate reductase gene with the Klebsiella pneumoniae to knock out the lactate dehydrogenase gene and the fumarate reductase gene.

The invention also provides application of the genetic engineering bacteria in co-production of 1, 3-propylene glycol and polyhydroxybutyrate.

In the present invention, 1, 3-propanediol and polyhydroxybutyrate are prepared by culturing the genetically engineered bacterium of claim 1 in a fermentation medium containing glycerol.

In the invention, the fermentation temperature is 35-39 ℃, the ventilation amount is 2.5-3.5L/min, the stirring speed is 200-300rpm, the pH value is maintained at 6.3-6.8 in the fermentation process, and when OD is reached₆₀₀And when the concentration of the glycerol reaches 0.6-0.8, adding IPTG (isopropyl-beta-thiogalactoside) for induction, and in the fermentation process, supplementing the glycerol when the concentration of the glycerol is less than 2 g/L so as to maintain the concentration of the glycerol in the fermentation liquid at 20-30 g/L.

In the present invention, the fermentation medium contains the following components: k₂HPO₄·3H₂O 5-7g/L，KH₂PO₄1.5-2.5g/L，MgSO₄·7H₂0.25-0.4 g/L O, 4-6 g/L ammonium sulfate, 0.8-1.2 g/L yeast extract, 0.5-0.7 g/L sodium citrate, 15-25 g/L glycerol and 0.8-1.2 ml/L trace element solution, wherein the trace element solution contains CaCl₂·2H₂O 3.2mg/L，ZnCl₂3.8mg/L，FeCl₃·6H₂O 30.0mg/L，MnCl₂·4H₂O 11.14mg/L，CuCl₂·2H₂O 0.96mg/L，CoCl₂·6H₂O 2.64mg/L，H₃BO₃0.35mg/L，Na₂MoPO₄·2H₂O24.5. mu.g/L, chloramphenicol 20ug/m L.

In the invention, the seed liquid is inoculated into a fermentation culture medium for fermentation culture, and the seed liquid culture medium contains the following components: k₂HPO₄·3H₂O 6-8g/L，KH₂PO₄1.5-2.5g/L，MgSO₄·7H₂0.05-0.15 g/L of O, 6.5-7.5 g/L of yeast extract, 1-1.5 g/L of ammonium sulfate, 18-22 g/L of glycerol and 0.8-1.2 ml/L of trace element solution, wherein the trace element solution contains CaCl₂·2H₂O 3.2mg/L，ZnCl₂3.8mg/L，FeCl₃·6H₂O 30.0mg/L，MnCl₂·4H₂O 11.14mg/L，CuCl₂·2H₂O 0.96mg/L，CoCl₂·6H₂O 2.64mg/L，H₃BO₃0.35mg/L，Na₂MoPO₄·2H₂O24.5. mu.g/L, chloramphenicol 25. mu.g/m L.

The invention has the beneficial effects that: the novel genetic engineering strain provided by the invention is used for co-producing 1, 3-propylene glycol and Polyhydroxybutyrate (PHB). The strain is obtained by taking klebsiella pneumoniae ATCC 25955 as a starting strain, deleting aldehyde/alcohol dehydrogenase gene (adhE), lactate dehydrogenase gene (ldhA) and fumarate reductase gene (frdA), and introducing cuprividusnecator-derived phbCBA gene. The genetic engineering strain adopts fed-batch fermentation method to finally realize biomass (OD) of thallus₆₀₀) The yield of 1, 3-propanediol is increased greatly and improved by about 2.76 times (figure 3), the yield of 1, 3-propanediol reaches 76.21 g/L, the conversion rate is 0.56mol/mol, the contents of ethanol and lactic acid are effectively controlled below 1 g/L, and simultaneously the PHB accumulated by thalli reaches 42.36 percent of the dry weight of the thalli.

Drawings

FIG. 1 is the identification diagram of Klebsiella pneumoniae Q3-PHB electrophoresis, M is DNA marker; the control is Klebsiella pneumoniae ATCC 25955; knocking out the recombinant Klebsiella pneumoniae Q3-PHB strain; delta adhE indicates PCR validation of the adhE gene with primers adhE-F and adhE-R1; Δ ldhA indicates that the ldhA gene was verified by PCR using the primers ldhA-F and ldhA-R1; Δ frdA indicates that the frdA gene was verified by PCR using frdA-F and frdA-R1; PHB represents that primers PHB-F and PHB-R are used for carrying out PCR verification on phbCBA gene in Klebsiella pneumoniae Q3-PHB strain.

FIG. 2 is an electrophoretic identification chart of Klebsiella pneumoconiae Δ adhE Δ ldhA Δ frdA, and M is DNAker; the control is Klebsiella pneumoniae ATCC 25955; the knockout is Klebsiella pneumoconiae delta adhE delta ldhA delta frdA strain; delta adhE indicates PCR validation of the adhE gene with primers adhE-F and adhE-R1; Δ ldhA indicates that the ldhA gene was verified by PCR using the primers ldhA-F and ldhA-R1; Δ frdA indicates that the frdA gene was verified by PCR using frdA-F and frdA-R1.

FIG. 3 shows the growth curve of Klebsiella pneumoniae Q3-PHB against the original strain.

FIG. 4 suicide plasmid pKR6K backbone.

Detailed Description

The invention is further illustrated by the following examples

The experimental procedures used in the following examples are all conventional ones unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Klebsiella pneumoniae ATCC 25955 was purchased from American type culture Collection.

pKR6K plasmid vector is shown in FIG. 4, and its construction method is described in Wang Y, Tao F, Xu P. Glyceroaldehydrogense a dual role in glycerol metabolism and 2, 3-butanol purification in Klebsiella pneumoniae [ J ]. Journal of Biological Chemistry 2014,289(9): 6080-.

pDK7 plasmid constructs open papers: kleiner D, Paul W, Merrick M J. construction of multicopy expression vectors for the regulation of over-production of proteins including Klebsiella pneumoniae and other bacteria [ J ] Microbiology,1988,134(7): 1779-.

publication of the pBHR68 plasmid: spiekermann P, Rehm B H A, Kalscheuer R, et al, sensitive, usable-color stabilizing method using nickel red for direct screening of bacteria that are not important in the practice of microbial science and other lipid storage compositions [ J ]. Archives of microbiology,1999,171(2):73-80.

Example 1: construction of aldehyde/alcohol dehydrogenase Gene (adhE), lactate dehydrogenase Gene (ldhA) and fumaric acid reductase Gene (frdA) deleted recombinant Klebsiella pneumoniae

(1) Cloning of homologous sequences of upper and lower parts of aldehyde/alcohol dehydrogenase gene (adhE)

The genome of Klebsiella pneumoniae ATCC 25955 is used as a template, primers are respectively designed, and the upstream homology arm and the downstream homology arm of the adhE gene are amplified by high-fidelity enzyme PCR.

Primers for PCR amplification of upstream homology arms include adhE-F and adhE-F1:

adhE-F：ctatgacatgattacgaattcATGGCTGTTACTAATATCGCTGAACT；

adhE-F1：cattcgcgttatagcAGAGAAAATGATGGCGTTACGC。

primers for PCR amplification of downstream homology arm part homologous sequences include adhE-R and adhE-R1:

adhE-R：tctctGCTATAACGCGAATGACAACCC；

adhE-R1：gccaagcttgcatgcctgcagTTTTTTCTCAGCCTTTACCGGA。

the PCR amplification system is as follows:

components	Volume of
		PrimerSTAR Max Premix	25ul
Form panel	1ul
		Primer1	2ul
Primer2	2ul
		Distilled water	20ul

PCR amplification was carried out using the above primers, and the PCR enzyme used in the above PCR reaction was PrimeSTAR Max DNA polymerase from Taozi technology (Beijing) Ltd.

The procedure for the above PCR was as follows: denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, extension at 72 ℃ (extension time ═ length of target fragment/1 kb in min), and 30 cycles of repetition.

The primers are used for PCR amplification, and the conditions for amplifying the homologous sequences of the upstream homologous arm parts are as follows: denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, extension at 72 ℃ (extension time ═ length of target fragment/1 kb in min), and 30 cycles of repetition. The amplification conditions of the homologous sequences of the downstream homologous arm portions are as follows: denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, extension at 72 ℃ (extension time ═ length of target fragment/1 kb in min), and 30 cycles of repetition. And after the PCR reaction is finished, carrying out 1.0% agarose gel electrophoresis on the PCR amplification product, and recovering and purifying to obtain the target fragment.

(2) Construction of aldehyde/alcohol dehydrogenase Gene (adhE) suicide plasmid pKR 6K-delta adhE

The pKR6K plasmid was digested with restriction enzymes PstI and EcoRI, and the linearized pKR6K plasmid vector was recovered and purified by 1.0% agarose gel electrophoresis.

The upstream homology arm (the One amplified in title (1) of this example), the downstream homology arm (the One amplified in title (1) of this example) and the linearized pKR6K plasmid vectors of adhE gene were ligated together using Clon express Multi S One Step Cloning Kit of Biotechnology Ltd of Nanjing Novowed.

The ligation product was transformed into E.coli S17-1(E.coli S17-1), positive transformants were selected on L B plates containing chloramphenicol resistance (25. mu.g/m L), and the suicide plasmid pKR 6K-. DELTA.adhe was obtained after colony PCR and sequencing verification.

(3) And (3) carrying out double-parent hybridization on the Escherichia coli S17-1 (donor bacteria) containing the suicide plasmid pKR 6K-delta adhE obtained in the step (2) and Klebsiella pneumoniae ATCC 25955 (recipient bacteria), and carrying out homologous recombination on the upstream and downstream homologous arms of the adhE gene carried on the pKR 6K-delta adhE and the genome of the Klebsiella pneumoniae ATCC 25955 so as to delete the 412 th-2248 th nucleotides of the aldehyde/alcohol dehydrogenase gene (adhE) gene (SEQ ID NO.1) and obtain Klebsiella pneumoniae delta adhE.

The specific method for double-parent hybridization comprises the following steps:

a. inoculating the activated donor and recipient into 10m L L B culture medium, respectively, culturing at 37 deg.C and 200rpm until OD of the donor and recipient₆₀₀0.6-0.8, respectively taking 5m L donor bacteria and 1m L recipient bacteria, centrifuging at 6500rpm, discarding supernatant, respectively washing twice with sterile physiological saline, mixing the donor bacteria and the recipient bacteria with sterile physiological saline not more than 100ul, re-suspending, dripping into the middle of a L B solid culture medium plate, and culturing at 37 ℃ for overnight for joint transfer.

b. Washing the thallus on the L B solid plate in the step a twice by using 5M L sterile physiological saline, diluting the thallus twice, taking 100u L, coating the thallus on an M9 solid plate culture medium with the final concentration of chloramphenicol being 25ug/M L, and culturing at 37 ℃ overnight.

c. A single colony grown on M9 solid medium in step B was picked up, transferred to L B liquid medium containing 25ug/M L chloramphenicol, and cultured overnight at 37 ℃ and 200 rpm.

d. The bacterial liquid obtained in step c is diluted appropriately, 100u L is taken and spread on L AS solid medium, and the culture is carried out overnight at 25 ℃.

e. And (d) selecting a single colony on the plate in the step d, streaking and preserving on a nonreactive L B solid plate, and simultaneously carrying out colony PCR (polymerase chain reaction) verification by using primers adhE-F and adhE-R1 to obtain a correct clone through gel electrophoresis verification, wherein the correct clone is the engineering bacterium knocked out by aldehyde/alcohol dehydrogenase gene (adhE), and is named as Klebsiella pneumoniae delta adhE.

L B solid culture medium composed of peptone 10 g/L, NaCl 10 g/L, yeast powder 5 g/L, and agar powder 20 g/L, and sterilizing at 121 deg.C for 20 min.

M9 solid medium: na (Na)₂HPO₄·12H₂O 1.7g/L、KH₂PO₄0.3 g/L、NaCl 0.05g/L、NH₄Cl0.1g/L, trisodium citrate 0.5 g/L and agar powder 15 g/L, and sterilizing at 121 ℃ for 20 min.

L AS solid culture medium, peptone 10 g/L, yeast powder 5 g/L, sucrose 150 g/L, agar powder 15 g/L, sterilized at 115 deg.C for 20 min.

(4) Cloning of upstream and downstream homology arms of lactate dehydrogenase Gene (ldhA)

The primers were designed using the genome of Klebsiella pneumoniae ATCC 25955 as a template, and the upstream homology arm and the downstream homology arm of ldhA gene were amplified by high fidelity enzymatic PCR, and the primers were as follows:

primers for PCR amplification of upstream homology arms:

ldhA-F：ctatgacatgattacgaattcTTTTCAGCCGCTTTCTCTCTCC，

ldhA-F1：gtttcgCCGTTGGCGGTTTTGGCA。

primers for PCR amplification of downstream homology arms:

ldhA-R：aaaaccgccaacggCGAAACCTGTCCGAACGCC，

ldhA-R1：gccaagcttgcatgcctgcagAGGGTATTGAGCTGGGCGTC。

the upstream and downstream homology arms of the ldhA gene were PCR-amplified using the above primers using the Klebsiella pneumoniae ATCC 25955 genome as a template. The PCR amplification system was the same as that described in the title (1) of this example. Upstream homology arm amplification procedure: denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, extension at 72 ℃ (extension time ═ length of target fragment/1 kb in min), and 30 cycles of repetition. Downstream homology arm amplification procedure: denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, extension at 72 ℃ (extension time ═ length of target fragment/1 kb in min), and 30 cycles of repetition. And after the PCR reaction is finished, carrying out 1.0% agarose gel electrophoresis on the PCR amplification product, and recovering and purifying to obtain the target fragment.

(5) Construction of lactate dehydrogenase Gene (ldhA) suicide plasmid pKR 6K-DeltaldhA

The plasmid pKR6K was digested with restriction enzymes PstI and EcoRI, and the linearized pKR6K plasmid vector was recovered and purified by 1.0% agarose gel electrophoresis gel, the upstream homology arm, the downstream homology arm and the linearized pKR6K plasmid vector were ligated together with the Clon express MultiS OnS Onstep Cloning Kit of Nanjing Nonakai Biotech Co., Ltd, the ligation product was transformed into E.coli S17-1(E.coli S17-1), positive transformants were selected on L B plates containing chloramphenicol resistance (25ug/m L), and the suicide plasmid pKR 6K-. DELTA.ldhA was obtained after colony PCR and sequencing verification.

(6) Carrying out parental hybridization on the Escherichia coli S17-1 containing the suicide plasmid pKR 6K-delta ldhA obtained in the step (5) and Klebsiella pneumoniae delta adhE, and carrying out homologous recombination on upstream and downstream homology arms of an ldhA gene carried on the pKR 6K-delta ldhA and the genome of the Klebsiella pneumoniae delta adhE so as to delete the 132 th-962 th nucleotides of a lactate dehydrogenase gene (ldhA, SEQ ID NO.2) and obtain the Klebsiella pneumoniae delta adhE. The specific method is the same as the step (3), primers ldhA-F and ldhA-R1 are used for colony PCR verification, and transformants which are correct through electrophoretic verification are engineering strains with deletion of lactate dehydrogenase genes (ldhA) and aldehyde/alcohol dehydrogenase genes (adhE) and are named as Klebsiella pneumoniae delta adhE delta ldhA.

(7) Cloning of upstream and downstream homologous sequences of fumarate reductase Gene (frdA)

Respectively designing primers by taking the genome of Klebsiella pneumoniae ATCC 25955 as a template, and amplifying an upstream homology arm and a downstream homology arm of the frdA gene by using high-fidelity enzyme PCR, wherein the required primers are as follows:

primers for PCR amplification of upstream homology arms:

frdA-F：ctatgacatgattacgaattcCGGTAAAGAAACGGCGGAT，

frdA-F1：atattccagacggCGTATGGCTGCGCATCGG。

primers for PCR amplification of downstream homology arms:

frdA-R：ccatacgCCGTCTGGAATATGGCGATG，

frdA-R1：gccaagcttgcatgcctgcagGGTCTGCTTGTTTGGCCCC。

the PCR amplification system and amplification conditions were the same as in the title (1) of this example.

(8) Construction of fumaric reductase Gene (frdA) suicide plasmid pKR6K- Δ frdA

The plasmid pKR6K was digested with restriction enzymes PstI and EcoRI, and the linearized pKR6K plasmid vector was recovered and purified by 1.0% agarose gel electrophoresis gel, the upstream and downstream homology arms of the frdA gene and the linearized pKR6K plasmid vector were ligated together with the Clon express MultiS OneStep Cloning Kit of Nanjing Nonakai Biotech Co., Ltd, the ligation product was transformed into E.coli S17-1(E.coli S17-1), positive transformants were selected on L B plates containing chloramphenicol resistance (25ug/m L), and the suicide plasmid pKR 6K-. DELTA.frdA was obtained after colony PCR and sequencing verification.

(9) Carrying out parental hybridization on the Escherichia coli S17-1 containing the suicide plasmid pKR 6K-delta frdA obtained in the step (8) and Klebsiella pneumoniae delta adhE delta ldhA to ensure that the upstream and downstream homologous arms of the frdA gene carried by the pKR 6K-delta ldhA are subjected to homologous recombination with the genome of Klebsiella pneumoniae delta adhE delta ldhA, so that the 139 th to 1685 th nucleotides of a fumarate reductase gene (frdA, SEQ ID NO.3) are deleted to obtain the recombinant Klebsiella pneumoniae delta adhE delta ldhA delta frdA. The specific method is the same as the step (3), the primers used in colony PCR verification are frdA-F and frdA-R1, the transformants with correct electrophoretic verification are engineering strains with lactate dehydrogenase gene (ldhA), aldehyde/alcohol dehydrogenase gene (adhE) and fumarate reductase gene (frdA) deleted at the same time, and the transformants are named Klebsiella pneumoniae delta adhE delta ldhA delta frdA, and the electrophoretic verification result is shown in FIG. 2.

Example 2: construction of recombinant plasmid pDK7-phbCBA

(1) The phbCBA gene (SEQ ID NO.4) from cupriavidius necato is amplified by taking pBHR68 plasmid as a template, and primers PHB-F and PHB-R required by PCR are designed.

PHB-F：ggtacccggggatcctctagaGGGCAAGTACCTTGCCGAC，

PHB-R：tccgccaaaacagccaagcttCTTCTGAATCCATGACCAGCTGC。

The PCR system and the amplification conditions are the same as in step (1).

(2) Construction of expression plasmid pDK7-phbCBA

The pDK7 plasmid was digested with restriction enzymes HindIII and XbaI, the linearized pDK7 plasmid vector was recovered and purified by 1.0% agarose gel electrophoresis gel, the phbCBA gene fragment and the linearized pDK7 plasmid were ligated together with the Clon express MultiS OnSttep Cloning Kit of Nanjing Novone Biotech Ltd, the ligation product was transformed into E.coli DH5 α (E.coli DH5 α), positive transformants were selected with L B plates containing chloramphenicol resistance (25. mu.g/m L), and the transformants which were positive by colony PCR and sequencing verification were named transformant pDK7-phbCBA plasmid.

Example 3: the recombinant plasmid pDK7-phbCBA is electrically transformed to recombine Klebsiella pneumoniae delta adhE delta ldhA delta frdA to construct Klebsiella pneumoniae Q3-PHB

The recombinant plasmid pDK7-phbCBA is transformed into Klebsiella pneumoniae delta adhE delta ldhA delta frdA by an electroporator, the parameters of the electrical transformation conditions are that voltage is 2.5kv, resistance is 200 omega, pulse time is 6.0msec, after the electrical transformation is finished, Klebsiella pneumoniae delta adhE delta ldhA delta frdA which is treated by electric shock is washed out by using a precooled liquid L B culture medium, the Klebsiella pneumoniae delta adhE delta ldhA delta frdA is cultured for 1-2 hours at 37 ℃ in a shaker at 200rpm and then is coated on a L B plate containing 25 mu g/m L chloramphenicol, and primers PHB-F are used for carrying out colony PCR verification, the obtained positive clone is Klebsiella pneumoniae Q3-PHB, the results of the Klebsiella pneumoniae Q3-PHB gene adbC frhA gene, the PHB gene and the PHBA gene are shown in a figure, and the correct PCR results are shown in the figure 1.

Klebsiella pneumoniae Q3-PHB is sent to China center for type culture Collection to be preserved, and the preservation information is as follows:

and (3) classification and naming: klebsiella pneumoniae (Klebsiella pneumoniae) Q3-PHB

The preservation number is: CCTCC NO: M20191090;

the preservation date is as follows: 12 months and 23 days 2019;

the preservation unit: china Center for Type Culture Collection (CCTCC), address: wuhan university in Wuhan City, China.

Example 4: application of genetically engineered bacterium Klebsiella pneumoniae Q3-PHB in co-production of 1, 3-propylene glycol and Polyhydroxybutyrate (PHB)

(1) The strain is as follows: klebsiella pneumoniae Q3-PHB.

(2) Media composition

Seed culture medium: k₂HPO₄·3H₂O 7g/L，KH₂PO₄2g/L，MgSO₄·7H₂0.1 g/L O, 7 g/L yeast extract, 1.25 g/L ammonium sulfate, 20 g/L glycerol, 1.0m L/L trace elements and water as solvent, wherein the trace elements are water solution containing CaCl₂·2H₂O 3.2mg/L，ZnCl₂3.8mg/L，FeCl₃·6H₂O 30.0mg/L，MnCl₂·4H₂O11.14mg/L，CuCl₂·2H₂O 0.96mg/L，CoCl₂·6H₂O 2.64mg/L，H₃BO₃0.35mg/L，Na₂MoPO₄·2H₂O24.5. mu.g/L, chloramphenicol 25. mu.g/m L.

Fermentation medium: k₂HPO₄·3H₂O 6g/L，KH₂PO₄2g/L，MgSO₄·7H₂0.32 g/L O, 5 g/L ammonium sulfate, 1 g/L yeast extract, 0.6 g/L sodium citrate, 20 g/L glycerol and 1.0 ml/L trace elements, wherein the trace elements are aqueous solution containing CaCl₂·2H₂O 3.2mg/L，ZnCl₂3.8mg/L，FeCl₃·6H₂O 30.0mg/L，MnCl₂·4H₂O11.14mg/L，CuCl₂·2H₂O 0.96mg/L，CoCl₂·6H₂O 2.64mg/L，H₃BO₃0.35mg/L，Na₂MoPO₄·2H₂O24.5. mu.g/L, chloramphenicol 20. mu.g/m L.

(3) The fermentation method comprises the following steps:

1) seed activation

Klebsiella pneumoniae Q3-PHB deposited in glycerol was inoculated into L B medium of 5m L and cultured in a shaker at 37 ℃ and 200rpm for 12 hours to activate Klebsiella pneumoniae Q3-PHB.

2) Seed culture

The activated Klebsiella pneumoniae Q3-PHB obtained in the step 1) was transferred to a 250m L flask containing 50m L seed medium at an inoculation amount of 1% (by volume) and cultured in a shaker at 37 ℃ and 200rpm for 12 hours to obtain a seed solution.

3) Fermentation culture

1, 3-propanediol and Polyhydroxybutyrate (PHB) are co-produced by fed-batch fermentation. At 5% inoculum size (body)Volume ratio), inoculating the seed liquid into fermentation medium, performing fed-batch fermentation in 5L fermenter with initial liquid loading of 2L, fermentation temperature of 37 deg.C, aeration rate of 3.0L/min, stirring speed of 250rpm, adjusting pH of the fermentation liquid with 2M NaOH solution during fermentation to 6.5, and adjusting OD to 6.5₆₀₀When the concentration of the glycerol in the fermentation medium reaches 2 g/L, adding the glycerol in a fed-batch manner to maintain the concentration of the glycerol at 20 g/L, and when the fermentation time reaches 30h, harvesting the fermentation liquor.

In addition, Klebsiella pneumoniae ATCC 25955 was cultured as a control in the same manner as described above.

4) Biomass detection and product detection

Klebsiella pneumoniae Q3-PHB and Klebsiella pneumoniae ATCC 25955 were tested as follows:

(I) biomass detection

In the fermentation process, the fermentation liquid is taken at regular intervals, and OD is detected₆₀₀To examine the change in biomass during fermentation.

(II) detecting the components of the fermentation liquor:

centrifuging the fermentation broth sample at 12000r/min for 5min when the fermentation time is 30H, collecting supernatant, filtering with 0.22 μm filter membrane, and detecting the components of the fermentation broth by high performance liquid chromatography (HP L C) under the conditions of a detector, a differential detector, a chromatographic column, a Bio-Rad Aminex HPX-87H chromatographic column (300 × 7.8.8 mM), and a mobile phase, 5mM H₂SO₄A solution; flow rate: 0.6 ml/min; column temperature: and 65 ℃.

(III) PHB detection:

taking a fermentation liquid sample with the fermentation time of 30 hours, centrifuging for 10 minutes at 6500r/min, discarding the supernatant, cleaning for three times by using ultrapure water under the same centrifugation condition, collecting thalli, drying to balance weight, weighing a certain amount of dry thalli, adding 150 mu L concentrated sulfuric acid, treating for one hour in a boiling water bath, adjusting the pH to 2.5 by using 15% ammonia water, filtering by using a 0.22 mu m filter membrane, taking the filtrate, and adopting high performance liquid detection, wherein the detection condition comprises that a detector is an ultraviolet detector, the ultraviolet wavelength is 210nm, a chromatographic column is a C18 column, and the mobile phase is 1 per mill formic acid solution.

5) Results of fermentation

The fermentation time was 30 hours, and the fermentation broth components and PHB production were as shown in Table 1.

TABLE 1 comparison of fermentation components of Klebsiella pneumoniae Q3-PHB strain, Q3 strain and original strain

Note: "ND" in Table 1 means no detection; "Q3" refers to Klebsiella pneumoconiae. DELTA. adhE. DELTA. ldhA. DELTA. frdA obtained by removing the adhE gene, ldhA gene and frdA gene from the original strain ATCC 25955 (example 1).

As can be seen from Table 1, the yield of the target product 1, 3-propanediol is improved from 68.73 g/L to 76.21 g/L, the conversion rate is also increased from 0.52mol/mol to 0.56mol/mom, simultaneously, the deletion of aldehyde/alcohol dehydrogenase gene (adhE) and lactate dehydrogenase gene (ldhA) controls the content of ethanol and lactic acid in the fermentation liquor to be below 1 g/L, and the knock-out of fumarate reductase gene (frdA) reduces the content of succinic acid, meanwhile, the acetic acid accumulation in the culture medium is obviously reduced along with the introduction of PHB synthesis route, and is reduced from 10.77 g/L to 6.28 g/L, on the basis, the PHB accumulation reaches 42.36%₆₀₀) The biomass is increased greatly, and the biomass is increased to about 2.76 times of the original strain at the fermentation time of 30 hours.

The experimental results show that the introduction of the heterogeneous PHB synthesis approach can not only simultaneously accumulate the extracellular product 1, 3-propanediol and the intracellular product PHB, but also effectively pull the carbon metabolic flow and relieve the excessive overflow phenomenon of acetic acid caused by the deletion of the byproduct approach, thereby obviously improving the biomass of the thalli, reducing the production cost, greatly reducing the downstream separation cost, having higher comprehensive economic benefit and having industrial application value.

SEQUENCE LISTING

<110> Changzhou New east chemical development Co., Ltd

NANJING TECH University

<120> gene engineering bacterium for co-production of 1, 3-propylene glycol and polyhydroxybutyrate and construction method and application thereof

<130>20200323

<160>4

<170>PatentIn version 3.3

<210>1

<211>2676

<212>DNA

<213>Klebsiella pneumoniae ATCC 25955

<400>1

atggctgtta ctaatatcgc tgaactgaac gcgcttgttg agcgtgtcaa gaaagcccag 60

cgtgaatatg ccagtttcac tcaagaacaa gtcgacaaaa tcttccgcgc cgccgctctg 120

gccgctgcag atgctcgaat ccctctcgcc aaaatggccg tagccgaatc cggcatgggc 180

atcgttgaag acaaagtgat caaaaaccac tttgcttccg aatacattta caacgcttat 240

aaagacgaaa aaacctgtgg cgttctgtca gaagacgaca catttggcac catcactatt 300

gctgagccta tcggcatcat ctgcggtatc gtaccgacca ctaacccgac ttcaacagcc 360

attttcaaat cgctcatcag tctgaaaacg cgtaacgcca tcattttctc tccgcacccg 420

cgtgcaaaag aagccaccaa caaagcggct gatatcgtgc tgcaggctgc catcgcggcc 480

ggcgcgccga aggacctgat tggctggatc gatcagccgt ctgtagaact gtctaacgcg 540

ctgatgcatc acccggacat caacctgatc ctggcgaccg gtggtccagg catggtgaaa 600

gcagcataca gctccggtaa acctgctatc ggcgtcggcg ccggtaacac cccggtagtc 660

attgatgaaa ccgccgacat caaacgtgcc gtagcgtccg tactgatgtc taaaaccttc 720

gataacggcg tgatctgtgc ttctgagcag tccgttgtgg tggttgattc agtctatgac 780

gcggttcgcg aacgtttcgc cagccacggc ggctacctgc tgcagggtaa agagctgaaa 840

gccgttcagg acattatcct gaaaaatggc gcgctgaacg cggctatcgt tggtcagcca 900

gcggccaaaa tcgctgaact ggcaggcttc accgtgccgg ccaccaccaa aattctgatt 960

ggtgaagtga ccaacgttga cgagagcgag ccgtttgctc acgaaaaact gtctccgacg 1020

ctggctatgt atcgtgcaaa agatttcgaa gacgcggtag ccaaagcaga aaaactggtc 1080

gccatgggcg gtatcggcca cacctcttgc ctgtacaccg accaggacaa ccagccggct 1140

cgcgtggctt acttcggcca aatgatgaaa accgcgcgta tcctgatcaa caccccggct 1200

tctcagggtg gtatcggtga cctgtacaac ttcaaactcg cgccttccct gactctgggt 1260

tgtggttcct ggggtggtaa ctccatctct gaaaacgttg gtccgaaaca cctgatcaac 1320

aagaaaaccg ttgctaagcg agctgaaaac atgttgtggc acaaacttcc gaaatctatc 1380

tacttccgtc gtggctccct gccaatcgca ctggatgaag tgattaccga tggtcacaaa 1440

cgcgcgctga tcgtgactga ccgcttcctg ttcaacaacg gctatgccga tcagatcact 1500

tccgtactga aagctgcggg tgttgaaacg gaagtcttct tcgaagttga agctgacccg 1560

acgctgacca tcgtacgtaa aggtgccgac ctggcgaact cctttaaacc agacgtgatc 1620

atcgccctgg gcggcggttc cccgatggat gcggcgaaaa tcatgtgggt catgtacgaa 1680

catccggaaa cccacttcga agaactggcg ctgcgcttta tggatatccg taaacgtatc 1740

tacaagttcc cgaaaatggg cgtcaaagcc aagatggtgg cgatcactac cacttccggt 1800

accggttctg aagtgacgcc gttcgccgtg gtgaccgatg acgcgaccgg tcagaaatac 1860

ccgctggcag actacgcgct gaccccggat atggccattg tcgatgccaa cctggtgatg 1920

gatatgccga aatcgctgtg tgcgttcggt ggtctggatg cggtgactca cgccctggaa 1980

gcttacgttt ccgtactggc ttctgagttc tctgacggcc aggcgctgca ggcgctgaaa 2040

ctgctgaaag agtacctgcc ggcttcctat cacgaaggtt ccaagaaccc ggttgcccgt 2100

gagcgcgtac acagtgccgc gactatcgcc ggtatcgcgt tcgctaacgc cttcctcggc 2160

gtgtgtcact cgatggcgca caaactgggc tcccagttcc atattccgca cggtctggcc 2220

aacgccctgc tgatctgcaa cgttatccgc tataacgcga atgacaaccc gaccaagcag 2280

accgcgttca gccagtacga ccgtccgcag gctcgtcgtc gttacgctga aatcgccgat 2340

cacctgggcc tctccgcacc aggcgaccgc accgcagcga aaatcgagaa actgctggca 2400

tggctggaaa gcattaaagc tgagctgggt attccgaaat ctatccgcga agctggcgtt 2460

caggaagctg acttcctggc ccacgttgat aaactgtctg aagatgcatt cgatgaccag 2520

tgcaccggcg ctaacccgcg ctacccgctg atctccgaac tgaaacagat cctgctggat 2580

acctactacg gccgcgaatt cgtcgaaggc gaagccggtg cgaaagccga agttgctccg 2640

gtaaaggctg agaaaaaagc gaaaaaatcc gcttaa 2676

<210>2

<211>990

<212>DNA

<213>Klebsiella pneumoniae ATCC 25955

<400>2

atgaaaatcg cggtttatag tacgaagcag tacgataaaa agtacctgca gcacgttaat 60

gatgcatacg gctttgaact ggaattcttc gatttcctgc tgacagcgaa gactgccaaa 120

accgccaacg gttgcgaagc ggtatgtatc ttcgtcaatg acgacggcag ccgcccggtg 180

ctggaagagc tgaaggccca cggggtgaaa tatatcgccc tgcgctgcgc cgggtttaac 240

aacgtcgacc ttgaggcggc aaaggagctt ggcctgcgcg tcgtgcgcgt tccagcttac 300

tctccggaag cggtcgctga gcatgcgatc ggtatgatga tgtcgctcaa ccgccgcatc 360

caccgcgctt accagcgtac ccgcgatgcc aatttctccc tcgaaggcct caccggcttc 420

accatgtacg gcaaaaccgc cggggtgatc ggcaccggga aaattggcgt agcgatgttg 480

cggatcctca aaggcttcgg catgcgcctg ctggcgttcg acccgtaccc aagcgccgcc 540

gcgctggagc tgggggtgga atatgttgac ctcgccacgc tgtacaagga atcggacgtg 600

atctccctgc actgtccgct gaccgacgaa aactaccacc tgctcaatcg cgaagccttc 660

gatcagatga aagacggggt gatggtgatc aacaccagcc gcggcgccct gatcgactct 720

caggcggcca tcgacgccct gaagcaccag aaaattggcg cgctggggct ggacgtttat780

gagaacgaac gcgatctgtt ctttgaagac aaatccaacg acgtgatcca ggacgatgtc 840

ttccgccgcc tctccgcctg ccataacgtg ctgtttaccg gccaccaggc gttcctcacc 900

gccgaggcgc tgatcagcat ttcggagacc actctgggta acctgcagca ggtcgccaac 960

ggcgaaacct gtccgaacgc catcgtctaa 990

<210>3

<211>1791

<212>DNA

<213>Klebsiella pneumoniae ATCC 25955

<400>3

gtgcaaactt ttcaagccga tctcgccgta attggcgccg gcggggcagg ccttcgtgcc 60

gctatcgccg ccgctcaggc caaccccaac gctaaaatcg ccctgatttc taaagtctat 120

ccgatgcgca gccatacggt cgctgcagaa ggggggtccg cagccgttgc ccaggatcac 180

gatagctttg agtaccattt tcacgacacc gtcgcgggag gcgactggct gtgcgagcag 240

gatgtcgtcg actattttgt tcatcactgc ccgacggaaa tgacccagct ggagcagtgg 300

ggatgcccat ggagccgccg ccccgacggc agcgtcaacg tccggcgctt cggtggcatg 360

aaaatcgaac gcacctggtt tgccgccgac aaaaccggct tccacatgct gcataccctc 420

ttccagacct cccttcagtt tccccaaatt cagcgctttg acgaacattt cgtccttgac 480

ctgctggtcg atgacgggca ggcccgcggc ctggtggcga tgaatatgat ggaaggcacc 540

ctggtgcaga tccgcgctaa cgccgtggtg ctggctaccg gcggcgccgg gcgcgtctac 600

cgctacaaca ccaacggcgg catcgtcacc ggtgacggga tggggatggc cctgggacac 660

ggcgtgccgc tgcgcgacat ggaattcgtt cagtatcacc cgaccggcct gccgggctcc 720

gggatcctga tgaccgaagg ctgccgcggc gaaggcggga ttctggtcaa caaaaacggc 780

taccgctatc tgcaagatta cggcatgggc ccggaaaccc cgctcggcga accgaagaat 840

aaatatatgg agctgggccc acgcgacaaa gtgtcccagg ccttctggca cgagtggcgg 900

aaagggaaca ccatcccgac gccgcgcggc gacgtggtct acctcgatct ccgccacctc 960

ggcgagaaaa aactgctgga gcgtctgccg tttatctgtg aactggcgaa agcctacgtc 1020

ggcgtcgatc cggtgaaaga gccgatcccg gtgcgcccga ccgcgcacta caccatgggc 1080

ggtattgaaa ccgatcagca gtgcgaaacc cggatcaaag ggctgtttgc cgtgggcgaa 1140

tgctcgtccg ttggcttaca tggcgccaac cgtctgggct ccaactcgct ggccgagctg 1200

gtggtcttcg gccgcctggc cggggagcag gccatgcagc gcgccgctca ggcgggcgag 1260

gcaaacggcg ccgccctcga tgcgcaggct gccgatgtgg agcagcgcct gaaagatctc 1320

gtcaaccagg aaggtaacga aaactgggcg aaaattcgcg atgaaatggg gctgtcgatg 1380

gaggaaggct gcggcatcta ccgcacgccg gagctgatgc agaaaacgat cgacaagctg 1440

gctgaactgc aggagcgctt taagcgcgtg cgcattaccg ataattccag cgtgttcaat 1500

accgatctgc tttacaccat tgagctgggg cacggtttaa acgttgccga atgtatggcg 1560

cactcggcca tcgcccgtaa agaatcgcgc ggggcgcacc agcgtctgga tgaaggctgt 1620

accgaacgcg acgacgtcaa cttcctcaag cacaccctcg ccttccgcga ggccgatggc 1680

actacccgtc tggaatatgg cgatgtgaag atcaccacgc tgccgccggc aaaacgtgtc 1740

tatggcgcag aagcggacgc ggctgagaag aaggagacca cccatggctg a 1791

<210>4

<211>5253

<212>DNA

<213>Cupriavidus necator

<400>4

gggcaagtac cttgccgaca tctatgcgct ggcgcgcacg cgcctggcgc gcgccggctg 60

taccgaggtc tacggcggcg acgcctgcac cgtggccgac gccggtcgct tctactccta 120

tcggcgcgat ggcgtgaccg gccgcatggc cagcctggtc tggctggcgg actgagcccg 180

ccgctgcctc actcgtcctt gcccctggcc gcctgcgcgc gctcggcttc agccttgcgt 240

cggcggcggc cgggcgtgcc catgatgtag agcaccagcg ccaccggcgc catgccatac 300

atcaggaagg tggcaacgcc tgccaccacg ttgtgctcgg tgatcgccat catcagcgcc 360

acgtagagcc agccaatggc cacgatgtac atcaaaaatt catccttctc gcctatgctc 420

tggggcctcg gcagatgcga gcgctgcata ccgtccggta ggtcgggaag cgtgcagtgc 480

cgaggcggat tcccgcattg acagcgcgtg cgttgcaagg caacaatgga ctcaaatgtc 540

tcggaatcgc tgacgattcc caggtttctc cggcaagcat agcgcatggc gtctccatgc 600

gagaatgtcg cgcttgccgg ataaaagggg agccgctatc ggaatggacg caagccacgg 660

ccgcagcagg tgcggtcgag ggcttccagc cagttccagg gcagatgtgc cggcagaccc 720

tcccgctttg ggggaggcgc aagccgggtc cattcggata gcatctcccc atgcaaagtg 780

ccggccaggg caatgcccgg agccggttcg aatagtgacg gcagagagac aatcaaatca 840

tggcgaccgg caaaggcgcg gcagcttcca cgcaggaagg caagtcccaa ccattcaagg 900

tcacgccggg gccattcgat ccagccacat ggctggaatg gtcccgccag tggcagggca 960

ctgaaggcaa cggccacgcg gccgcgtccg gcattccggg cctggatgcg ctggcaggcg 1020

tcaagatcgc gccggcgcag ctgggtgata tccagcagcg ctacatgaag gacttctcag 1080

cgctgtggca ggccatggcc gagggcaagg ccgaggccac cggtccgctg cacgaccggc 1140

gcttcgccgg cgacgcatgg cgcaccaacc tcccatatcg cttcgctgcc gcgttctacc 1200

tgctcaatgc gcgcgccttg accgagctgg ccgatgccgt cgaggccgat gccaagaccc 1260

gccagcgcat ccgcttcgcg atctcgcaat gggtcgatgc gatgtcgccc gccaacttcc 1320

ttgccaccaa tcccgaggcg cagcgcctgc tgatcgagtc gggcggcgaa tcgctgcgtg 1380

ccggcgtgcg caacatgatg gaagacctga cacgcggcaa gatctcgcag accgacgaga 1440

gcgcgtttga ggtcggccgc aatgtcgcgg tgaccgaagg cgccgtggtc ttcgagaacg 1500

agtacttcca gctgttgcag tacaagccgc tgaccgacaa ggtgcacgcg cgcccgctgc 1560

tgatggtgcc gccgtgcatc aacaagtact acatcctgga cctgcagccg gagagctcgc 1620

tggtgcgcca tgtggtggag cagggacata cggtgtttct ggtgtcgtgg cgcaatccgg 1680

acgccagcat ggccggcagc acctgggacg actacatcga gcacgcggcc atccgcgcca 1740

tcgaagtcgc gcgcgacatc agcggccagg acaagatcaa cgtgctcggc ttctgcgtgg 1800

gcggcaccat tgtctcgacc gcgctggcgg tgctggccgc gcgcggcgag cacccggccg 1860

ccagcgtcac gctgctgacc acgctgctgg actttgccga cacgggcatc ctcgacgtct 1920

ttgtcgacga gggccatgtg cagttgcgcg aggccacgct gggcggcggc gccggcgcgc 1980

cgtgcgcgct gctgcgcggc cttgagctgg ccaatacctt ctcgttcttg cgcccgaacg 2040

acctggtgtg gaactacgtg gtcgacaact acctgaaggg caacacgccg gtgccgttcg 2100

acctgctgtt ctggaacggc gacgccacca acctgccggg gccgtggtac tgctggtacc 2160

tgcgccacac ctacctgcag aacgagctca aggtaccggg caagctgacc gtgtgcggcg 2220

tgccggtgga cctggccagc atcgacgtgc cgacctatat ctacggctcg cgcgaagacc 2280

atatcgtgcc gtggaccgcg gcctatgcct cgaccgcgct gctggcgaac aagctgcgct 2340

tcgtgctggg tgcgtcgggc catatcgccg gtgtgatcaa cccgccggcc aagaacaagc 2400

gcagccactg gactaacgat gcgctgccgg agtcgccgca gcaatggctg gccggcgcca 2460

tcgagcatca cggcagctgg tggccggact ggaccgcatg gctggccggg caggccggcg 2520

cgaaacgcgc cgcgcccgcc aactatggca atgcgcgcta tcgcgcaatc gaacccgcgc 2580

ctgggcgata cgtcaaagcc aaggcatgac gcttgcatga gtgccggcgt gcgtcatgca 2640

cggcgccggc aggcctgcag gttccctccc gtttccattg aaaggactac acaatgactg 2700

acgttgtcat cgtatccgcc gcccgcaccg cggtcggcaa gtttggcggc tcgctggcca 2760

agatcccggc accggaactg ggtgccgtgg tcatcaaggc cgcgctggag cgcgccggcg 2820

tcaagccgga gcaggtgagc gaagtcatca tgggccaggt gctgaccgcc ggttcgggcc 2880

agaaccccgc acgccaggcc gcgatcaagg ccggcctgcc ggcgatggtg ccggccatga 2940

ccatcaacaa ggtgtgcggc tcgggcctga aggccgtgat gctggccgcc aacgcgatca 3000

tggcgggcga cgccgagatc gtggtggccg gcggccagga aaacatgagc gccgccccgc 3060

acgtgctgcc gggctcgcgc gatggtttcc gcatgggcga tgccaagctg gtcgacacca 3120

tgatcgtcga cggcctgtgg gacgtgtaca accagtacca catgggcatc accgccgaga 3180

acgtggccaa ggaatacggc atcacacgcg aggcgcagga tgagttcgcc gtcggctcgc 3240

agaacaaggc cgaagccgcg cagaaggccg gcaagtttga cgaagagatc gtcccggtgc 3300

tgatcccgca gcgcaagggc gacccggtgg ccttcaagac cgacgagttc gtgcgccagg 3360

gcgccacgct ggacagcatg tccggcctca agcccgcctt cgacaaggcc ggcacggtga 3420

ccgcggccaa cgcctcgggc ctgaacgacg gcgccgccgc ggtggtggtg atgtcggcgg 3480

ccaaggccaa ggaactgggc ctgaccccgc tggccacgat caagagctat gccaacgccg 3540

gtgtcgatcc caaggtgatg ggcatgggcc cggtgccggc ctccaagcgc gccctgtcgc 3600

gcgccgagtg gaccccgcaa gacctggacc tgatggagat caacgaggcc tttgccgcgc 3660

aggcgctggc ggtgcaccag cagatgggct gggacacctc caaggtcaat gtgaacggcg 3720

gcgccatcgc catcggccac ccgatcggcg cgtcgggctg ccgtatcctg gtgacgctgc 3780

tgcacgagat gaagcgccgt gacgcgaaga agggcctggc ctcgctgtgc atcggcggcg 3840

gcatgggcgt ggcgctggca gtcgagcgca aataaggaag gggttttccg gggccgcgcg 3900

cggttggcgc ggacccggcg acgataacga agccaatcaa ggagtggaca tgactcagcg 3960

cattgcgtat gtgaccggcg gcatgggtgg tatcggaacc gccatttgcc agcggctggc 4020

caaggatggc tttcgtgtgg tggccggttg cggccccaac tcgccgcgcc gcgaaaagtg 4080

gctggagcag cagaaggccc tgggcttcga tttcattgcc tcggaaggca atgtggctga 4140

ctgggactcg accaagaccg cattcgacaa ggtcaagtcc gaggtcggcg aggttgatgt 4200

gctgatcaac aacgccggta tcacccgcga cgtggtgttc cgcaagatga cccgcgccga 4260

ctgggatgcg gtgatcgaca ccaacctgac ctcgctgttc aacgtcacca agcaggtgat 4320

cgacggcatg gccgaccgtg gctggggccg catcgtcaac atctcgtcgg tgaacgggca 4380

gaagggccag ttcggccaga ccaactactc caccgccaag gccggcctgc atggcttcac 4440

catggcactg gcgcaggaag tggcgaccaa gggcgtgacc gtcaacacgg tctctccggg 4500

ctatatcgcc accgacatgg tcaaggcgat ccgccaggac gtgctcgaca agatcgtcgc 4560

gacgatcccg gtcaagcgcc tgggcctgcc ggaagagatc gcctcgatct gcgcctggtt 4620

gtcgtcggag gagtccggtt tctcgaccgg cgccgacttc tcgctcaacg gcggcctgca 4680

tatgggctga cctgccggcc tggttcaacc agtcggcagc cggcgctggc gcccgcgtat 4740

tgcggtgcag ccagcgcggc gcacaaggcg gcgggcgttt cgtttcgccg cccgtttcgc 4800

gggccgtcaa ggcccgcgaa tcgtttctgc ccgcgcggca ttcctcgctt tttgcgccaa 4860

ttcaccgggt tttccttaag ccccgtcgct tttcttagtg ccttgttggg catagaatca 4920

gggcagcggc gcagccagca ccatgttcgt gcagcgcggc cctcgcgggg gcgaggctgc 4980

aggccgccac gcgcagccat gcgcgaacgg gccaccagat ggccggcacg acaacaagca 5040

gatggcgcgg gcgataccga tttgcgcact gcaccccatg cggtgcagca gcgcgcaaac 5100

agcgatgaca caaggacaga gcaccgatgg ccacgaccaa aaaaggcgca gagcgactga 5160

tcaaaaagta tccgaaccgt aggctctacg acacccagac cagcacctac atcaccctgg 5220

ccgacgtcaa gcagctggtc atggattcag aag 5253

Claims

1. A genetically engineered bacterium for co-production of 1, 3-propylene glycol and polyhydroxybutyrate, which is classified and named as Klebsiella pneumoniae Q3-PHB (PHB)Klebsiella pneumoniaeQ3-PHB) with the preservation registration number of CCTCC M20191090.

2. The method of constructing a genetically engineered bacterium according to claim 1, wherein the aldehyde/alcohol dehydrogenase gene, lactate dehydrogenase gene and fumarate reductase gene of Klebsiella pneumoniae ATCC 25955 are deleted and introduced into the bacteriumCupriavidus necatorAnd obtaining the genetic engineering bacteria after the derived phbCBA gene.

3. The construction method according to claim 2, characterized by comprising the steps of:

4. The method according to claim 3, wherein the recombinant Klebsiella pneumoniae deleted three genes in step (1) is constructed by the following method: homologous recombination is carried out on the upstream and downstream homologous arms of the Klebsiella pneumoniae aldehyde/alcohol dehydrogenase gene and the Klebsiella pneumoniae ATCC 25955 by taking a suicide plasmid pKR6K as a vector, so as to knock out the aldehyde/alcohol dehydrogenase gene; and then, taking a suicide plasmid pKR6K as a vector to carry out homologous recombination on the upstream and downstream homologous arms of the lactate dehydrogenase gene and the upstream and downstream homologous arms of the fumarate reductase gene with the Klebsiella pneumoniae to knock out the lactate dehydrogenase gene and the fumarate reductase gene.

5. The use of the genetically engineered bacterium of claim 1 for the co-production of 1, 3-propanediol and polyhydroxybutyrate.

6. The use according to claim 5, wherein the genetically engineered bacterium of claim 1 is cultured in a fermentation medium containing glycerol to produce 1, 3-propanediol and polyhydroxybutyrate.

7. The use according to claim 6, wherein the fermentation temperature is 35-39 ℃, the aeration rate is 2.5-3.5L/min, the stirring speed is 200-300rpm, the pH is maintained at 6.3-6.8 during the fermentation process, and the OD is determined as₆₀₀And when the concentration of the glycerol reaches 0.6-0.8, adding IPTG (isopropyl-beta-thiogalactoside) for induction, and in the fermentation process, supplementing the glycerol when the concentration of the glycerol is less than 2 g/L so as to maintain the concentration of the glycerol in the fermentation liquid at 20-30 g/L.

8. Use according to claim 7, characterized in that the fermentation medium contains the following components: k₂HPO₄·3H₂O 5-7g/L，KH₂PO₄1.5-2.5g/L，MgSO₄·7H₂0.25-0.4 g/L O, 4-6 g/L ammonium sulfate, 0.8-1.2 g/L yeast extract, 0.5-0.7 g/L sodium citrate, 15-25 g/L glycerol and 0.8-1.2 ml/L trace element solution, wherein the trace element solution contains CaCl₂·2H₂O 3.2 mg/L， ZnCl₂3.8 mg/L， FeCl₃·6H₂O 30.0 mg/L， MnCl₂·4H₂O11.14 mg/L， CuCl₂·2H₂O 0.96 mg/L， CoCl₂·6H₂O 2.64 mg/L， H₃BO₃0.35 mg/L，Na₂MoPO₄·2H₂O24.5. mu.g/L, chloramphenicol 20ug/m L.

9. The use of claim 8, wherein the seed liquid is inoculated into a fermentation medium for fermentation culture, and the seed liquid medium comprises the following components: k₂HPO₄·3H₂O 6-8g/L，KH₂PO₄1.5-2.5g/L，MgSO₄·7H₂0.05-0.15 g/L of O, 6.5-7.5 g/L of yeast extract, 1-1.5 g/L of ammonium sulfate, 18-22 g/L of glycerol and 0.8-1.2 ml/L of trace element solution, wherein the trace element solution contains CaCl₂·2H₂O 3.2 mg/L， ZnCl₂3.8 mg/L，FeCl₃·6H₂O 30.0 mg/L，MnCl₂·4H₂O 11.14 mg/L，CuCl₂·2H₂O 0.96 mg/L，CoCl₂·6H₂O 2.64 mg/L，H₃BO₃0.35mg/L，Na₂MoPO₄·2H₂O24.5. mu.g/L, chloramphenicol 25. mu.g/m L.