CN114015633A - Method for synthesizing propionic acid by using threonine and recombinant bacterium used in method - Google Patents

Abstract

The invention discloses a recombinant bacterium which expresses 2-ketoisovalerate dehydrogenase (also called ketoacid decarboxylase). The recombinant bacterium is obtained by modifying the genome of a receptor bacterium, wherein the modification comprises the step of enabling the receptor bacterium to express the 2-ketoisovalerate deacase, and the receptor bacterium is pseudomonas or escherichia coli. The approach for synthesizing the propionic acid by the recombinant bacteria is to synthesize the propionic acid by catalyzing 2-ketobutyric acid by 2-ketoisovalerate deacidification enzyme. Provides a new metabolic pathway for propionic acid synthesis. The conversion rate of the pseudomonas recombinant strain PS32 obtained by transformation in the invention for catalyzing threonine to synthesize propionic acid for 24 hours is 96.67%; the conversion of threonine to propionic acid catalyzed by PS32 after feeding may be 98.47%. The recombinant Escherichia coli obtained by transformation in the invention can be transformed into 393mM of propionate L-threonine with the concentration of 400mM in 24 hours, and the transformation rate is 98.25%.

Description

Method for synthesizing propionic acid by using threonine and recombinant bacterium used in method

Technical Field

The invention relates to the fields of microorganisms and genetic engineering, in particular to a method for synthesizing propionic acid by using threonine and a recombinant bacterium used by the same.

Background

Pseudomonas putida (Pseudomonas putida) is the first gram-negative bacterium recognized as environmentally safe by the Recombinant DNA Committee (RAC) of the United states department of health (1982), and permits KT2440 as a genetically engineered host bacterium. Pseudomonas putida KT2440(P.putida KT2440) can decompose some organic matters in the environment, has the functions of biocatalysis, biological pollution discharge and the like, and is a microorganism with extremely high industrial catalytic value.

Coli is one of the most common biocatalytic hosts at present, and mass production of various biochemical products is realized, but the production of propionic acid by using the e coli is not common.

The propionic acid and the derivatives thereof have wide industrial application fields, and are mainly applied to food preservation, feed storage, synthesis of medical intermediates, synthesis of agricultural herbicides, organic synthesis intermediates and the like. At present, propionic acid is produced mainly by utilizing the anaerobic fermentation of propionibacterium, the fermentation period is generally more than 10 days, and byproducts, such as acetic acid, succinic acid and other impurity organic acids, have the defects of rigorous reaction conditions, difficulty in separation and purification and the like. The problems of reducing the production cost and improving the conversion rate are urgently needed to be solved in the industrial production of the propionic acid.

L-threonine is an essential amino acid, is mainly prepared by a microbial fermentation method, has a mature production process at present, and has excessive risks in global production capacity. Comprehensively analyzes the metabolic pathway of the pseudomonas putida KT2440, utilizes the L-threonine to produce high-purity propionic acid with higher added value, and has wider market and value. In previous patents, L-threonine has been explored as a substrate for the catalytic production of 2-ketobutyrate followed by the production of propionyl CoA in the presence of coenzyme CoA to further produce propionic acid.

Disclosure of Invention

The invention aims to provide a new construction way to obtain a recombinant bacterium, and the recombinant bacterium can be used for catalyzing threonine to obtain propionic acid.

In order to achieve the above objects, in a first aspect, the present invention provides a recombinant bacterium expressing 2-ketoisovalerate dehydrogenase (also called ketoacid decarboxylase).

The 2-ketoisovalerate deacidification enzyme may be selected from any of the following proteins:

A1) protein coded by DNA molecule with the coding sequence of the coding chain as shown in SEQ ID No. 3;

A2) a protein having an identity of 80% or more to the protein represented by A1) and having 2-ketoisovalerate deacidification enzyme activity, which is obtained by substituting and/or deleting and/or adding amino acid residues in the protein represented by A1);

A3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2).

Further, the recombinant bacterium can be obtained by modifying the genome of a recipient bacterium, wherein the modification comprises the step of enabling the recipient bacterium to express the 2-ketoisovalerate deacase, and the recipient bacterium can be pseudomonas or escherichia coli.

Furthermore, in the recombinant bacterium, the expression of the 2-ketoisovalerate deacetylase by the recipient bacterium can be realized by introducing a gene kivD encoding the 2-ketoisovalerate deacetylase into the recipient bacterium.

kivD may be any of g1) -g 3):

g1) the coding sequence of the coding chain is shown as SEQ ID No. 3;

g2) the nucleotide sequence of the coding chain is shown as SEQ ID No. 3;

g3) a nucleotide sequence which has 80% or more identity with g1) or g2) and encodes the 2-ketoisovalerate deacetylase.

Further, in the above recombinant bacterium, the recipient bacterium may be pseudomonas, and the modification may include:

a1) inhibiting or reducing threonine aldolase activity in the pseudomonas genome;

a2) inhibiting or reducing the activity of methyl citrate synthase in the pseudomonas genome;

a3) inhibiting or reducing the activity of propionyl-coa synthetase in the pseudomonas genome;

a4) increasing or enhancing the activity of a threonine transporter in the pseudomonas;

a5) increasing or enhancing the activity of threonine deaminase in said Pseudomonas sp.

Further, in the above recombinant bacterium, the threonine transporter in a4) may be selected from any one of the following proteins:

B1) a protein encoded by a DNA molecule whose coding sequence is represented by nucleotide 617-1648 of SEQ ID No. 1;

B2) a protein having threonine transporter activity, which is obtained by substituting and/or deleting and/or adding amino acid residues to the protein represented by B1), and which has an identity of 80% or more to the protein represented by B1);

B3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of B1) or B2).

a5) The threonine deaminase of (1) can be selected from any one of the following proteins:

C1) a protein coded by a DNA molecule with the coding sequence of the coding chain as shown in the 617-2161 th nucleotide of SEQ ID No. 2;

C2) a protein having threonine deaminase activity, which is identical to or higher than 80% to the protein represented by C1) and which is obtained by substituting and/or deleting and/or adding an amino acid residue to the protein represented by C1);

C3) a fusion protein obtained by connecting protein tags at the N-terminal or/and C-terminal of C1) or C2).

In the recombinant bacterium, the coding gene of threonine aldolase is an ltaE gene, the coding gene of methyl citrate synthase is an ilvA gene, the coding gene of propionyl-CoA synthetase is a prpE gene, the coding gene of threonine transport enzyme is a tdcC gene, and the coding gene of threonine deaminase is an ilvA gene.

Further, in the recombinant bacterium described above, in a1), the inhibition or reduction of the activity of threonine aldolase in the pseudomonas genome can be achieved by gene knockout or gene silencing of a gene encoding threonine aldolase named ltaE gene in the pseudomonas genome;

in the a2), the inhibiting or reducing the activity of the methyl citrate synthetase in the pseudomonas genome can be realized by gene knockout or gene silencing of a gene encoding the methyl citrate synthetase named as a prpC gene in the pseudomonas genome;

said a3), said inhibiting or reducing the activity of propionyl-coa synthetase in the pseudomonas genome can be a gene knock-out or gene silencing of the gene encoding propionyl-coa synthetase designated as the prpE gene in the pseudomonas genome;

in the a4), the improvement or enhancement of the activity of threonine transporter in pseudomonas may be the introduction of a gene encoding the threonine transporter, named tdcC gene, and a promoter thereof into pseudomonas;

in the a5), the improvement or enhancement of the activity of threonine deaminase of pseudomonas may be achieved by introducing a gene coding for threonine deaminase named ilvA gene and a promoter thereof into pseudomonas.

Further, the recombinant bacterium described above, D1) may be modified by replacing the ltaE gene in the pseudomonas genome with a fragment containing the ilvA gene and its promoter to achieve the a1) and the a5), and/or D2) may be modified by replacing the prpC gene in the pseudomonas genome with a fragment containing the tdcC gene and its promoter to achieve the a2) and the a 4).

Further, in the recombinant bacterium, the tdcC gene may be any one of g4) -g 6):

g4) the coding sequence of the coding strand is shown as the 617-1648 th site of SEQ ID No. 1;

g5) the nucleotide sequence of the coding strand is shown as the 617-1648 th site of SEQ ID No. 1;

g6) a nucleotide sequence which has 80% or more identity to g4) or g5) and encodes the threonine transporter.

Further, in the recombinant bacterium, the ilvA gene may be any one of g7) to g 9):

g7) the coding sequence of the coding chain is shown as the 617-2161 th site of SEQ ID No. 2;

g8) the nucleotide sequence of the coding strand is shown as 617-2161 bit of SEQ ID No. 2;

g9) nucleotide sequence which has more than 80% of identity with g7) or g8) and codes the threonine deaminase.

Furthermore, in the above recombinant bacteria, the tdcC gene and its promoter can be DNA molecules with nucleotide sequences as shown in the 501 st-1648 th site of SEQ ID No. 1; and/or the ilvA gene and the promoter thereof can be DNA molecules with nucleotide sequences shown in the 501 st-2161 th site of SEQ ID No. 2.

Further, in the recombinant bacterium, the segment containing the tdcC gene and the promoter thereof can be a DNA molecule with a nucleotide sequence shown as SEQ ID No. 1; and/or the segment containing the ilvA gene and the promoter thereof can be a DNA molecule with a nucleotide sequence shown as SEQ ID No. 2.

Herein, the pseudomonas putida may be pseudomonas putida KT 2440.

Herein, the threonine aldolase may be a protein encoded by an ltaE Gene, and the threonine aldolase Gene may be ltaE (Gene ID: 1044018, discontinated on 2-Apr-2020; genome: NC-002947.4: c 386535-385495).

Herein, the methyl citrate synthetase can be a protein encoded by a prpC Gene, which can be prpC (Gene ID:1045332, discontinated on 2-Apr-2020; genome: NC-002947.4: c2662848-2663975,20200402).

Here, the propionyl-CoA synthetase may be a protein encoded by a prpE Gene, which may be prpE (Gene ID:1045356, discontinated on 2-Apr-2020; genome NC-002947.4: c 2681435-2683324).

Further, in the above recombinant bacterium, the recipient bacterium may be escherichia coli, and the modification may include:

b1) inhibiting or reducing the activity of threonine aldolase in the genome of E.coli;

b2) inhibiting or reducing the activity of propionyl-coa synthetase in the genome of escherichia coli;

b3) increasing or enhancing the activity of threonine deaminase in said E.coli;

b4) increasing or enhancing the activity of aldehyde dehydrogenase in said E.coli.

Further, the threonine deaminase of b3) is selected from any one of the following proteins:

B1) a protein coded by a DNA molecule with the coding sequence of the coding chain as shown in the 617-2161 th nucleotide of SEQ ID No. 2;

B2) a protein having threonine transporter activity, which is obtained by substituting and/or deleting and/or adding amino acid residues to the protein represented by B1), and which has an identity of 80% or more to the protein represented by B1);

B3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of B1) or B2).

b4) The aldehyde dehydrogenase is selected from any one of the following proteins:

E1) protein coded by DNA molecule with the coding sequence of the coding chain as shown in SEQ ID No. 4;

E2) a protein having threonine transporter activity, which is obtained by substituting and/or deleting and/or adding amino acid residues to the protein represented by E1), and which has an identity of 80% or more to the protein represented by E1);

E3) a fusion protein obtained by attaching a protein tag to the N-terminus or/and C-terminus of E1) or E2).

Further, in the recombinant bacterium, in b1), the inhibiting or reducing the activity of threonine aldolase in the escherichia coli genome can be realized by gene knockout or gene silencing of a coding gene named ltaE gene threonine aldolase in the escherichia coli genome;

said b2), said inhibiting or reducing the activity of propionyl-CoA synthetase in the genome of E.coli; the gene of the coding gene of propionyl coenzyme A synthetase named as prpE gene in the genome of the Escherichia coli can be subjected to gene knockout or gene silencing;

in b3), the improvement or enhancement of the activity of threonine deaminase in the genome of escherichia coli may be the introduction of a gene encoding the threonine transporter named ilvA gene and its promoter into escherichia coli;

in the b4), the increasing or enhancing the activity of the aldehyde dehydrogenase in the genome of escherichia coli may be introducing a gene encoding the aldehyde dehydrogenase, named PpadH gene, into escherichia coli.

Further, in the recombinant bacterium, the ilvA gene in b3) may be any one of g7) to g 9):

g7) the coding sequence of the coding chain is shown as the 617-2161 th site of SEQ ID No. 2;

g8) the nucleotide sequence of the coding strand is shown as 617-2161 bit of SEQ ID No. 2;

g9) nucleotide sequence which has more than 80% of identity with g7) or g8) and codes the threonine deaminase.

The coding sequence of the coding strand of the PpadH gene in b4 may be any one of g10) -g 12):

g10) the coding sequence of the coding chain is shown as SEQ ID No. 4;

g11) the nucleotide sequence of the coding chain is shown as SEQ ID No. 4;

g12) a nucleotide sequence which has 80% or more identity to g10) or g11) and encodes the above aldehyde dehydrogenase.

Herein, the escherichia coli may be escherichia coli MG 1655.

The threonine aldolase of E.coli May be a protein encoded by the ltaE Gene, which May be ltaE (Gene ID:944955, updated on 6-May-2021; genome: NC-000913.3: c 909294-908293).

Here, the propionyl-CoA synthetase of Escherichia coli May be a protein encoded by a prpE Gene, which May be prpE (Gene ID:946891, updated on 6-May-2021; genome: NC-000913.3: c 352706-354592).

In order to achieve the above object, in a second aspect, the present invention provides a method for preparing a recombinant bacterium, which may include M1) or/and M2):

m1) transforming pseudomonas according to the method to obtain recombinant bacteria;

m2) transforming the escherichia coli according to the method to obtain the recombinant bacteria.

Wherein, the protein coded by the DNA molecule shown in SEQ ID No.3 consists of 548 amino acid residues; the protein encoded by the DNA molecule represented by the 617-1648 th nucleotide of SEQ ID No.1 consists of 343 amino acid residues; the protein coded by the DNA molecule shown as 617-2161 th nucleotide of SEQ ID No.2 consists of 514 amino acid residues; the protein encoded by the DNA molecule shown in SEQ ID No.4 consists of 497 amino acid residues.

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

The protein-tag refers to a polypeptide or protein which is expressed by fusion with a target protein by using a DNA in vitro recombination technology so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag, and/or a SUMO protein tag, etc.

Table 1: sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG
	8	DYKDDDDK
			Strep-tag II	8	WSHPQFEK
c-myc	10	EQKLISEEDL

In the recombinant bacteria, identity refers to identity of amino acid sequences or nucleotide sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the recombinant bacterium, the 80% or more identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

In order to achieve the above object, the present invention provides, in a third aspect, use of the above recombinant bacterium for producing or producing propionic acid using threonine.

In order to achieve the above object, in a third aspect, the present invention provides a method for producing propionic acid, comprising the steps of: and catalyzing threonine by using the recombinant bacteria to obtain propionic acid.

In the invention, the direct product synthesized by converting the recombinant bacteria by using threonine is propionate, and the propionate can be calcium propionate, ammonium propionate or sodium propionate.

The propionate can be reacted to produce pure propionic acid, and can also be directly used in the form of propionate.

In the content measurement, the content measurement method of the propionic acid and the propionate is the same.

In the invention, the pseudomonas can be pseudomonas putida, such as pseudomonas putida KT 2440.

In the present invention, the Escherichia coli may be Escherichia coli MG 1655.

In the invention, the threonine aldolase plays a role in catalyzing threonine to synthesize glycine, and the purpose of knocking out threonine aldolase coding gene ltaE is to block a pathway from threonine to glycine.

In the invention, the function of the methyl citrate synthetase is to catalyze propionic acid coenzyme A to 2-methyl isocitrate, and the aim of knocking out the prpC encoding gene of the methyl citrate synthetase is to block the degradation of the branch path of the propionic acid coenzyme A to 2-methyl isocitrate.

In the invention, the propionyl-CoA synthetase is used for catalyzing propionic acid to propionic acid CoA, and the purpose of knocking out the coding gene prpE of the propionyl-CoA synthetase is to block the synthetic pathway of propionic acid to propionic acid CoA.

In the present invention, the threonine transporter functions to positively regulate the transport of threonine through a membrane, and the threonine transporter-encoding gene tdcC is introduced for the purpose of enhancing the ability of threonine to transport through a membrane.

In the present invention, the threonine transporter encoding gene is derived from Escherichia coli.

In the invention, the threonine deaminase plays a role in catalyzing the dehydration and decomposition of threonine into ammonia and 2-ketobutyrate (also called alpha-ketobutyrate), and the purpose of introducing the threonine deaminase coding gene ilvA is to improve the capability of thalli to catalyze the dehydration and generation of 2-ketobutyrate.

In the present invention, the coding gene ilvA of threonine transporter is derived from Escherichia coli.

In the invention, the 2-ketoisovalerate deacase has the effect of realizing the decarboxylation of 2-ketobutyrate to synthesize propionaldehyde, and the purpose of introducing the 2-ketoisovalerate deacase coding gene kivD is to improve the capability of thalli to catalyze the decarboxylation of 2-ketobutyrate to synthesize propionaldehyde.

In the invention, the 2-ketoisovalerate deacidification enzyme coding gene kivD is from lactococcus lactis.

In the present invention, the aldehyde dehydrogenase (Ppadh) is used to dehydrogenate propionaldehyde to form propionic acid.

In the present invention, the aldehyde dehydrogenase (Ppadh) is derived from pseudomonas putida.

In the recombinant bacterium, the ilvA gene promoter may be any one of lac promoter, lacUV5 mutation, tac promoter and trc promoter.

In the recombinant bacterium, the promoter of the tdcBC gene may be any one of a lac promoter, a lacUV5 mutation, a tac promoter and a trc promoter.

The invention has the beneficial effects that:

the pyruvate decarboxylase and the branched ketoacid decarboxylase are utilized to catalyze the 2-ketobutyrate to generate the propionaldehyde without the participation of coenzyme CoA in the reaction process. The approach for synthesizing propionic acid by the recombinant bacteria is to synthesize propionic acid by catalyzing 2-ketobutyric acid with 2-ketoisovalerate deacidification enzyme, and provides a new metabolic approach for synthesizing propionic acid (as shown in figure 1 and figure 2).

The conversion rate of the pseudomonas recombinant strain PS32 obtained by transformation in the invention for catalyzing threonine to synthesize propionic acid for 24 hours is 96.67%; the conversion of threonine to propionic acid catalyzed by PS32 after feeding may be 98.47%. The recombinant Escherichia coli obtained by transformation in the invention can be transformed into 393mM of propionate L-threonine with the concentration of 400mM in 24 hours, and the transformation rate is 98.25%.

Drawings

FIG. 1 shows the synthesis of propionate from L-threonine with a keto acid decarboxylase, 2-ketoisovalerate deacetylase.

FIG. 2 is a schematic diagram of recombinant bacterium transformation.

FIG. 3 shows the yield of propionic acid synthesized by recombinant Pseudomonas using threonine as a substrate.

FIG. 4 shows the yield of propionic acid synthesized by recombinant Escherichia coli using threonine as a substrate.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Coli S17-1 is competent (Shanghai Weidi Biotechnology Co.Ltd. CAT #: DL 2010).

Plasmid pUCP18 was purchased from Biovector under the trade name Cloning vector pUCP 18).

TABLE 2 primer sequence Listing used in this patent

The way for synthesizing the propionic acid by the recombinant bacteria is to synthesize the propionic acid by catalyzing 2-ketobutyric acid with 2-ketoisovalerate deacidification enzyme, and provides a new metabolic way for synthesizing the propionic acid (as shown in figure 1). The pyruvate decarboxylase and the branched ketoacid decarboxylase are utilized to catalyze the 2-ketobutyrate to generate the propionaldehyde without the participation of coenzyme CoA in the reaction process. Recipient bacteria were engineered according to the schematic shown in FIG. 2.

Example 1 preparation of recombinant bacterium PS30

1.1 deletion of threonine Aldolase ltaE

Starting from Pseudomonas putida KT2440(ATCC 47054) strain, the threonine aldolase Gene ltaE (Gene ID: 1044018, discontinated on 2-Apr-2020; genome: NC-002947.4: c386535-385495) is knocked out by a homologous recombination method, and the pathway from threonine to glycine is blocked. The amino acid sequence of threonine aldolase is shown in NCBI as WP _010951681.1 (2017-5-14). The method comprises the following specific steps:

1.1.1 construction of knock-out plasmid pK18-ltaE

Using pK18 plasmid (ATCC 87097) as a template, BamHI and HindIII restriction enzymes were used for cleavage to obtain a 6022bp DNA linearized fragment.

500bp (1-500 bit of SEQ ID No.2 and 2163-2662 bit of SEQ ID No. 2) of the upstream and downstream of the ltaE gene are selected, primers ltaE-up-F/ltaE-up-R and ltaE-down-F/ltaE-down-R are designed, genome DNA of pseudomonas putida KT2440 is taken as a template, the primers are respectively used for PCR amplification, and 500bp upstream and downstream knockout homologous arm DNA fragments are respectively obtained.

A500 bp upstream knockout homology arm DNA fragment, a 500bp downstream knockout homology arm DNA fragment and a 6022bp pK18 plasmid DNA linear fragment are connected together by using a Gibson method (Gibson DG, Young L, Chuang RY, et al. enzymic assembly of DNA molecules up to segmented cloned plasmid Meth,2009,6(5): 343-345.), transformed Escherichia coli DH5 alpha (trans biotech, CD201-01) is identified by using a primer pK18-F/pK18-R, and a positive clone with a correct target fragment sequence is selected to extract a plasmid pK 18-ltaE.

1.1.2 transformation of plasmid pK18-ltaE into KT2440 by the use of the Combined transduction technique

Transforming the plasmid pK18-ltaE into an Escherichia coli S17-1 competent cell to obtain a strain named S17-1/pK 18-ltaE;

the S17-1/pK18-ltaE strain and the pseudomonas putida KT2240 are respectively inoculated into LB liquid culture medium and activated at 37 ℃ overnight. 1mL of the bacterial solution was collected, centrifuged, and the supernatant was discarded and resuspended in 500. mu.L of LB liquid. Dropping 10 μ L of Pseudomonas putida liquid on a nonresistant LB solid medium, and air-drying. A15-microliter of S17-1/pK18-ltaE bacterial solution is covered on KT2440 and dried. Culturing at 37 deg.C for 12 h.

1.1.3 two homologous recombinations

And scraping the mixed colonies by using a coating rod, uniformly coating the mixed colonies on an LB solid culture medium containing final concentration of 50 micrograms/ml gentamicin and 25 micrograms/ml chlorophenol, and inversely culturing the mixed colonies at 37 ℃ for 18 hours to obtain grown colonies which are strains with pK18-ltaE after the first homologous recombination is completed. And selecting a single colony, streaking the single colony on an LB solid culture medium containing 25 microgram/milliliter chlorophenol and 200g/L sucrose at the final concentration, and culturing at 37 ℃ until the single colony grows out, wherein the grown-out colony is a knockout strain which completes the second homologous recombination. And (3) carrying out PCR verification by using an ltaE-yz-F/ltaE-yz-R primer to obtain a 1040bp fragment single colony which is a strain for completing ltaE knockout, and selecting the strain with a positive knockout result and naming the strain as KT2440 delta ltaE.

1.2 knock-out of the methyl citrate synthetase Gene prpC

Based on KT2440 delta ltaE, a homologous recombination method is utilized to knock out a methyl citrate synthetase Gene prpC (Gene ID:1045332, discontinated on 2-Apr-2020; genome: NC-002947.4: c2662848-2663975), and degradation of propionic acid coenzyme A to a 2-methyl isocitrate branch path is blocked. The amino acid sequence of the methyl citrate synthetase is shown in NCBI as WP-003250313.1 (2019-5-31). The method comprises the following specific steps:

1.2.1 construction of knock-out plasmid pK18-prpC

Cutting by using BamHI and HindIII restriction enzymes with pK18 plasmid as a template to obtain a 6022bp DNA linearized fragment;

selecting 500bp (1-500 bit of SEQ ID No.1 and 1649-2148 bit of SEQ ID No. 1) at the upstream and downstream of the prpC gene, designing primers prpC-up-F/prpC-up-R and prpC-down-F/prpC-down-R, taking genome DNA of pseudomonas putida KT2440 as a template, and respectively carrying out PCR amplification by using the primers to respectively obtain 500bp upstream and downstream knockout homologous arm DNA fragments;

connecting the 500bp upstream knockout homologous arm DNA fragment, the 500bp downstream knockout homologous arm DNA fragment and the 6022bp pK18 plasmid DNA linear fragment together by using a Gibson method, transforming Escherichia coli DH5 alpha (transgen biotech, CD201-01), identifying by using a primer pK18-F/pK18-R, selecting a positive clone with a correct target fragment sequence, extracting a plasmid, and obtaining a knockout plasmid pK 18-prpC.

1.2.2 transfer of plasmid pK18-prpC into KT 2440. delta. ltaE using the Combined transduction technique

Transforming the plasmid pK18-prpC into S17-1 competence to obtain a strain of S17-1/pK 18-prpC;

the S17-1/pK18-prpC strain and KT 2440. delta. ltaE were inoculated into LB liquid medium, respectively, and activated overnight at 37 ℃. 1mL of the bacterial solution was collected, centrifuged, and the supernatant was discarded and resuspended in 500. mu.L of LB liquid. 10 mu L of KT2440 delta ltaE bacterial liquid is spotted on an antibiotic-free LB solid culture medium and dried in the air. A spot of 15. mu.L of S17-1/pK18-prpC bacterial solution was overlaid on KT 2440. delta. ltaE, and air-dried. Culturing at 37 deg.C for 12 h.

1.2.3 two homologous recombinations

And scraping the mixed colony by using a coating rod, uniformly coating the mixed colony on an LB solid culture medium containing final concentration of 50 micrograms/milliliter of gentamicin and 25 micrograms/milliliter of chlorophenol, and inversely culturing the mixed colony at 37 ℃ for 18 hours to obtain a colony which is a strain with pK18-prpC after the first homologous recombination is completed. And selecting a single colony, streaking the single colony on an LB solid culture medium containing 25 microgram/milliliter chlorophenol and 200g/L sucrose at the final concentration, and culturing at 37 ℃ until the single colony grows out, wherein the grown-out colony is a knockout strain which completes the second homologous recombination. PCR verification is carried out by using a prpC-yz-F/prpC-yz-R primer, a single colony of a 1040bp fragment is a bacterial strain for completing prpC knockout, and a bacterial strain with a positive knockout result is selected and named as KT2440 delta ltaE delta prpC.

1.3 knock-out of propionyl-CoA synthetase prpE

Starting from a KT2440 delta ltaE delta prpC strain, a propionyl coenzyme A synthetase Gene prpE (Gene ID:1045356, discontinated on 2-Apr-2020; genome NC-002947.4: c2681435-2683324) is knocked out by a homologous recombination method, and the synthetic pathway from propionic acid to propionic acid coenzyme A is blocked. The amino acid sequence of propionyl-CoA synthetase is shown in NCBI as WP _010953311.1 (2019-7-27). The method comprises the following specific steps:

1.3.1 construction of knock-out plasmid pK18-prpE

Cutting by using BamHI and HindIII restriction enzymes with pK18 plasmid as a template to obtain a 6022bp DNA linearized fragment;

selecting 500bp upstream and downstream of the prpE gene, designing primers prpE-up-F/prpE-up-R and prpE-down-F/prpE-down-R, respectively using genome DNA of pseudomonas putida KT2440 as a template, and respectively carrying out PCR amplification by using the primers to respectively obtain a 500bp upstream knockout homologous arm DNA fragment and a 500bp downstream knockout homologous arm DNA fragment (nucleotide sequences are respectively shown in 1-500 and 501-1000 bits of SEQ ID No. 5);

the DNA fragment of the 500bp upstream knockout homologous arm, the DNA fragment of the 500bp downstream knockout homologous arm and the pK18 plasmid DNA linear fragment of 6022bp are connected together by a Gibson method, transformed Escherichia coli DH5 alpha (Transgen biotech, CD201-01) is transformed, identified by a primer pK18-F/pK18-R, and a positive clone with a correct sequence of a target fragment is selected to extract a plasmid, so that a knockout plasmid pK18-prpE is obtained.

1.3.2 transformation of plasmid pK18-prpE into KT 2440. delta. ltaE. delta. prpC by means of the Combined transduction technique

Transforming the plasmid pK18-prpE into S17-1 competence to obtain a strain of S17-1/pK 18-prpE;

the S17-1/pK18-prpE strain and KT 2440. delta. ltaE. delta. prpC were inoculated into LB liquid medium, respectively, and activated overnight at 37 ℃. 1mL of the bacterial solution was collected, centrifuged, and the supernatant was discarded and resuspended in 500. mu.L of LB liquid. 10 mu L of KT 2440. delta. ltaE. delta. prpC bacterial solution was spotted on an antibiotic-free LB solid medium, and air-dried. A spot of 15. mu.L of S17-1/pK18-prpE bacterial suspension was overlaid on KT 2440. delta. ltaE. delta. prpC, and air-dried. Culturing at 37 deg.C for 12 h.

1.3.3 two homologous recombination

And scraping the mixed colony by using a coating rod, uniformly coating the mixed colony on an LB solid culture medium containing final concentration of 50 micrograms/ml gentamicin and 25 micrograms/ml chlorophenol, and inversely culturing the mixed colony at 37 ℃ for 18 hours to obtain a colony which is a strain with pK18-prpE after the first homologous recombination is completed. And selecting a single colony, streaking the single colony on an LB solid culture medium containing 25 microgram/milliliter chlorophenol and 200g/L sucrose at the final concentration, and culturing at 37 ℃ until the single colony grows out, wherein the grown-out colony is a knockout strain which completes the second homologous recombination. PCR verification is carried out by using a prpE-yz-F/prpE-yz-R primer, a single colony of 1040bp fragment is a bacterial strain for completing the prpE knockout, a bacterial strain with positive knockout result is selected and named as KT2440 delta ltaE delta prpC delta prpE, and the bacterial strain is marked as PS 30.

Example 2 preparation of recombinant bacterium PS31

2.1 genome overexpression of the threonine deaminase Gene ilvA derived from Escherichia coli

Amino acid sequences of threonine deaminases of Escherichia coli (Escherichia coli) origin are as described in EEV5737228.1(20200406) in NCBI. The preparation steps are as follows:

2.1.1 construction of over-expressed ilvA plasmid

Genomic DNA was extracted from E.coli (ATCC 700926) and ilvA gene was amplified using the primers ilvA-F/ilvA-R to obtain a 1545bp DNA fragment (the nucleotide sequence of the coding strand is shown in SEQ ID No.2 at position 617-2161). Plasmid pUCP18(Biovector, Cloning vector pUCP18) was cleaved with restriction enzymes BamHI and HindIII to give a 5465bp DNA linearized fragment. The 1545bp DNA fragment ilvA and the 5465bp pUCP18 plasmid DNA linear fragment are connected together by a Gibson method, transformed into Escherichia coli DH5 alpha (transgen biotech, CD201-01), identified by a primer pUCP18-F/pUCP18-R, and a positive clone with correct target fragment sequence is selected to extract a plasmid, so that an over-expression plasmid pUCP18-ilvA is obtained.

2.1.2 insertion integration of lac promoter and ilvA Gene into the genome

Starting from HS30 strain, a lac promoter and an ilvA gene derived from escherichia coli are inserted and integrated into the ltaE site of the PS30 genome by a homologous recombination method, so that a pathway from threonine to 2-ketobutyrate is enhanced. The method comprises the following specific steps:

2.1.2.1 construction of the integration plasmid pK 18-ilvA::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::

designing a primer pK18-ltaE-F/pK18-ltaE-R for PCR amplification by taking pK18-ltaE plasmid as a template to obtain a 7022bp plasmid DNA linear fragment; using plasmid pUCP18-ilvA as a template, designing a primer lac-ilvA-F/lac-ilvA-R for PCR amplification to obtain a DNA fragment (nucleotide sequence is shown as 501-2161 bit of SEQ ID No. 2) with the size of 1661 bp; the two DNA fragments are connected together by a Gibson method, transformed into Escherichia coli DH5 alpha (trans biotech, CD201-01), identified by a primer pK18-F/pK18-R, and a positive clone with correct target fragment sequence is selected to extract a plasmid, so as to obtain an integrated plasmid pK18-ilvA:: ltaE.

2.1.2.2, transferring the plasmid pK18-ilvA into the original strain PS30 by utilizing the combined transduction technology

Transforming the plasmid pK18-ilvA:: ltaE into S17-1 competence to obtain a strain of S17-1/pK18-ilvA:: ltaE;

the S17-1/pK18-ilvA strain and the PS30 strain are respectively inoculated into LB liquid culture medium and activated at 37 ℃ overnight. 1mL of the bacterial solution was collected, centrifuged, and the supernatant was discarded and resuspended in 500. mu.L of LB liquid. mu.L of PS30 bacterial suspension was spotted on an antibiotic-free LB solid medium, and air-dried. 15 mu L of S17-1/pK18-ilvA is added, and the ltaE bacterial solution is covered on PS30 and dried. Culturing at 37 deg.C for 12 h.

2.1.2.3, double homologous recombination

The mixed colony is scraped by a coating rod and evenly coated on LB solid culture medium containing final concentration of 50 micrograms/ml gentamicin and 25 micrograms/ml chlorophenol, the mixture is inversely placed at 37 ℃ for 18 hours of culture, and the grown colony is the strain which completes the first homologous recombination and has pK18-ilvA:: ltaE. And selecting a single colony, streaking the single colony on an LB solid culture medium containing 25 microgram/milliliter chlorophenol and 200g/L sucrose at the final concentration, and culturing at 37 ℃ until the single colony grows out, wherein the grown-out colony is a knockout strain which completes the second homologous recombination. PCR verification is carried out by using ltaE-yz-F/ltaE-yz-R primers, and a single colony of 2701bp fragment (the nucleotide sequence is shown as SEQ ID No. 2) is a strain PS30 delta ltaE:: ilvA which completes lac-ilvA replacement.

2.2 genome overexpression of threonine Transporter Gene tdcC derived from Escherichia coli (Escherichia coli)

Amino acid sequences of threonine transporters from E.coli (Escherichia coli) such as WP _136764442.1(20191013) in NCBI. The preparation steps are as follows:

2.2.1 construction of an overexpression tdcC plasmid

Genomic DNA was extracted from Escherichia coli (ATCC 700926) and tdcC gene was amplified using the primer tdcC-F/tdcC-R to obtain a 1332bp DNA fragment (the nucleotide sequence of the coding strand is shown in SEQ ID No.1, position 617-1948). Plasmid pUCP18(Biovector, Cloning vector pUCP18) was cleaved with restriction enzymes BamHI and HindIII to give a 5465bp DNA linearized fragment. A1332 bp DNA fragment tdcC and a 5465bp pUCP18 plasmid DNA linear fragment are connected together by a Gibson method, escherichia coli DH5 alpha (transgen biotech, CD201-01) is transformed, a positive clone with correct target fragment sequence is selected for identification by a primer pUCP18-F/pUCP18-R, and a plasmid is extracted to obtain an over-expression plasmid pUCP 18-tdcC.

2.2.2 insertion integration of lac promoter and tdcC Gene into the genome

Starting from PS30 DeltaltaE:: ilvA strain, a lac promoter and a tdcC gene from Escherichia coli are inserted and integrated into the prpC site of the PS30 DeltaltaE:: ilvA genome by a homologous recombination method, so that a threonine transport pathway is enhanced. The method comprises the following specific steps:

2.2.2.1 construction of the integration plasmid pK18-tdcC prpC

Designing a primer pK18-prpC-F/pK18-prpC-R by using pK18-prpC plasmid as a template for PCR amplification to obtain a 7022bp plasmid DNA linear fragment; using plasmid pUCP18-tdcC as a template, designing a primer lac-tdcC-F/lac-tdcC-R for PCR amplification to obtain a DNA fragment with the size of 1448bp (the nucleotide sequence of a coding strand is shown as 501-1948 of SEQ ID No. 1); the two DNA fragments are connected together by a Gibson method, transformed into Escherichia coli DH5 alpha (transgen biotech, CD201-01), identified by a primer pK18-F/pK18-R, and a positive clone with correct target fragment sequence is selected to extract a plasmid, so as to obtain an integrated plasmid pK18-tdcC:: prpC.

2.2.2.2.2 transformation of plasmid pK18-tdcC prpC into the starting strain PS30 DeltaltaE ilvA by means of the combined transduction technique

prpC was transformed into S17-1 competence with plasmid pK18-tdcC:, resulting in S17-1/pK 18-tdcC:prpCstrain. S17-1/pK18-tdcC:: prpC strain and PS30 DeltaltaE:: ilvA were inoculated into LB liquid medium respectively and activated overnight at 37 ℃. 1mL of the bacterial solution was collected, centrifuged, and the supernatant was discarded and resuspended in 500. mu.L of LB liquid. mu.L of PS30 DeltaltaE strain solution is added to the LB solid medium without antibiotics, and then dried. 15 mu L of S17-1/pK18-tdcC bacterial liquid is covered on PS30 delta ltaE and ilvA is dried. Culturing at 37 deg.C for 12 h.

2.2.2.3, double homologous recombination

The mixed colony is scraped by a coating rod and evenly coated on LB solid culture medium containing final concentration of 50 micrograms/ml gentamicin and 25 micrograms/ml chlorophenol, and the mixture is inversely placed at 37 ℃ for 18 hours, and the grown colony is the strain which completes the first homologous recombination and has pK18-tdcC:: prpC. And selecting a single colony, streaking the single colony on an LB solid culture medium containing 25 microgram/milliliter chlorophenol and 200g/L sucrose at the final concentration, and culturing at 37 ℃ until the single colony grows out, wherein the grown-out colony is a knockout strain which completes the second homologous recombination. PCR verification was performed using the prpC-yz-F/prpC-yz-R primers to obtain a single colony of the 2488bp fragment (nucleotide sequence of coding strand is shown in SEQ ID No. 1) as strain PS 30. delta. ltaE:: ilvA. delta. prpC:: tdcC, denoted PS31, which completes the lac-tdcC substitution.

Example 3 preparation of recombinant bacterium PS32

3.1 overexpression of 2-ketoisovalerate dehydrogenase derived from Lactococcus lactis

The amino acid sequence of 2-ketoisovalerate deacetylase (2-ketoisovalerate decarboxylase, kivD) derived from Lactococcus lactis (Lactococcus lactis) such as WP _012897921.1(20190728) in NCBI was prepared as follows:

genomic DNA was extracted from lactococcus lactis (NCDO 2118), and the kivD gene was amplified using the primer kivD-F/kivD-R to give a 1647bp DNA fragment (the coding sequence of the coding strand is shown in SEQ ID No. 3). Plasmid pUCP18(Biovector, Cloning vector pUCP18) was cleaved with restriction enzymes BamHI and HindIII to give a 5465bp DNA linearized fragment. Connecting a 1647bp DNA fragment kivD and a 5465bp pUCP18 plasmid DNA linear fragment together by using a Gibson method, transforming Escherichia coli DH5 alpha (trans biotech, CD201-01), identifying by using a primer pUCP18-F/pUCP18-R, selecting a positive clone with correct target fragment sequence, extracting a plasmid, and obtaining an over-expression plasmid pUCP 18-kivD.

3.2 preparation of PS31 electrotransformation competence, transformation of plasmid pUCP18-kivD into PS31

The recombinant strain PS31 is inoculated into LB liquid culture medium and activated at 37 ℃ overnight. Transferring 100 mu L of activated bacteria liquid into 10mL of LB culture medium, culturing at 37 ℃ for 3 hours, centrifuging, removing supernatant, washing 3 times by using 5mL of 100g/L sucrose solution, finally re-suspending by using 300 mu L of 100g/L sucrose solution, completing electric transfer competence preparation, and taking 100 mu L of electric transfer competence for later use.

An appropriate amount of plasmid pUCP18-kivD was placed in PS31 electrotransfer competence, 2.5KV electrotransfer was performed, 1mL of LB medium was added after electrotransfer, incubation was performed at 37 ℃ for 2 hours, then the mixture was spread on LB solid medium containing final concentration of 50. mu.g/mL gentamicin and 25. mu.g/mL chlorophenol, and culture was performed at 37 ℃ for 24 hours. The grown single clone was identified with the primer pUCP18-F/pUCP18-R, the positive clone was designated as PS31/pUCP18-kivD, and the strain was designated as recombinant strain PS 32.

Example 4 Whole-cell catalytic production of propionic acid by recombinant bacteria PS32

4.1 culture of cells

The recombinant strain PS32 was inoculated into LB liquid medium and activated overnight at 37 ℃. Transferring 1mL of the activated bacterial liquid into 100mL of LB liquid culture medium containing gentamicin with the final concentration of 50 micrograms/mL, culturing at 30 ℃ for 24 hours, centrifuging and removing supernatant to obtain the thalli.

4.2 Whole cell catalytic production of propionic acid

The cells collected in step 1 were resuspended in a test tube with 50mM PBS buffer, pH7.0, OD_600nmA value of 30, a bacterial suspension was obtained. Adding L-threonine into the bacterial suspension until the content of L-threonine is 600mM, reacting in a shaking table at 37 ℃ and 150rpm, sampling for 8 hours, 16 hours and 24 hours respectively, centrifuging for 1min at 10000g, taking supernatant, and filtering by using a 0.22 mu m filter to obtain filtrate, namely the sample to be detected.

And (3) quantitatively analyzing the content of propionic acid in the sample to be detected by using propionic acid as a standard substance by using a standard curve method (an external standard method) and using HPLC. HPLC method: aminex HPX-87H column (300X 7.8 mm); diode Array Detectors (DADs); mobile phase: 6mM sulfuric acid; detection wavelength: 210 nm; sample introduction amount: 10 mu L of the solution; flow rate: 0.5 mL/min; column temperature: 35 ℃ is carried out.

600mM L-threonine can be converted to 378mM ammonium propionate (propionate) by strain PS32 for 8 hours, 530mM ammonium propionate (propionate) for 16 hours and 580mM ammonium propionate (propionate) for 24 hours.

Example 5 feeding technology for increasing content of propionic acid produced by recombinant bacterium PS32 whole cell catalysis

5.1 culture and Collection of cells

The recombinant bacterium PS32 was cultured and the cells were collected according to example 4.

5.2 Whole-cell catalytic production of propionic acid with feed supplement Process

The cells collected in step 1 were resuspended in a test tube with 50mM PBS buffer, pH7.0, OD₆₀₀The value was 30. Adding 600mM L-threonine into the bacterial suspension, reacting in a shaking table at 37 ℃ and 150rpm, adding 250mM L-threonine and PS32 thallus with the same initial amount after 22 hours, respectively sampling for 46, 51 and 58 hours, centrifuging for 1min at 10000g, taking supernatant, and filtering by using a 0.22 mu m filter to obtain filtrate, namely the sample to be detected.

850mM L-threonine was converted to 778mM ammonium propionate (propionate) by strain PS 3246 h conversion, to 828mM ammonium propionate (propionate) by 51 h conversion and to 837mM ammonium propionate (propionate) by 58 h conversion.

Example 6 construction of propionic acid Synthesis pathway in E.coli

The metabolic pathway of the escherichia coli is analyzed, and the complete pathway of generating the propionic acid by the L-threonine through CoA can be found, so that the foundation is laid for the 2-ketobutyrate designed by the invention to generate the propionic acid through decarboxylase without the CoA. By knocking out branch competition way, key enzyme is introduced, so that the Escherichia coli MG1655 can generate propionic acid with high efficiency, and no other organic acid is generated.

Reference to the Crispr-Cas knockout method employed "Multigene edition in the Escherichia coli Genome via the CRISPR-Cas9 System"; the pCAS and pTARGET plasmid construction methods in the following examples are described in the references "Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System. applied Environmental microbiology.2015,81(7): 2506-.

6.1 knock-out of threonine aldolase ltaE of Escherichia coli

Threonine aldolase ltaE (Gene ID:944955, updated on 6-May-2021; genome: NC-000913.3: c909294-908293) was knocked out by homologous recombination using Escherichia coli MG1655 as an original strain.

The pCAS plasmid (kanamycin-resistant) was introduced into MG1655(ZOMANBIO, ZC1040-2), and cultured at 30 ℃ for 15 hours to obtain a positive clone MG1655/pCAS, since the pCAS plasmid is a temperature-sensitive plasmid.

The plasmid containing 2118bp of ltaE gene recognition N20 is obtained by carrying out PCR amplification by using pTARGET as a template and pTARGET-ltaE-F/R as a primer, and is named as pTARGET-ltaE, wherein the plasmid contains a coding gene of ltaE gene gRNA, and the nucleotide sequence of N20 is CGCGGCGAAGAGTATATTGT.

Amplifying an ltaE-up-F/R primer and an ltaE-down-F/R primer by taking MG1655 genome DNA as a template to respectively obtain a 500bp ltaE site upstream homology arm and a 500bp ltaE site downstream homology arm; then, overlapping pcr is carried out by taking the upstream and downstream homologous arms as a template, and a 1000bp targeting fragment (the nucleotide sequence is shown as SEQ ID No. 6) containing the upstream and downstream homologous arms of the ltaE gene is obtained by amplification.

Culturing MG1655/pCAS at 30 ℃ for 3h, washing with 10% (V/V) glycerol for 3 times, electrically transferring pTARGET-ltaE plasmid (streptomycin resistance) and a targeting fragment containing upstream and downstream homology arms of the ltaE gene into the plasmid under the condition of 2.5kv, carrying out PCR verification by using kanamycin and streptomycin double-resistance screening to obtain gene knockout or insertion positive clones, and obtaining 1000bp positive clone MG1655/pCAS delta ltaE by using ltaE-up-F and ltaE-down-R primers; the clones were cultured at 42 ℃ for 5 hours, streaked on LB non-resistant plates, and the grown clones were subjected to control verification on LB non-resistant and kanamycin-resistant plates to confirm the elimination of pCAS plasmid. The strain MG 1655/. DELTA.ltaE was obtained.

6.2 knock-out of propionyl-CoA synthetase prpE

Propionyl coenzyme A synthetase prpE (Gene ID:946891, updated on 6-May-2021; genome: NC _000913.3: c352706-354592) is knocked out by a homologous recombination method by taking Escherichia coli MG 1655/. DELTA.ltaE as an initial strain.

The plasmid containing 2118bp and prpE gene recognition N20 is obtained by PCR amplification with pTARGET as a template and pTARGET-prpE-F/R as a primer and is named as pTARGET-prpE, the plasmid contains a coding gene of prpE gene gRNA, and the nucleotide sequence of N20 is TTTACGAAGGATTGCCGACC.

Amplifying the prpE-up-F/R and prpE-down-F/R primers by taking MG1655 genome DNA as a template to respectively obtain a 500bp prpE site upstream homology arm and a 500bp prpE site downstream homology arm; then the upstream and downstream homologous arms are taken as a template to carry out overlap pcr, and 1000bp of targeted fragments containing the upstream and downstream homologous arms of the prpE gene are obtained by amplification (the nucleotide sequence of the coding chain is shown as SEQ ID No. 7).

Culturing MG1655 delta ltaE/pCAS at 30 ℃ for 3h, washing with 10% (V/V) glycerol for 3 times, electrically transferring pTARGET-prpE plasmid (streptomycin resistance) and targeted fragments containing upstream and downstream homologous arms of the prpE gene into the plasmid under the condition of 2.5kv, screening by using kanamycin and streptomycin double resistance, carrying out PCR verification to obtain gene knockout or insertion positive clone, and obtaining 1000bp positive clone MG1655 delta ltaE/pCAS delta prpE; the clones were cultured at 42 ℃ for 5 hours, streaked on LB non-resistant plates, and the grown clones were subjected to control verification on LB non-resistant and kanamycin-resistant plates to confirm the elimination of pCAS plasmid. The strain MG 1655/. DELTA.ltaE.DELTA.prpE was obtained and designated MG 1655. DELTA.2.

6.3 overexpression of threonine deaminase gene ilvA derived from Escherichia coli, overexpression of 2-ketoisovalerate deacidification enzyme kivD derived from lactococcus lactis and aldehyde dehydrogenase Ppadh derived from Pseudomonas putida

Amino acid sequence of threonine deaminase ilvA derived from Escherichia coli (Escherichia coli) such as EEV5737228.1 in NCBI (20200406); amino acid sequences of 2-ketoisovalerate deacetylase (2-ketoisovalerate decarboxylase, kivD) derived from Lactococcus lactis (Lactococcus lactis) such as WP _012897921.1 in NCBI (20190728); amino acid sequence of aldehyde dehydrogenase (Ppadh) derived from Pseudomonas putida such as WP _010955785.1 in NCBI, (20160128)

Genomic DNA was extracted from E.coli (ATCC 700926) and the ilvA gene was amplified using the primer pTrc99a-ilvA-F/pTrc99a-ilvA-R to obtain a 1545bp DNA fragment (nucleotide sequence is shown in 617-2161 of SEQ ID No. 2). Genomic DNA was extracted from lactococcus lactis (NCDO 2118), and the kivD gene was amplified using the primer pTrc99a-kivD-F/pTrc99a-kivD-R to give a 1647bp DNA fragment (nucleotide sequence shown in SEQ ID No. 3). A genome is extracted from pseudomonas putida KT2440(ATCC 47054), and a Ppadh gene is amplified by using a primer pTrc99a-Ppadh-F/pTrc99a-Ppadh-R to obtain a 1494bp DNA fragment (the nucleotide sequence is shown as SEQ ID No. 4). Plasmid pTrc99a (Biovector, Cloning vector pTrc99a) was cleaved with restriction enzymes NcoI and HindIII to obtain a DNA linearized fragment of 4120 bp. The DNA fragments ilvA, kivD, Ppadh and pTrc99a plasmid DNA linear fragments are connected together by a Gibson method, transformed into Escherichia coli DH5 alpha (transgen biotech, CD201-01), identified by primers pTrc99a-F/pTrc99a-R, and a positive clone with correct sequence of the target fragment is selected to extract a plasmid, so that an over-expression plasmid pTrc99a-ilvA-kivD-Ppadh is obtained.

Plasmid pTrc99a-ilvA-kivD-Ppadh was transferred into MG 1655. delta.2 competent cells, heat-shocked at 42 ℃ for 90 seconds, ice-washed for 2 minutes, added with 1mL of LB medium, incubated at 37 ℃ for 1 hour, spread evenly on LB solid medium containing ampicillin at a final concentration of 100. mu.g/mL, and cultured at 37 ℃ for 12 hours. The single clone that grows out is identified by primer pTrc99a-F/pTrc99a-R, and the positive clone is recorded as MG 1655. delta.2/pTrc 99 a-ilvA-kivD-Ppadh.

6.4 recombinant strain MG1655 delta 2/pTrc99a-ilvA-kivD-Ppadh whole-cell catalytic production of propionic acid

The recombinant strain MG1655/pTrc99a-ilvA-kivD-Ppadh was inoculated into LB liquid medium and activated overnight at 37 ℃. Transferring 1mL of the activated bacterial solution into 100mL of LB liquid medium containing 100 microgram/mL of ampicillin at the final concentration, culturing at 37 ℃ for 2 hours, adding IPTG at the final concentration of 1 millimole/liter, culturing at 30 ℃ for 16 hours, centrifuging, and removing supernatant to obtain the bacteria.

The collected cells were resuspended in a test tube with 50mM PBS buffer (pH7.0), OD_600nmA value of 30 resulted in a bacterial suspension. Adding L-threonine into the bacterial suspension until the content of L-threonine is 400mM, reacting in a shaking table at 37 ℃ and 150rpm, sampling for 6, 12, 18 and 24 hours respectively, centrifuging at 10000g for 1min, taking supernatant, and filtering with a 0.22 mu m filter to obtain filtrate, namely the sample to be detected.

And (3) quantitatively analyzing the content of propionic acid in the sample to be detected by using propionic acid as a standard substance by using a standard curve method (an external standard method) and using HPLC. HPLC method: aminex HPX-87H column (300X 7.8 mm); diode Array Detectors (DADs); mobile phase: 6mM sulfuric acid; detection wavelength: 210 nm; sample introduction amount: 10 mu L of the solution; flow rate: 0.5 mL/min; column temperature: 35 ℃ is carried out.

400mM L-threonine were converted to 393mM ammonium propionate (propionate) by transformation of the strain MG 1655. delta.2/pTrc 99a-ilvA-kivD-Ppadh for 46 h. The sampling results in the period show that: conversion to 92mM ammonium propionate (propionate) at 6 hours, 209mM ammonium propionate (propionate) at 12 hours, 298mM ammonium propionate (propionate) at 18 hours, and 393mM ammonium propionate (propionate) at 24 hours (see FIG. 4).

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> institute of microbiology of Chinese academy of sciences

<120> method for synthesizing propionic acid by using threonine and recombinant bacterium used in same

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2448

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgacagctt cgtgatcatg gcgcgtaccg atgcgctggc cgtcgaaggc ctgaacgccg 60

ccctggaccg tgcccaggcg tgtgtcgaag ctggcgccga catgatcttc ccggaagcca 120

tcaccgaact gcagatgtac aagactttcg ctgatcgggt gaaggcaccg atcctggcca 180

acatcaccga gttcggtgcc acgccgctgt acacaaccga agagctggcc tcggtcgacg 240

tgtcgttggt gctgtacccg ctgtcggcgt tccgcgccat gaacaaagca gccgagaacg 300

tgtataccgc gctgcgccgt gacggcacgc aaaagaatgt gatcgacacc atgcagaccc 360

gcatggagct ttacgatgcc attggttatc acgccttcga gcagagcctc gatgcactgt 420

ttgcccagaa gaagggctaa gcgctagcgc ccgaattagc cagaaagcat ccataacaaa 480

ttcaagaaag gagaaacacc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt 540

gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacga attcgagctc 600

ggtacccggg gatcctatga gtacttcaga tagcattgta tccagccaga caaaacaatc 660

gtcctggcgt aaatcagata ccacatggac gttaggcttg tttggtacgg caatcggcgc 720

cggggtgctg ttcttcccta tccgcgcagg ttttggcgga ctgatcccga ttcttctgat 780

gttggtattg gcatacccca tcgcgtttta ttgccaccgg gcgctggcgc gtctgtgtct 840

ttctggctct aacccttccg gcaacattac ggaaacggtg gaagagcatt ttggtaaaac 900

tggcggcgtg gttatcacgt tcctgtactt cttcgcgatt tgcccactgc tgtggattta 960

tggcgttact attaccaata cctttatgac gttctgggaa aaccagctcg gctttgcacc 1020

gctgaatcgc ggctttgtgg cgctgttcct gttgctgctg atggctttcg tcatctggtt 1080

tggtaaggat ctgatggtta aagtgatgag ctacctggta tggccgttta tcgccagcct 1140

ggtgctgatt tctttgtcgc tgatccctta ctggaactct gcagttatcg accaggttga 1200

cctcggttcg ctgtcgttaa ccggtcatga cggtatcctg atcactgtct ggctggggat 1260

ttccatcatg gttttctcct ttaacttctc gccaatcgtc tcttccttcg tggtttctaa 1320

gcgtgaagag tatgagaaag acttcggtcg cgacttcacc gaacgtaaat gttcccaaat 1380

catttctcgt gccagcatgc tgatggttgc agtggtgatg ttctttgcct ttagctgcct 1440

gtttactctg tctccggcca acatggcgga agccaaagcg cagaatattc cagtgctttc 1500

ttatctggct aaccactttg cgtccatgac cggtaccaaa acaacgttcg cgattacact 1560

ggaatatgcg gcttccatca tcgcactcgt ggctatcttc aaatctttct tcggtcacta 1620

tctgggaacg ctggaaggtc tgaatggcct ggtcctgaag tttggttata aaggcgacaa 1680

aactaaagtg tcgctgggta aactgaacac tatcagcatg atcttcatca tgggctccac 1740

ctgggttgtt gcctacgcca acccgaacat ccttgacctg attgaagcca tgggcgcacc 1800

gattatcgca tccctgctgt gcctgttgcc gatgtatgcc atccgtaaag cgccgtctct 1860

ggcgaaatac cgtggtcgtc tggataacgt gtttgttacc gtgattggtc tgctgaccat 1920

cctgaacatc gtatacaaac tgttttaaat agctatagag aggcaacccg tttcccttga 1980

gggagcgggc gtgcccgcga atgcacccgg ccttgtaacc actgtgcacc tgctgacgca 2040

ttcgcgggca cgcccgctcc cacaggtacg gtgaaacctt ccgaatcctg tgaccgagcc 2100

tgattccatg atgaacactg cataccgcaa gcacctgcca ggcaccgacc tggactactt 2160

cgacgcccgc gcggcggtcg aggcgatcaa gcccggcgcc tacgacggct tgccatacac 2220

gtcccgcgtg ctcgccgaga acctggtgcg ccgctgcgac cctgctacgc tcgacgcctc 2280

gctgagccag ctgatcgaac gcaagcgcga cctcgacttc ccgtggttcc cggcccgcgt 2340

ggtgtgccat gacatccttg gccagactgc gctggtcgac cttgccggcc tgcgtgacgc 2400

cattgccgac aaaggcggcg acccggccca ggtcaacccg gtggtgcc 2448

<210> 2

<211> 2661

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gctcgcgttg aacgctgcca tcgaggccgc gcgggcaggg gagatggggc gcgggttcgc 60

tgtggtggcc gatgaagtac gtacgttggc ccggcgcaca cagctgtcca ccgatgaagt 120

gcagcagatg atccagcgcc tgaagctagg tgcaggttcg gcggtgagtt caatgcaggc 180

agggcagcag gcgaccggca gtggcgtgga atcgagccag cgcaccgggg catcgctaag 240

cgcgattacc gaccaggtag agcacatcag cgacatgaac catcaggtgg ccacggctac 300

cgaggagcag tcggcggtga ccgaggaaat caaccggacg gtgcagggga tttccgattt 360

ggcgcgtgag acggcggcgg aggtgcaagg gtgccgcgag gagtgccagg cgttgcgtgg 420

gttggctgat gacctggcgc ggcagatggg tgggttcagg ctctagatcg caggggccgc 480

acggcggccc cggctctcgc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt 540

gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacga attcgagctc 600

ggtacccggg gatcctatgg ctgactcgca acccctgtcc ggtgctccgg aaggtgccga 660

atatttaaga gcagtgctgc gcgcgccggt ttacgaggcg gcgcaggtta cgccgctaca 720

aaaaatggaa aaactgtcgt cgcgtcttga taacgtcatt ctggtgaagc gcgaagatcg 780

ccagccagtg cacagcttta agctgcgcgg cgcatacgcc atgatggcgg gcctgacgga 840

agaacagaaa gcgcacggcg tgatcactgc ttctgcgggt aaccacgcgc agggcgtcgc 900

gttttcttct gcgcggttag gcgtgaaggc cctgatcgtt atgccaaccg ccaccgccga 960

catcaaagtc gacgcggtgc gcggcttcgg cggcgaagtg ctgctccacg gcgcgaactt 1020

tgatgaagcg aaagccaaag cgatcgaact gtcacagcag caggggttca cctgggtgcc 1080

gccgttcgac catccgatgg tgattgccgg gcaaggcacg ctggcgctgg aactgctcca 1140

gcaggacgcc catctcgacc gcgtatttgt gccagtcggc ggcggcggtc tggctgctgg 1200

cgtggcggtg ctgatcaaac aactgatgcc gcaaatcaaa gtgatcgccg tagaagcgga 1260

agactccgcc tgcctgaaag cagcgctgga tgcgggtcat ccggttgatc tgccgcgcgt 1320

agggctattt gctgaaggcg tagcggtaaa acgcatcggt gacgaaacct tccgtttatg 1380

ccaggagtat ctcgacgaca tcatcaccgt cgatagcgat gcgatctgtg cggcgatgaa 1440

ggatttattc gaagatgtgc gcgcggtggc ggaaccctct ggcgcgctgg cgctggcggg 1500

aatgaaaaaa tatatcgccc tgcacaacat tcgcggcgaa cggctggcgc atattctttc 1560

cggtgccaac gtgaacttcc acggcctgcg ctacgtctca gaacgctgcg aactgggcga 1620

acagcgtgaa gcgttgttgg cggtgaccat tccggaagaa aaaggcagct tcctcaaatt 1680

ctgccaactg cttggcgggc gttcggtcac cgagttcaac taccgttttg ccgatgccaa 1740

aaacgcctgc atctttgtcg gtgtgcgcct gagccgcggc ctcgaagagc gcaaagaaat 1800

tttgcagatg ctcaacgacg gcggctacag cgtggttgat ctctccgacg acgaaatggc 1860

gaagctacac gtgcgctata tggtcggcgg acgtccatcg catccgttgc aggaacgcct 1920

ctacagcttc gaattcccgg aatcaccggg cgcgctgctg cgcttcctca acacgctggg 1980

tacgtactgg aacatttctt tgttccacta tcgcagccat ggcaccgact acgggcgcgt 2040

actggcggcg ttcgaacttg gcgaccatga accggatttc gaaacccggc tgaatgagct 2100

gggctacgat tgccacgacg aaaccaataa cccggcgttc aggttctttt tggcgggtta 2160

gggcacggtc ctgtgaacga cgcagatcag cactctaaac catcgattca gcacttgcct 2220

atacaacccg tgcagcgatt tctgttcatt actgtgccgg ccccttcgcg ggcgcgcccg 2280

cccccacaat ggtcctgcaa acgccaatca ctgtaggagc gggttcaccc gcgaaagggc 2340

cagcgcaaac agcacaacag cgaatgtcgt aaacgcgcca ttgcaaggcg tgcgcaggca 2400

tctgacccgg ccccaccagt cataacatcg ccacaaaggg ccacagtgcc ccacgacaaa 2460

aaacgatcgc tgccgggaga tacacaatgt tcagcaagca agaccagatc cagggttacg 2520

acgatgcact gctggcggcg atgaatgccg aagaacagcg ccaggaagat cacatcgagc 2580

tgatcgcctc ggagaactac accagcaagc gcgtcatgca ggcccaaggc agcggcctga 2640

ccaacaagta cgccgaaggc t 2661

<210> 3

<211> 1647

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgtatacag taggagatta cctattagac cgattacacg agttaggaat tgaagaaatt 60

tttggagtcc ctggagacta taacttacaa tttttagatc aaattatttc ccgcaaggat 120

atgaaatggg tcggaaatgc taatgaatta aatgcttctt atatggctga tggctatgct 180

cgtactaaaa aagctgccgc atttcttaca acctttggag taggtgaatt gagtgcagtt 240

aatggattag caggaagtta cgccgaaaat ttaccagtag tagaaatagt gggatcacct 300

acatcaaaag tccaaaatga aggaaaattt gttcatcata cgctggctga cggtgatttt 360

aaacacttta tgaaaatgca cgaacctgtt acagcagctc gaactttact gacagcagaa 420

aatgcaaccg ttgaaattga ccgagtactt tctgcactac taaaagaaag aaaacctgtc 480

tatatcaact taccagttga tgttgctgct gcaaaagcag agaaaccctc actccctttg 540

aaaaaagaaa atccaacttc aaatacaagt gaccaagaga ttttgaataa aattcaagaa 600

agcttgaaaa atgccaaaaa accaatcgtg attacaggac atgaaataat tagctttggc 660

ttagaaaata cagtcactca atttatttca aagacaaaac tccctattac gacattaaac 720

tttggaaaaa gttcagttga tgaaactctc ccttcatttt taggaatcta taatggtaaa 780

ctctcagagc ctaatcttaa agaattcgtg gaatcagccg acttcatcct gatgcttgga 840

gttaaactca cagactcttc aacaggagca tttacccatc atttaaatga aaataaaatg 900

atttcactga acatagacga aggaaaaata tttaacgaaa gcatccaaaa ttttgatttt 960

gaatccctca tctcctctct cttagaccta agcggaatag aatacaaagg aaaatatatc 1020

gataaaaagc aagaagactt tgttccatca aatgcgcttt tatcacaaga ccgcctatgg 1080

caagcagttg aaaacctaac tcaaagcaat gaaacaatcg ttgctgaaca agggacatca 1140

ttctttggcg cttcatcaat tttcttaaaa ccaaagagtc attttattgg tcaaccctta 1200

tggggatcaa ttggatatac attcccagca gcattaggaa gccaaattgc agataaagaa 1260

agcagacacc ttttatttat tggtgatggt tcacttcaac ttacagtgca agaattagga 1320

ttagcaatca gagaaaaaat taatccaatt tgctttatta tcaataatga tggttataca 1380

gtcgaaagag aaattcatgg accaaatcaa agctacaatg atattccaat gtggaattac 1440

tcaaaattac cagaatcatt tggagcaaca gaagaacgag tagtctcgaa aatcgttaga 1500

actgaaaatg aatttgtgtc tgtcatgaaa gaagctcaag cagatccaaa tagaatgtac 1560

tggattgagt tagttttggc aaaagaagat gcaccaaaag tactgaaaaa aatgggtaaa 1620

ctatttgctg aacaaaataa atcatga 1647

<210> 4

<211> 1494

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgaccaccc tgacccgtgc ggactgggaa caacgtgccc agcaactgaa gatcgaaggc 60

cgtgccttca tcaacggcga atacaccgat gccgtatccg gtgaaacctt cgagtgcctg 120

agcccggtcg acggacgctt cctggccaag gttgccagct gcgacctggc cgatgccaac 180

cgcgctgttg aaaacgcccg tgccaccttc aactccggcg tgtggtcgca gctggccccc 240

gccaagcgca aggccaagct gatccgcttc gccgacctgc tgcgcaagaa cgtcgaagag 300

ctggcgctgt tggaaacgct ggacatgggc aagccgatcg gcgactcctc cagcatcgac 360

atcccaggtg cggcccaagc cattcactgg actgccgaag ccatcgacaa ggtctacgac 420

gaagttgccc cgaccccgca tgaccagctt ggcctggtta cccgcgaacc cgtgggtgtg 480

gttggtgcca tcgtgccgtg gaacttcccg ttgctgatgg cctgctggaa actcggccct 540

gcactggcca ccggtaactc ggtcgtgctc aagccgtccg aaaaatcgcc gctgaccgcc 600

atccgcatcg cccaactggc gatcgaggcc ggtatccccg ctggcgtgct gaacgtgctg 660

ccaggctatg gccacaccgt gggcaaggca ctggccctgc acatggacgt ggacaccctg 720

gtgttcaccg gttcgaccaa gatcgccaag caactgatgg tttacgcggg cgagtcgaac 780

atgaaacgca tctggctgga agccggtggc aagagcccga acatcgtctt tgccgacgcc 840

ccggacctgc aagcagccgc cgaggccgca gccagcgcca tcgccttcaa ccagggcgaa 900

gtgtgcactg caggctcccg cctgctggtc gagcgttcga tcaaggacaa gttcctgccg 960

atggtggtag aggccctgaa gggctggaag ccaggcaacc cgctggaccc gcagaccact 1020

gtcggtgcct tggtcgatac ccagcagatg aacaccgtgc tgtcgtacat cgaggctggc 1080

cacaaggacg gcgccaagct gctggccggc ggcaaacgca ccctggaaga gaccggcggc 1140

acctacgtcg agccgaccat cttcgacggc gtgactaacg ccatgcgcat tgcccaggaa 1200

gaaatcttcg gcccggtgct gtcggtgatt gccttcgaca ccgccgaaga agccgtggcc 1260

attgccaacg acaccccgta tggcctggcc gctggtatct ggacttcgga catttccaag 1320

gcccacaaga ccgcccgtgc cgtgcgtgca ggcagcgtct gggtcaacca gtacgatggt 1380

ggcgacatga ccgcgccgtt cggtggcttc aaacagtcgg gcaacggccg tgacaagtcg 1440

ctgcacgcac tggagaagta caccgagctg aaggcgacct ggatcaagct gtaa 1494

<210> 5

<211> 1000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tcgagcatca tcgggttgcc caggctcacc tcgccgccca catgcagcac ctgctggcaa 60

ccttcaggca acggcaggtc gcggcgcgcg gcttcccagg tttgcaccaa ctggttggca 120

taactgacga acgcttcgcc atcgctggtc aggctggcgc cgctgcggct acgcacgaac 180

agctggcaac ccagttgctg ttccaggcgc tgcacgcgcg cggtgatcgc ggtttgtgac 240

acgaacaggc gctcggccgc ggcgaccagg ctgccgcaac gcacaatttc caggaaggta 300

cgggcctgct cgatgtccat gggctgctcg gtggttgaag gggaggccta ttctagaggc 360

tttgcaccgt tatggggcgt aattgcgttg tctgcaaatt tgcaggtcgg ttgtgacttt 420

agtcccatgg cggcgtgggt atagcggtcc atactcaagt ttcgcccttc aataacaaca 480

agtaccgggt ttctaacgac tggtttgacc tcgatggccc tttcgcgggc ggcacgcgca 540

tcggtggttc ggcagaggcc gtcgtgtagg agcgggttta cccgcgaaag ggccatctca 600

ggcactgcag gctcaggggg caatctggtg cagttgcccc aaccggctgc gggtacgcat 660

caggtcggcc agcgggccgc ccaggctctc ttccagcgga cgtttaccta tgacaatcac 720

ccgtcgatcc tggtcataca gcacatccac caggttgacg aaacgctgct gagccgccag 780

cgaacacgcc gagaggtcat ccagcccatc gatcacccac tcgtcatact ccctggccag 840

caccaggtag tcgatgaccg cagtggcctt ctcgcacaga tcgtcaaagc ccagcaccac 900

ccgccggcca tcgatggcca gcgctcgcag cgggcgcttg ttcacgtcca gtatcaccgg 960

ttgttcgttc ggcacgttca gcgcctggcg ctgcgccgca 1000

<210> 6

<211> 1000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

agaccatatc gcgcatgact tcgaacgccg cggaacctgc gccaacgata attccggccc 60

gcagttcggt cacgggtaca ttcgcttcac gcagaatgtc cgccgtagcc tgacgagcac 120

gcagatgatc cgactgctca tgtggcgggg cctgcaacga actaagaaag attaattgct 180

taactggtac ttcacgtagc gcatcgcgga cgttgagagc cacctggcgc tcctgagcga 240

taaaatcgcc gccttcgccc atgctgtgca ccagaaaata gaccgtatcg atatcctgca 300

acagggccgg taggttatcc ggccagctga gatcgacttt atggcaactg acgtttgcca 360

gttgcagctt tgcaagcctg tcgacatgac gtgccgccgc caggatctga tgcccttgct 420

ggctgagtgt gcgcaccaga tgctgaccaa tgtagccact ggcaccgaga actaaaatgc 480

gttgcggcac gtctctctcc ggcatgtcct tattatgacg ggaaatgcca ccctttttac 540

cttagccagt ttgttttcgc cagttcgatc acttcatcac cgcgtccgct gatgattgcg 600

cgcagcatat acaggctgaa acctttggcc tgttcgagtt tgatctgcgg tggaatggct 660

aactcttctt tggcgaccac cacatccacc aacaccggac cgtcgatgga gaaggcgcgt 720

tgcagggctt catcaacttc agacgctttt tctacacgga tacccgtaat gccgcacgct 780

tcggcaatgc gggcaaagtt tgtgtcgtgt agttcggtgc cgtcagtcaa atagccacca 840

gctttcatct ccatcgccac aaagcccagc acgctgttgt taaagacgac aattttcact 900

ggcagtttca tctgcactac tgagaggaaa tcgcccatca acatgctaaa accgccatcg 960

ccgcacatgg cgaccacctg acgttctggc tctgtcgcct 1000

<210> 7

<211> 1000

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tcatcgacaa aaaagggccg ctcaataacc cggcagaccg cgatcactgc attcagtaca 60

tggtggcgat cccgctgcta ttcgggcgct taacggcggc agattacgag gacaacgttg 120

cgcaagataa acgcattgac gccctgcgcg agaagatcaa ttgctttgaa gatccggcat 180

ttaccgctga ctaccacgac ccggaaaaac gcgccatcgc caatgccatt acccttgagt 240

tcaccgacgg cacacgattt gaagaagtgg tggtggagta ccccattggt catgctcgcc 300

gccgtcagga tggtattccg aaactggtcg ataaattcaa aatcaatctc gcgcgccagt 360

tcccgactcg ccaacagcag cgcattctgg aggtttctct cgacagagct cgcctggaac 420

agatgccggt caatgagtat ctcgacctgt acgtcattta agtaaacggc ggtaaggcgt 480

aagttcaaca ggagagcatt gttattgtcg gatgcgtcgc gcggtgcatc cggcactgtg 540

tgccgatgcc tgatgcgacg ctgacgcgtt ttatcatgcc tacggacctg aaccgtaggt 600

cggataaggc gctcgcgtcg catccgacac catgctcaga tgcctgatgc gacgctgacg 660

cgtcttatca ggcctaccca ctgtttttac accgataatt tttcccccac ctttttgcac 720

tcattcatat aaaaaatata tttccccacg aaaacgattg ctttttatct tcagatgaat 780

agaatgcggc ggattttttg ggtttcaaac agcaaaaagg gggaatttcg tgtcgcaaga 840

taacaacttt agccaggggc cagtcccgca gtcggcgcgg aaaggggtat tggcattgac 900

gttcgtcatg ctgggattaa ccttcttttc cgccagtatg tggaccggcg gcactctcgg 960

aaccggtctt agctatcatg atttcttcct cgcagttctc 1000

Claims (10)

1. The recombinant strain is characterized in that: the recombinant strain expresses 2-ketoisovalerate dehydrogenase (also called ketoacid decarboxylase).

2. The recombinant bacterium according to claim 1, wherein: the recombinant bacterium is obtained by modifying the genome of a receptor bacterium, wherein the modification comprises the step of enabling the receptor bacterium to express the 2-ketoisovalerate deacase, and the receptor bacterium is pseudomonas or escherichia coli.

3. The recombinant bacterium according to claim 1 or 2, wherein: the recipient bacterium is pseudomonas and the modification comprises the following steps:

a1) inhibiting or reducing threonine aldolase activity in the pseudomonas genome;

a2) inhibiting or reducing the activity of methyl citrate synthase in the pseudomonas genome;

a3) inhibiting or reducing the activity of propionyl-coa synthetase in the pseudomonas genome;

a4) increasing or enhancing the activity of a threonine transporter in the pseudomonas genome;

a5) increasing or enhancing the activity of threonine deaminase in the pseudomonas genome.

4. The recombinant bacterium according to claim 3, wherein:

the a1), the inhibition or reduction of the activity of threonine aldolase in the pseudomonas genome is to subject the coding gene of threonine aldolase named ltaE gene in the pseudomonas genome to gene knockout or gene silencing;

the a2), wherein the inhibition or reduction of the activity of the methyl citrate synthetase in the pseudomonas genome is the gene knockout or gene silencing of the gene coding for the methyl citrate synthetase named as the prpC gene in the pseudomonas genome;

the a3), the inhibiting or reducing the activity of methyl citrate synthetase in the pseudomonas genome is to knock out or silence the gene encoding propionyl-coa synthetase named as prpE gene in the pseudomonas genome;

in the a4), the improvement or enhancement of the expression of the threonine transporter by the pseudomonas is that a coding gene of the threonine transporter named tdcC gene and a promoter thereof are introduced into the pseudomonas;

in a5), the improvement or enhancement of the expression of threonine deaminase by pseudomonas is to introduce a coding gene of threonine deaminase named ilvA gene and a promoter thereof into pseudomonas.

5. The recombinant bacterium according to claim 3 or 4, wherein:

D1) the modification of said a1) and said a5) is achieved by replacing the ltaE gene in the Pseudomonas genome with a fragment containing the ilvA gene and its promoter, and/or,

D2) the transformation of said a2) and said a4) is achieved by replacing the prpC gene in the pseudomonas genome with a fragment containing the tdcC gene and its promoter.

6. The recombinant bacterium according to claim 1 or 2, wherein: the recipient bacterium is escherichia coli, and the modification comprises the following steps:

b1) inhibiting or reducing the activity of threonine aldolase in the genome of E.coli;

b2) inhibiting or reducing the activity of propionyl-coa synthetase in the genome of escherichia coli;

b3) increasing or enhancing the activity of threonine deaminase in the genome of escherichia coli;

b4) increasing or enhancing the activity of aldehyde dehydrogenase in the genome of said E.coli.

7. The recombinant bacterium according to claim 6, wherein:

in b1), the inhibition or reduction of the threonine aldolase activity in the E.coli genome is the gene knockout or gene silencing of the encoding gene named ltaE gene threonine aldolase in the E.coli genome;

said b2), said inhibiting or reducing the activity of propionyl-CoA synthetase in the genome of E.coli; carrying out gene knockout or gene silencing on a coding gene of propionyl coenzyme A synthetase named as a prpE gene in the genome of the escherichia coli;

in b3), the improvement or enhancement of the activity of threonine deaminase in the genome of escherichia coli is to introduce a coding gene of the threonine transporter named ilvA gene and a promoter thereof into escherichia coli;

in b4), the increasing or enhancing the activity of the aldehyde dehydrogenase in the genome of E.coli is to introduce a gene encoding the aldehyde dehydrogenase, named PpadH gene, into E.coli.

8. A method for preparing a recombinant bacterium is characterized in that: the method for preparing the recombinant bacteria is M1) or/and M2):

m1) carrying out the modification on pseudomonas as described in any one of claims 2-5 to obtain a recombinant bacterium;

m2) carrying out the transformation of the Escherichia coli as described in claim 2, 6 or 7 to obtain a recombinant bacterium.

9. Use of the recombinant bacterium of any one of claims 1-7 for the production or preparation of propionic acid from threonine.

10. A process for preparing propionic acid, characterized by: the method comprises the following steps: catalyzing threonine to obtain propionic acid by using the recombinant bacterium of any one of claims 1-7.

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