CN111197054B - Construction of recombinant pseudomonas putida and application thereof in production of propionic acid - Google Patents

Construction of recombinant pseudomonas putida and application thereof in production of propionic acid Download PDF

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CN111197054B
CN111197054B CN201811381133.2A CN201811381133A CN111197054B CN 111197054 B CN111197054 B CN 111197054B CN 201811381133 A CN201811381133 A CN 201811381133A CN 111197054 B CN111197054 B CN 111197054B
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马超
于波
马延和
陶勇
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Abstract

The invention discloses a construction of recombinant pseudomonas putida and application thereof in propionic acid production. The invention provides a method for preparing recombinant bacteria, which comprises the following steps: deleting or inactivating the prpC gene on the pseudomonas genome to obtain the recombinant bacterium. The invention takes threonine as raw material and takes recombinant pseudomonas putida KT2440 as engineering bacteria to produce propionic acid. The method can avoid the pollution of the chemical synthesis method to the environment due to high temperature and high pressure and strong acid and strong alkali, and can overcome the defect that the microbial fermentation method generates byproducts such as formic acid, acetic acid and the like, thereby providing a technical route for synthesizing propionic acid by a biotransformation method with important prospect.

Description

Construction of recombinant pseudomonas putida and application thereof in production of propionic acid
Technical Field
The invention belongs to the technical field of biology, and particularly relates to construction of recombinant pseudomonas putida and application of the recombinant pseudomonas putida in production of propionic acid.
Background
Propionic Acid (PA), a colorless and transparent liquid with a strong pungent odor, can be dissolved in water, ethanol and other solvents in any proportion, has the typical chemical properties of carboxylic acid, and has the molecular formula of C3H6O2. 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. (1) The food such as bread, cake and the like can also play a good role in corrosion prevention by adding sodium propionate, fresh grain grains have a large water content, and calcium propionate is used as a preservative, so that the food can be prevented from corrosion and caking, can also be prevented from being decomposed, and can keep nutritional ingredients; (2) monochloropropaneic acid and sodium monochloropropaneate synthesized by propionic acid can play a good role in weeding when being used as agricultural herbicide; (3) the isoamyl propionate synthesized by propionic acid and isoamylol can be used as an intermediate for synthesizing essence and can also be used for synthesizing artificial flower essential oil; (4) Calcium propionate and sodium propionate are used as medicinal components for treating dermatosis caused by skin parasitic mold; (5) methyl propionate, 3-chloropropionic acid, ethyl propionate and the like synthesized by propionic acid can be used as intermediates of organic synthesis; (6) cellulose acetate propionate synthesized from propionic acid can be used in television, automobile parts, etc.
The production method of propionic acid includes chemical synthesis method and microbial fermentation method, the chemical synthesis method uses chemical products of petroleum, etc. as raw material, and utilizes catalyst to synthesize propionic acid by heating and pressurizing, and said method is a main production method of propionic acid on industrial scale. The commonly used chemical synthesis methods include propionaldehyde oxidation, light hydrocarbon oxidation, ethanol carbonylation, ethylene carbonylation, acrylonitrile, and the like. (1) Propionaldehyde oxidation: the method takes propionaldehyde as a raw material, cobalt acetate as a catalyst and sodium hexametaphosphate as a cocatalyst, and propionaldehyde is oxidized to prepare propionic acid; (2) light hydrocarbon oxidation process: the method takes light naphtha, liquefied natural gas or C4-C8 alkane with the boiling point lower than 100 ℃ as raw materials, takes oil soluble salts such as manganese naphthenate and the like as catalysts, and carries out oxidation reaction at the temperature of 160-170 ℃ and the pressure of 2.03-4.05M Pa to generate acetic acid and simultaneously produce propionic acid, formic acid and the like as byproducts; (3) ethanol carbonylation method: the method takes Ni-Cu bimetal loaded by active carbon as a reaction catalyst and takes ethanol as a raw material to carry out gas-phase carbonylation; (4) the acrylonitrile method: the method uses sulfide of metal in VI-VIII family or its mixture as catalyst, and makes acrylonitrile react with hydrogen and water at proper temp. and pressure to produce propionic acid.
The propionic acid produced by microbial fermentation can reduce the dependence on non-renewable energy sources such as petroleum and the like, and reduce the pressure on the environment. Therefore, under the severe conditions of environmental pollution and energy shortage around the world, the production of propionic acid by a microbial fermentation method provides a new idea for the synthesis of propionic acid, and is also concerned by more researchers. The microbial fermentation method is characterized in that general nutrient sources are metabolized by microorganisms to generate propionic acid, and the propionic acid is biosynthesized mainly through the following three ways. (1) Succinic acid pathway: also called as a dicarboxylic acid pathway, propionic acid is produced by fermentation starting from glucose, glycerol, lactic acid and the like as raw materials. The key step is the production of oxaloacetate and propionyl CoA by the catalysis of oxaloacetate transcarboxylase (EC6.4.1.1) by pyruvate and methylmalonyl CoA. Another key step is the catalysis of succinic acid and propionyl-CoA by CoA transferase (EC2.8.3.5) to produce succinyl-CoA and the final product propionic acid. The disadvantage of this route is the production of acetate as a by-product, pyruvate being subjected to the action of pyruvate dehydrogenase to produce acetyl CoA and then further acetate; (2) acrylic acid route: is the process of finally producing propionic acid by catalyzing lactic acid with propionyl-CoA transferase (EC2.8.3.1), lactyl-CoA dehydrogenase (EC4.2.1.54) and acryloyl-CoA reductase (EC1.3.1.95). The disadvantage of this route is that there is insufficient energy, and the reduction of lactic acid to propionic acid requires energy; (3) the propylene glycol pathway: in some bacteria, the carbon source is first fermented to produce 1, 2-propanediol, then the 1, 2-propanediol is subjected to a propanediol dehydrogenase (EC4.2.1.28) to produce propionaldehyde, which is then passed through a propionaldehyde dehydrogenase (EC1.2.1.87) to produce propionyl CoA and finally the final product propionic acid. The disadvantage of this route is the production of propanol as a by-product, and the direct production of propanol from propionaldehyde by the action of a propanol dehydrogenase.
In summary, the chemical synthesis method for producing propionic acid generally faces the problems of harsh reaction conditions, difficulty in separation and purification, easy environmental pollution and the like. The production of propionic acid by the biotransformation method needs to establish a synthetic route with high transformation rate, reduce the accumulation of byproducts, reduce the production cost and form a production mode with popularization prospect. Threonine is an essential amino acid, is mainly prepared by a microbial fermentation method, has a simple process and low cost, and can be used as a raw material for producing propionic acid.
Pseudomonas putida (Pseudomonas putida) is a gram-negative bacterium, widely grows in soil, water environment and plant roots, can decompose some organic matters in the environment, can play a role in purifying the environment, has functions of biocatalysis, biological pollution discharge and the like beneficial to human beings, and is called as a large group of environmental probiotics with the most development potential. Pseudomonas putida KT2440(P.putida KT2440) is a strain which is extensively studied and applied worldwide, and is the first gram-negative bacterium (1982) which is recognized as environmentally safe by the Recombinant DNA Advisory Committee (RAC) of the United states department of health, and which permits KT2440 to be used as a host bacterium for genetic engineering.
Disclosure of Invention
An object of the present invention is to provide a method for producing a recombinant bacterium.
The method provided by the invention comprises the following steps: deleting or inactivating the prpC gene on the pseudomonas genome to obtain the recombinant bacterium.
The method also comprises the following steps: deleting or inactivating the ltaE gene on the pseudomonas genome.
In the above method, the deletion or inactivation of the prpC gene on the pseudomonas genome is achieved by replacing the prpC gene on the pseudomonas genome with the tdcBC gene and the lac promoter driving its expression.
The method also comprises the following steps: replacing the promoter of the bkd gene cluster in the pseudomonas genome with a lac promoter.
Another object of the present invention is to provide a recombinant bacterium.
The recombinant bacterium provided by the invention is obtained by replacing a prpC gene on a pseudomonas genome with a tdcBC gene and a lac promoter for driving the expression of the tdcBC gene, knocking out an ltaE gene on the genome, replacing an bkd gene cluster promoter in the genome with the lac promoter, and keeping other sequences unchanged;
or, the recombinant bacterium is obtained by replacing a prpC gene on a pseudomonas genome with a tdcBC gene, knocking out an ltaE gene on the genome and keeping other sequences unchanged;
or, the recombinant bacterium is obtained by replacing the prpC gene on the pseudomonas genome with a tdcBC gene and a lac promoter for driving the gene expression of the tdcBC gene, knocking out the ltaE gene on the genome and keeping other sequences unchanged;
or, the recombinant bacterium is obtained by knocking out the prpC gene on the pseudomonas genome, knocking out the ltaE gene on the genome and keeping other sequences unchanged;
or, the recombinant bacterium is obtained by knocking out the prpC gene on the pseudomonas genome and keeping other sequences unchanged.
In the above, the knockout or the substitution is carried out by means of homologous recombination;
or, the knockout or the replacement employs a lambda-red homologous recombination system or homologous recombination for sacB gene mediated screening;
in the above, the pseudomonas is pseudomonas putida.
In the above, the pseudomonas putida is pseudomonas putida KT 2440.
The application of the recombinant bacterium in producing or preparing propionic acid by utilizing threonine is also within the protection scope of the invention.
The 3 rd object of the present invention is to provide a method for producing propionic acid.
The method provided by the invention comprises the following steps: catalyzing threonine by using the recombinant bacteria to obtain propionic acid.
The nucleotide sequences of the tdcBC gene and a lac promoter for driving the expression of the tdcBC gene are sequence 1;
the nucleotide sequence of the lac promoter is sequence 2.
The threonine is L-threonine.
The invention takes threonine as raw material and takes recombinant pseudomonas putida KT2440 as engineering bacteria to produce propionic acid. The method can avoid the pollution of the chemical synthesis method to the environment due to high temperature and high pressure and strong acid and strong alkali, and can overcome the defect that the microbial fermentation method generates byproducts such as formic acid, acetic acid and the like, thereby providing a technical route for synthesizing propionic acid by a biotransformation method with important prospect.
Drawings
FIG. 1 is a diagram showing a route for producing propionic acid from threonine.
Figure 2 shows the propionic acid standard HPLC results.
FIG. 3 shows the HPLC results of the sample.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The primer sequences used in the following examples are listed in Table 1:
table 1 shows the sequence listing of the primers used
Figure BDA0001871911720000041
Figure BDA0001871911720000051
Example 1 construction of recombinant Pseudomonas putida engineered Strain PS04
This example prepared a basic strain PS04 that was used to produce propionic acid, and was prepared as follows, using primers as shown in Table 1.
Preparation of recombinant bacterium PS01
(1) Knockout of the prpC gene of the methyl citrate synthase.
Starting from pseudomonas putida KT2440(ATCC 47054), a homologous recombination method is utilized to knock out a methyl citrate synthetase gene prpC, so that the degradation of propionic acid is reduced, and an engineering strain PS01 is obtained, and the specific steps are as follows:
(1-a) construction of a knock-out plasmid pK 18-prpC.
Designing a primer (a primer is pK18-F/pK18-R) by taking pK18 plasmid (ATCC 87097) as a template for PCR amplification to obtain a plasmid DNA linear fragment of about 6000 bp;
selecting 500bp of each upstream and downstream of a prpC gene ORF, designing primers (the primers are 18-prpCup-F/prpCup-down-R and prpCup-down-F/prpDown-18-R respectively), taking genome DNA of pseudomonas putida KT2440 as a template, and carrying out PCR amplification by using the primers to obtain a 500bp upstream knockout homologous arm DNA fragment and a 500bp downstream knockout homologous arm DNA fragment;
the 500bp upstream knockout homology arm DNA fragment, the 500bp downstream knockout homology arm DNA fragment and the plasmid DNA linear fragment of about 6000bp are ligated together by the Gibson method (Gibson DG, Young L, Chuang RY, et al. enzymatic analysis of DNA molecules up to segmented humanized plasmids. Nat. Meth,2009,6(5):343 and 345.), to obtain the knockout plasmid pK 18-prpC.
(1-b) the plasmid pK18-prpC was transferred into the starting strain KT2440 using the combined transduction technique.
E.coli S17-1(ATCC 47055) was used as a donor for binding the transduced plasmid to prepare S17-1 for electrotransformation;
transforming the obtained knock-out plasmid pK18-prpC into S17-1 competent cells to obtain a strain containing S17-1 of the knock-out plasmid;
respectively inoculating the strain containing the S17-1 with the knockout plasmid and the starting strain KT2440 into LB liquid culture medium, and activating overnight at 37 ℃ to obtain S17-1 bacterial liquid and KT2440 bacterial liquid. 1mL of the suspension was centrifuged, and the supernatant was resuspended in 500. mu.L of LB liquid. 10 mu L of KT2440 bacterial liquid is spotted on an antibiotic-free LB solid culture medium and dried in the air. And (3) covering 15 mu L of S17-1 bacterial liquid on KT2440, and drying. Culturing at 37 deg.C for 16 h.
(1-c) two homologous recombinations.
The mixed colonies were scraped off with a coating rod and spread on LB solid medium containing gentamicin and chlorophenol at a final concentration of 25 mg/L. And coating a solid medium plate on every 4 colonies, and culturing at 37 ℃ for 24h, wherein the grown colonies are strains which complete the first homologous recombination and have the gentamicin resistance. And selecting a single colony, streaking the single colony on an LB solid culture medium containing 25mg/L chlorophenol and 20% sucrose at the final concentration, and culturing the single colony at 37 ℃ for 24 hours, wherein the grown colony is a knockout strain for completing the second homologous recombination. Single colonies were picked and cultured at 37 ℃ for 12 hours on LB solid medium containing chlorophenol at a final concentration of 25mg/L and LB solid medium containing gentamicin at a final concentration of 25mg/L, respectively. Single colonies that grew on the former and did not grow on the latter were picked for PCR validation.
PCR verification is carried out by using the prpC-YF/prpC-YR primers for amplification and identification, a strain without knockout of the prpC is amplified to obtain a fragment of about 2400bp, a strain with knockout of the prpC is amplified to obtain a fragment of about 1270bp, and a strain with a positive knockout result is selected and named as PS 01.
Sequencing analysis results show that the genome of PS01 has no prpC gene, PS01 knocks out the prpC gene on the genome of Pseudomonas putida KT2440, and other sequences are not changed, so that the recombinant Pseudomonas putida KT2440 mutant is obtained.
Second, preparation of recombinant bacterium PS02
(2) Deletion of threonine aldolase ltaE.
Starting from a PS01 strain, a threonine aldolase gene ltaE is knocked out by using a homologous recombination method, a pathway from threonine to glycine is blocked, and an engineering strain PS02 is obtained, and the method comprises the following specific steps:
(2-a) construction of a knock-out plasmid pK 18-ltaE.
Designing a primer (a primer is pK18-F/pK18-R) by taking pK18 plasmid (ATCC 87097) as a template for PCR amplification to obtain a plasmid DNA linear fragment of about 6000 bp;
selecting 500bp of each upstream and downstream of an ltaE gene ORF, designing primers (the primers are respectively 18-ltaEup-F/ltaEup-down-R and ltaEup-down-F/ltaEdown-18-R), and respectively carrying out PCR amplification by using the genomic DNA of pseudomonas putida KT2440 as a template to obtain a 500bp upstream knockout homologous arm DNA fragment and a 500bp downstream knockout homologous arm DNA fragment;
and connecting the 500bp upstream knockout homology arm DNA fragment, the 500bp downstream knockout homology arm DNA fragment and the plasmid DNA linear fragment of about 6000bp together by using a Gibson method to obtain a knockout plasmid pK 18-ltaE.
(2-b) the plasmid pK18-ltaE was transferred into the starting strain PS01 using the binding transduction technique.
E.coli S17-1 is used as a plasmid donor for combined transduction to prepare the electrotransformation competence of S17-1;
transforming the plasmid pK18-ltaE into S17-1 electrotransformation competence to obtain a strain containing S17-1 with a knockout plasmid;
respectively inoculating the strain containing the S17-1 with the knockout plasmid and the original strain PS01 to an LB liquid culture medium, and activating overnight at 37 ℃ to obtain S17-1 bacterial liquid and PS01 bacterial liquid. 1mL of the suspension was centrifuged, and the supernatant was resuspended in 500. mu.L of LB liquid. mu.L of PS01 bacterial suspension was spotted on an antibiotic-free LB solid medium, and air-dried. And covering 15 mu L of S17-1 bacterial liquid on the PS01, and airing. Culturing at 37 deg.C for 16 h.
(2-c) double homologous recombination
The mixed colonies were scraped off with a coating rod and spread on LB solid medium containing gentamicin and chlorophenol at a final concentration of 25 mg/L. And coating a solid medium plate on every 4 colonies, and culturing at 37 ℃ for 24h, wherein the grown colonies are strains which complete the first homologous recombination and have the gentamicin resistance. And selecting a single colony, streaking the single colony on an LB solid culture medium containing 25mg/L chlorophenol and 20% sucrose at the final concentration, and culturing the single colony at 37 ℃ for 24 hours, wherein the grown colony is a knockout strain for completing the second homologous recombination. Single colonies were picked and cultured at 37 ℃ for 12 hours on LB solid medium containing chlorophenol at a final concentration of 25mg/L and LB solid medium containing gentamicin at a final concentration of 25mg/L, respectively. Single colonies that grew on the former and did not grow on the latter were picked for PCR validation.
And (3) amplifying and identifying by using an ltaE-YF/ltaE-YR primer, amplifying strains without the ltaE knock-out to obtain a fragment of about 2250bp, amplifying the strains with the knock-out to obtain a fragment of about 1200bp, and selecting the strains with positive knock-out results to be named as PS 02.
Sequencing analysis results show that the PS02 is a strain obtained by knocking out the ltaE gene of the PS01 strain;
namely, the recombinant strain PS02 is obtained by knocking out the prpC gene and the ltaE gene on the pseudomonas putida KT2440 genome and keeping other sequences unchanged.
Preparation of recombinant bacterium PS03
(3) Construction of plasmids expressing the threonine deaminase gene tdcB and the threonine transporter gene tdcC of Escherichia coli (Escherichia coli).
Genomic DNA was extracted from E.coli (ATCC 700926) and tdcB and tdcC genes were amplified with primers tdcB (SacI) F/tdcC (BamHI) R to obtain a DNA fragment tdcBC of about 2500 bp. The fragment tdcBC and the vector pUCP18(Biovector, Cloning vector pUCP18) were digested with SacI and BamHI, respectively, and the digested product of the fragment tdcBC and the large fragment of the pUCP18 vector were recovered. The enzyme digestion product of the segment tdcBC and a pUCP18 vector large segment are subjected to ligation reaction by using T4 ligase, transformed escherichia coli DH5 alpha (transgen biotech, CD201-01) is identified by using a primer pUCP18-YF/pUCP18-YR, a positive clone with a correct target segment sequence is selected, a plasmid is extracted, and the obtained positive recombinant plasmid is named as pUCP 18-tdcBC.
(4) Integration of lac promoter and tdcBC gene insertion.
Starting from a PS02 strain, a lac promoter and an escherichia coli tdcBC gene are inserted and integrated into a prpC locus of a PS02 genome by using a homologous recombination method, a pathway from threonine to 2-ketobutyrate is enhanced, and an engineering strain PS03 is obtained, and the method specifically comprises the following steps:
(4-a) construction of integration plasmid pK18-tdcBC:: prpC.
Designing a primer (tdcBC-down-F/up-tdcBC-R) by taking the pK18-prpC plasmid as a template for PCR amplification to obtain a plasmid DNA linear fragment of about 7000 bp;
amplification of lac promoter and tdcBC gene: designing a primer (the primer is up-tdcBC-F/tdcBC-down-R), and carrying out PCR amplification by using a pUCP18-tdcBC plasmid as a template and the primer to obtain a DNA fragment (the tdcBC gene and a lac promoter for driving the gene expression thereof, sequence 1) with the size of about 2500 bp;
the above two DNA fragments were ligated together by the Gibson method to obtain the integrated plasmid pK18-tdcBC:: prpC.
(4-b) plasmid pK18-tdcBC:: prpC was transferred into the starting strain PS02 using the combined transduction technique.
E.coli S17-1 is used as a plasmid donor for combined transduction to prepare the electrotransformation competence of S17-1;
prpC is transformed into S17-1 to obtain a strain containing S17-1 with the knock-out plasmid;
respectively inoculating the strain containing the S17-1 with the knockout plasmid and the original strain PS02 to an LB liquid culture medium, and activating overnight at 37 ℃ to obtain S17-1 bacterial liquid and PS02 bacterial liquid. 1mL of the suspension was centrifuged, and the supernatant was resuspended in 500. mu.L of LB liquid. mu.L of PS02 bacterial suspension was spotted on an antibiotic-free LB solid medium, and air-dried. And covering 15 mu L of S17-1 bacterial liquid on the PS02, and airing. Culturing at 37 deg.C for 16 h.
(4-c) two homologous recombinations.
The mixed colonies were scraped off with a coating rod and spread on LB solid medium containing gentamicin and chlorophenol at a final concentration of 25 mg/L. And coating a solid medium plate on every 4 colonies, and culturing at 37 ℃ for 24h, wherein the grown colonies are strains which complete the first homologous recombination and have the gentamicin resistance. And selecting a single colony, streaking the single colony on an LB solid culture medium containing 25mg/L chlorophenol and 20% sucrose at the final concentration, and culturing the single colony at 37 ℃ for 24 hours, wherein the grown colony is a knockout strain for completing the second homologous recombination. Single colonies were picked and cultured at 37 ℃ for 12 hours on LB solid medium containing chlorophenol at a final concentration of 25mg/L and LB solid medium containing gentamicin at a final concentration of 25mg/L, respectively. Single colonies that grew on the former and did not grow on the latter were picked for PCR validation.
The prpC-YF/prpC-YR primers are used for amplification and identification, the bacterial strain without integration insertion is amplified to obtain a fragment of about 1200bp, the bacterial strain with integration insertion is amplified to obtain a fragment of about 3700bp, and the bacterial strain with positive selection result is named as PS 03.
Sequencing analysis results show that the PS03 is a strain obtained by inserting lac promoter and tdcBC gene into the PS02 strain at a position where prpC is deleted; namely, the recombinant strain PS03 is obtained by replacing a prpC gene on a pseudomonas putida KT2440 genome with a tdcBC gene and a lac promoter for driving the gene expression, knocking out an ltaE gene on the genome, and keeping other sequences unchanged.
Preparation of recombinant bacterium PS04
(5) Enhancement of bkd Gene expression by promoter replacement
Starting from a PS03 strain, a lac promoter is used for replacing a branched-chain ketoacid dehydrogenase complex gene bkd self promoter by utilizing a homologous recombination method, a pathway from 2-ketobutyrate to propionyl-CoA is enhanced, and an engineering strain PS04 is obtained, and the specific steps are as follows:
(5-a) construction of a promoter replacement plasmid pK18-lac:: bkdR.
Designing a primer (pK 18-F/pK18(CAPup) -R) by using pK18 plasmid as a template for PCR amplification to obtain a plasmid DNA linear fragment of about 6000 bp;
selecting 500bp upstream of a bkdR gene as an upstream homology arm, taking 500bp in front of ORF of a bkdAB gene as a downstream homology arm, designing primers (the primers are 18-bkdup-F/bkdup-lac-R and lac-bkdown-F/bkdown-18-R respectively), taking genomic DNA of pseudomonas putida KT2440 as a template, and performing PCR amplification by using the primers to obtain a 500bp upstream homology arm DNA fragment and a 500bp downstream homology arm DNA fragment;
using pUCP18 plasmid as a template, designing a primer (the primer is bkdup-lac-F/lac-bkdown-R) to perform PCR amplification, and obtaining a lac promoter DNA fragment (sequence 2) of about 160 bp;
the upstream homology arm DNA fragment of 500bp, the downstream homology arm DNA fragment of 500bp, the lac promoter and the plasmid DNA linear fragment of about 6000bp are connected together by a Gibson method to obtain a promoter replacement plasmid pK18-lac, bkdR.
(5-b) plasmid pK18-lac:: bkdR was transferred into the starting strain PS03 using the binding transduction technique.
E.coli S17-1 is used as a plasmid donor for combined transduction to prepare the electrotransformation competence of S17-1;
the plasmid pK18-lac is characterized in that bkdR is transformed into S17-1 to obtain a strain containing S17-1 of the knock-out plasmid;
respectively inoculating the strain containing the S17-1 with the knockout plasmid and the original strain PS03 to an LB liquid culture medium, and activating overnight at 37 ℃ to obtain S17-1 bacterial liquid and PS03 bacterial liquid. 1mL of the suspension was centrifuged, and the supernatant was resuspended in 500. mu.L of LB liquid. mu.L of PS03 bacterial suspension was spotted on an antibiotic-free LB solid medium, and air-dried. And covering 15 mu L of S17-1 bacterial liquid on the PS03, and airing. Culturing at 37 deg.C for 16 h.
(5-c) two homologous recombinations.
The mixed colonies were scraped off with a coating rod and spread on LB solid medium containing gentamicin and chlorophenol at a final concentration of 25 mg/L. And coating a solid medium plate on every 4 colonies, and culturing at 37 ℃ for 24h, wherein the grown colonies are strains which complete the first homologous recombination and have the gentamicin resistance. And selecting a single colony, streaking the single colony on an LB solid culture medium containing 25mg/L chlorophenol and 20% sucrose at the final concentration, and culturing the single colony at 37 ℃ for 24 hours, wherein the grown colony is a knockout strain for completing the second homologous recombination. Single colonies were picked and cultured at 37 ℃ for 12 hours on LB solid medium containing chlorophenol at a final concentration of 25mg/L and LB solid medium containing gentamicin at a final concentration of 25mg/L, respectively. Single colonies that grew on the former and did not grow on the latter were picked for PCR validation.
And (3) performing amplification identification by using bkd-YF/bkd-YR primers, amplifying the strain without replacing the promoter to obtain a fragment of about 2000bp, amplifying the strain replacing the promoter to obtain a fragment of about 1500bp, and selecting the strain with a positive result to be named as PS 04.
Sequencing analysis results show that PS04 is a strain obtained by replacing the bkd gene cluster promoter of the PS03 strain with a lac promoter;
namely, the recombinant strain PS04 is obtained by replacing a prpC gene on a pseudomonas putida KT2440 genome with a tdcBC gene and a lac promoter (sequence 1) for driving the gene expression, knocking out an ltaE gene on the genome, replacing a bkd gene cluster promoter in the genome with a lac promoter (sequence 2), and keeping other sequences unchanged.
Example 2 Whole cell catalytic production of propionic acid Using the PS04 Strain
1. Culture of bacterial cells
The strain KT2440, the strains PS01, PS02, PS03 and PS04 prepared in example 1 were inoculated in LB liquid medium, respectively, and activated overnight at 37 ℃. The activated bacterial strains are respectively inoculated into a shake flask containing 50mL LB liquid culture medium according to the inoculum size of 1 percent of the volume percentage, cultured for 16h at 37 ℃ to a stationary phase, centrifuged for 5min at 5000g and collected to obtain KT2440 thallus, PS01 thallus, PS02 thallus, PS03 thallus and PS04 thallus.
2. Whole cell catalytic production of propionic acid
Respectively re-suspending the thallus collected in the step 1 in a test tube by using 10mM PBS buffer solution to obtain a bacterial suspension, OD600The values are all 10.
Respectively adding 40mM L-threonine into the bacterial suspension, performing shake culture at 37 ℃ for 6h, centrifuging at 10000g for 1min, taking supernatant, and filtering with a 0.22 μm 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: 18mM sulfuric acid; detection wavelength: 210 nm; sample introduction amount: 10 mu L of the solution; flow rate: 0.6 mL/min; column temperature: 35 ℃ is carried out.
The HPLC result of the propionic acid standard substance is shown in figure 2, and the retention time of propionic acid is about 13.38 min; the HPLC results of the sample are shown in FIG. 3, in which 11.801min shows a peak of 2 ketobutyric acid and 18.316min shows a peak of propionic acid.
The quantitative detection result shows that the yield of the propionic acid prepared by KT2440 is 0 mM; the yield of propionic acid prepared using PS01 was 2 mM; the yield of propionic acid prepared using PS02 was 6 mM; the yield of propionic acid prepared using PS03 was 20 mM; the yield of propionic acid prepared using PS04 was 32 mM.
The results show that the recombinant bacterium capable of producing propionic acid is successfully constructed.
Therefore, the recombinant bacterium is used for catalyzing threonine to obtain propionic acid, the route is shown in figure 1, and the enzymes for catalyzing the reaction in the route are as follows: (a) EC4.3.1.19, respectively; (b) EC1.2.4.4, EC 23.1.168, EC1.8.1.4; (c) EC6.2.1.1/EC 6.2.1.17.
Sequence listing
<110> institute of microbiology of Chinese academy of sciences
<120> construction of recombinant Pseudomonas putida and application thereof in production of propionic acid
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 2458
<212> DNA
<213> Artificial sequence
<400> 1
taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc 60
ggataacaat ttcacacagg aaacagctat gaccatgatt acgaattcga gctcgatgca 120
tattacatac gatctgccgg ttgctattga tgacattatt gaagcgaaac aacgactggc 180
tgggcgaatt tataaaacag gcatgcctcg ctccaactat tttagtgaac gttgcaaagg 240
tgaaatattc ctgaagtttg aaaatatgca gcgtacgggt tcatttaaaa ttcgtggcgc 300
atttaataaa ttaagttcac tgaccgatgc ggaaaaacgc aaaggcgtgg tggcctgttc 360
tgcgggcaac catgcgcaag gggtttccct ctcctgcgcg atgctgggta tcgacggtaa 420
agtggtgatg ccaaaaggtg cgccaaaatc caaagtagcg gcaacgtgcg actactccgc 480
agaagtcgtt ctgcatggtg ataacttcaa cgacactatc gctaaagtga gcgaaattgt 540
cgaaatggaa ggccgtattt ttatcccacc ttacgatgat ccgaaagtga ttgctggcca 600
gggaacgatt ggtctggaaa ttatggaaga tctctatgat gtcgataacg tgattgtgcc 660
aattggtggt ggcggtttaa ttgctggtat tgcggtggca attaaatcta ttaacccgac 720
cattcgtgtt attggcgtac agtctgaaaa cgttcacggc atggcggctt ctttccactc 780
cggagaaata accacgcacc gaactaccgg caccctggcg gatggttgtg atgtctcccg 840
cccgggtaat ttaacttacg aaatcgttcg tgaattagtc gatgacatcg tgctggtcag 900
cgaagacgaa atcagaaaca gtatgattgc cttaattcag cgcaataaag tcgtcaccga 960
aggcgcaggc gctctggcat gtgctgcatt attaagcggt aaattagacc aatatattca 1020
aaacagaaaa accgtcagta ttatttccgg cggcaatatc gatctttctc gcgtctctca 1080
aatcaccggt ttcgttgacg cttaattaat tcgttgagga taggatatga gtacttcaga 1140
tagcattgta tccagccaga caaaacaatc gtcctggcgt aaatcagata ccacatggac 1200
gttaggcttg tttggtacgg caatcggcgc cggggtgctg ttcttcccta tccgcgcagg 1260
ttttggcgga ctgatcccga ttcttctgat gttggtattg gcatacccca tcgcgtttta 1320
ttgccaccgg gcgctggcgc gtctgtgtct ttctggctct aacccttccg gcaacattac 1380
ggaaacggtg gaagagcatt ttggtaaaac tggcggcgtg gttatcacgt tcctgtactt 1440
cttcgcgatt tgcccactgc tgtggattta tggcgttact attaccaata cctttatgac 1500
gttctgggaa aaccagctcg gctttgcacc gctgaatcgc ggctttgtgg cgctgttcct 1560
gttgctgctg atggctttcg tcatctggtt tggtaaggat ctgatggtta aagtgatgag 1620
ctacctggta tggccgttta tcgccagcct ggtgctgatt tctttgtcgc tgatccctta 1680
ctggaactct gcagttatcg accaggttga cctcggttcg ctgtcgttaa ccggtcatga 1740
cggtatcctg atcactgtct ggctggggat ttccatcatg gttttctcct ttaacttctc 1800
gccaatcgtc tcttccttcg tggtttctaa gcgtgaagag tatgagaaag acttcggtcg 1860
cgacttcacc gaacgtaaat gttcccaaat catttctcgt gccagcatgc tgatggttgc 1920
agtggtgatg ttctttgcct ttagctgcct gtttactctg tctccggcca acatggcgga 1980
agccaaagcg cagaatattc cagtgctttc ttatctggct aaccactttg cgtccatgac 2040
cggtaccaaa acaacgttcg cgattacact ggaatatgcg gcttccatca tcgcactcgt 2100
ggctatcttc aaatctttct tcggtcacta tctgggaacg ctggaaggtc tgaatggcct 2160
ggtcctgaag tttggttata aaggcgacaa aactaaagtg tcgctgggta aactgaacac 2220
tatcagcatg atcttcatca tgggctccac ctgggttgtt gcctacgcca acccgaacat 2280
ccttgacctg attgaagcca tgggcgcacc gattatcgca tccctgctgt gcctgttgcc 2340
gatgtatgcc atccgtaaag cgccgtctct ggcgaaatac cgtggtcgtc tggataacgt 2400
gtttgttacc gtgattggtc tgctgaccat cctgaacatc gtatacaaac tgttttaa 2458
<210> 2
<211> 120
<212> DNA
<213> Artificial sequence
<400> 2
caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg 60
ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc 120

Claims (11)

1. A method for preparing recombinant bacteria comprises the following steps: deleting or inactivating the prpC gene on the pseudomonas putida genome to obtain the recombinant bacterium.
2. The method of claim 1, wherein: the method further comprises the steps of: deleting or inactivating the ltaE gene on the pseudomonas putida genome.
3. The method according to claim 1 or 2, characterized in that: the deletion or inactivation of the prpC gene on the pseudomonas putida genome is realized by replacing the prpC gene on the pseudomonas putida genome with a tdcBC gene and a lac promoter driving the expression thereof.
4. The method according to claim 1 or 2, characterized in that: the method further comprises the steps of: replacing the bkd gene cluster promoter in the Pseudomonas putida genome with a lac promoter.
5. The method according to claim 1 or 2, characterized in that: the replacement is carried out by adopting a homologous recombination mode;
alternatively, the replacement employs a lambda-red homologous recombination system or homologous recombination for sacB gene mediated screening.
6. The method according to claim 1 or 2, characterized in that: the pseudomonas putida is pseudomonas putida KT 2440.
7. The recombinant strain is obtained by replacing a prpC gene on a pseudomonas putida genome with a tdcBC gene and a lac promoter for driving the expression of the tdcBC gene, knocking out an ltaE gene on the genome, replacing an bkd gene cluster promoter in the genome with the lac promoter, and keeping other sequences unchanged;
or, the recombinant bacterium is obtained by replacing a prpC gene on a pseudomonas putida genome with a tdcBC gene, knocking out an ltaE gene on the genome and keeping other sequences unchanged;
or, the recombinant bacterium is obtained by replacing a prpC gene on a pseudomonas putida genome with a tdcBC gene and a lac promoter for driving the gene expression of the tdcBC gene, knocking out an ltaE gene on the genome, and keeping other sequences unchanged;
or, the recombinant bacterium is obtained by knocking out the prpC gene on the pseudomonas putida genome, knocking out the ltaE gene on the genome and keeping other sequences unchanged;
or, the recombinant bacterium is obtained by knocking out the prpC gene on the pseudomonas putida genome and keeping other sequences unchanged.
8. The recombinant bacterium according to claim 7, wherein: the knockout or the replacement is carried out by means of homologous recombination;
or, the knockout or the replacement employs a lambda-red homologous recombination system or homologous recombination for sacB gene mediated screening.
9. The recombinant bacterium according to claim 7, wherein: the pseudomonas putida is pseudomonas putida KT 2440.
10. A recombinant bacterium produced by the method of any one of claims 1 to 6;
or, the use of the recombinant bacterium of any one of claims 7 to 9 for producing or preparing propionic acid by using threonine.
11. A process for preparing propionic acid comprising the steps of: catalyzing threonine with the recombinant bacterium of any one of claims 7-9 to obtain propionic acid.
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CN106086056A (en) * 2016-06-13 2016-11-09 江南大学 A kind of pseudomonas putida gene knockout and genome simplified system
CN108728471A (en) * 2017-04-14 2018-11-02 中国科学院微生物研究所 Produce the recombinant bacterium and the preparation method and application thereof of 3- hydracrylic acids

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CN106086056A (en) * 2016-06-13 2016-11-09 江南大学 A kind of pseudomonas putida gene knockout and genome simplified system
CN108728471A (en) * 2017-04-14 2018-11-02 中国科学院微生物研究所 Produce the recombinant bacterium and the preparation method and application thereof of 3- hydracrylic acids

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