CN114107356A

CN114107356A - Method for transforming pseudomonas putida to assimilate D-galactose

Info

Publication number: CN114107356A
Application number: CN202111226302.7A
Authority: CN
Inventors: 郑兆娟; 周凤; 刘鹏; 欧阳嘉; 赵沁源
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2021-10-21
Filing date: 2021-10-21
Publication date: 2022-03-01
Anticipated expiration: 2041-10-21
Also published as: CN114107356B

Abstract

The invention relates to a method for transforming pseudomonas putida to assimilate D-galactose, which leads exogenous galactonic acid transport module and metabolism module in the pseudomonas putida, and combines adaptive laboratory evolution to lead the pseudomonas putida to be capable of efficiently assimilating and metabolizing the D-galactose. Based on the strategy, the recombinant pseudomonas putida capable of rapidly growing by taking D-galactose as a sole carbon source is obtainedPseudomonas putida) ZF21, deposited at the China center for type culture Collection at 10.09.2021 with the deposition number: CCTCC NO: M20211155.

Description

Method for transforming pseudomonas putida to assimilate D-galactose

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a method for transforming pseudomonas putida to assimilate D-galactose.

Background

Most of the fuels and chemicals worldwide are produced from petroleum. However, petroleum is a limited resource, and the synthesis of biofuels and bio-based chemicals from renewable resources is the current research focus for the sustainable development and future of mankind. Among them, biomass resources are the most abundant and potential sustainable alternative resources. For example, lignocellulose from terrestrial sources can be pretreated to obtain various monosaccharides including D-glucose, D-xylose, D-galactose, L-arabinose, etc.; the marine algae biomass such as red algae such as Eucheuma Gelatinosum and herba Solani Lyrati is rich in D-galactose and D-glucose. The conversion of sugars rich in plant biomass into commodity fuels and chemicals by microbial fermentation is an important way to achieve green sustainable development. Currently, this process still faces many challenges. One of the bottlenecks is the lack of availability of other carbohydrate substrates than D-glucose by many important industrial microorganisms.

Pseudomonas putida ATCC 47054 (namely Pseudomonas putida KT2440) is a new synthetic biology chassis which has attracted extensive interest of researchers in recent years, and the strain has high tolerance and transformation capability to stress factors (organic acids, furans, phenols and other compounds) in pretreated biomass hydrolysate. However, the strain cannot grow by using monosaccharide derived from other biomass besides D-glucose as a unique carbon source, and the application of the strain in the field of biorefinery is greatly limited. The invention aims to provide a novel modification strategy for endowing pseudomonas putida with high-efficiency D-galactose assimilation capability.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the defects of the prior art, the invention provides a method for transforming pseudomonas putida to assimilate D-galactose, so as to realize the efficient utilization of D-galactose by the pseudomonas putida and widen the utilization substrate spectrum of biomass sugar of the pseudomonas putida.

The technical scheme is as follows: in order to achieve the above object, the present invention provides a method for engineering pseudomonas putida to assimilate D-galactose, wherein recombinant pseudomonas putida is constructed by introducing an exogenous galactic acid transport module and metabolic module into pseudomonas putida, wherein the galactic acid transport module is a sequence encoding a protein that transports galactic acid from the periplasm of the cell to the cytoplasm, and the galactic acid metabolic module is a sequence encoding a protein that converts galactic acid in the cytoplasm into pyruvate and glyceraldehyde-3-phosphate in the central metabolic pathway.

Wherein, the source strain of the exogenous module for transporting and metabolizing galactic acid can be any bacteria containing the corresponding module, such as Pseudomonas aeruginosa (Pseudomonas rhodesiae), Escherichia coli (Escherichia coli), Azotobacter vinelandii (Azotobacter vinelandii), Pseudomonas fluorescens (Pseudomonas fluorescens), Lactobacillus crescentus (Caulobacter creescens) and the like, preferably, the sequence of the module for transporting galactic acid of Pseudomonas aeruginosa is shown in SEQ ID NO.3, and the sequence of the module for metabolizing galactic acid of Pseudomonas aeruginosa is shown in SEQ ID NO. 4; the Escherichia coli is Escherichia coli (Escherichia coli) BL21(DE3), the sequence of a galactonic acid transport module is SEQ ID NO.1, and the sequence of a galactonic acid metabolism module is SEQ ID NO. 2.

Specifically, the method for introducing the exogenous galactosyl acid transport and metabolism module is any one of the following methods:

(1) placing the exogenous galactaric acid transport module and the exogenous galactaric acid transport module on different plasmids and converting the plasmids into the pseudomonas putida, wherein the two recombinant plasmids are self-replicating and compatible in the pseudomonas putida;

(2) placing the exogenous galactoside acid transport module and the exogenous metabolic module on the same plasmid and converting the galactoside acid transport module and the exogenous galactoside acid transport module into the pseudomonas putida, wherein the recombinant plasmid meets the self-replication requirement in the pseudomonas putida;

or (3) integrating all exogenous galactosyl acid transport module and metabolic module into the genome of the pseudomonas putida, so that the exogenous galactosyl acid transport module and the metabolic module can be synchronously replicated with the genome.

More preferably, after obtaining the recombinant pseudomonas putida, performing laboratory adaptive evolution, preferably, the laboratory adaptive evolution comprises the following steps:

(1) selecting 1-2 rings of recombinant pseudomonas putida cells cultured by an LB (lysogeny broth) culture medium solid inclined plane, inoculating the cells into a container filled with a sterilized liquid LB culture medium for culture, tying a breathable sealing film, placing the cells on a shaking table, and culturing the cells for 10 +/-2 hours at the rotating speed of 200 +/-10 r/min at the temperature of 25-35 ℃ to obtain a seed liquid culture of the strain;

(2) inoculating the liquid seed culture obtained in the step (1) into a culture bottle filled with a sterilized evolution culture medium according to the inoculation amount of 0.5-5 v/v% for culture, tying a breathable sealing film, placing on a shaking table, and culturing at the temperature of 25-35 ℃ at the rotating speed of 100-400 r/min until the OD of cells reaches 0.4-0.8 to obtain a first generation cell culture of the strain;

(3) centrifuging the first generation cell culture obtained in the step (2), transferring the first generation cell culture to a fresh evolution culture medium, wherein the formula of the culture medium is the same as that in the step (2), and the operation method is the same as that in the step (2), so as to obtain a second generation cell culture of the strain;

(4) continuously repeating the step (3) to obtain a 10 th-30 th generation cell culture of the strain;

(5) when the cell growth cycle was stable, passage was stopped.

Preferably, in step (1), the formulation of the LB liquid medium is: 1L of distilled water contained: 10g peptone, 5g yeast powder, 10g NaCl, 50mg kanamycin. Agar powder with the final concentration of 15g/L needs to be additionally added into the LB liquid culture medium.

In the step (2), the formula of the evolution culture medium is as follows: 1L of distilled water contained: 1-10 g of D-galactose; na (Na)₂HPO₄，1～5g；KH₂PO₄ 1～3g；NH₄Cl 0.1～1g；NaCl 0.1～1g；MgSO₄0.1-0.4 g; 2-3 mL of a trace element solution and 50mg of kanamycin. The formula of the trace element solution is as follows: 1L of distilled water contained: h₃BO₃，0.1～0.5g；ZnCl₂，0.01～0.1g；MnCl₂·4H₂O，0.01～0.05g；CoCl₂，0.1～0.5g；CuCl₂·2H₂O，0.01～0.5g；NiCl₂·6H₂O，0.01～0.1g；NaMoO₄·2H₂O，0.01～0.05g。

Preferably, the formula of the evolution culture medium is as follows: 1L of distilled water contained: 3-6 g of D-galactose; na (Na)₂HPO₄ 3～4g；KH₂PO₄ 1.5～2g；NH₄Cl 0.3～0.6g；NaCl 0.2～0.4g；MgSO₄0.2-0.3 g; 2.2-2.6 mL of trace element solution. The formula of the trace element solution is preferably as follows: 1L of distilled water contained: h₃BO₃，0.3g；ZnCl₂，0.05g；MnCl₂·4H₂O，0.03g；CoCl₂，0.2g；CuCl₂·2H₂O，0.01g；NiCl₂·6H₂O，0.02g；NaMoO₄·2H₂O，0.03g。

The number of cell passages in the step (4) is preferably 15-20.

In a preferred embodiment, the invention uses Pseudomonas putida ATCC 47054(Pseudomonas putida KT2440, accession number ATCC No.47054, purchased from ATCC) as an initial strain, introduces an exogenous galactonic acid transport and metabolism module to obtain recombinant Pseudomonas putida, and combines with laboratory adaptive evolution (ALE) to realize the efficient utilization of D-galactose by the Pseudomonas putida. Wherein, the strain obtained after the laboratory adaptive evolution is preserved in China center for type culture Collection at 10/09/2021 with the preservation number: CCTCC NO: M20211155.

Has the advantages that: the invention provides a novel simple strategy for transforming pseudomonas putida to assimilate D-galactose, and the strain can be endowed with the growth capacity of taking D-galactose as a unique carbon source only by introducing two modules from an external source. The preferred strain CCTCC NO of M20211155 can rapidly metabolize D-galactose by combining with the adaptive evolution of a laboratory.

Drawings

FIG. 1 is a growth curve of Pseudomonas putida NL910, which is the engineered Pseudomonas putida constructed in example 4, with D-galactose as the sole carbon source;

FIG. 2 is a growth curve of Pseudomonas putida NL911 constructed in example 5 with D-galactose as the sole carbon source;

FIG. 3 is a growth curve of the strain Pseudomonas putida ZF21 obtained after laboratory adaptive evolution of Pseudomonas putida NL910 in example 6 with D-galactose as the sole carbon source.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The examples will help to understand the present invention given the detailed embodiments and the specific operation procedures, but the scope of the present invention is not limited to the examples described below.

Example 1 Pseudomonas putida ATCC 47054 was cultured with D-galactose as the sole carbon source.

(1) Pseudomonas putida ATCC 47054 was stored in 15% volume fraction glycerol and kept in an ultra-low temperature refrigerator for a long period of time. When activated, the cells were inoculated at an inoculum size of 1 v/v% into a shake tube containing sterilized 5mLLB liquid medium, and cultured at 30 ℃ for 12 hours at a rotation speed of 200r/min to obtain a cell-activated culture.

(2) The prepared cell activation culture was centrifuged at 8000r/min for 5 minutes, the supernatant was discarded, the cells were washed with physiological saline, and then inoculated into a 250mL conical flask containing 50mL of a sterilized inorganic salt medium containing 2 g/LD-galactose as a sole carbon source, and the OD of the initial cells after inoculation was 0.2.

(3) Cell turbidity was measured every 1 hour until 24 hours. In this example, there was no significant change in cell turbidity of Pseudomonas putida ATCC 47054 in a medium containing D-galactose as a sole carbon source, i.e., Pseudomonas putida ATCC 47054 could not grow on D-galactose as a sole carbon source.

The LB liquid culture medium comprises the following components in percentage by weight: 1L of distilled water contained: 10g of tryptone, 5g of yeast extract powder and 10g of sodium chloride.

The inorganic salt culture medium comprises the following components in percentage by weight: 1L of distilled water contained: na (Na)₂HPO₄ 3.39g；KH₂PO₄ 1.5g；NH₄Cl 0.5g；NaCl 0.25g；MgSO₄0.24 g; 2.5mL of trace element solution.

The formula of the trace element solution is as follows: 1L of distilled water contained: h₃BO₃，0.3g；ZnCl₂，0.05g；MnCl₂·4H₂O，0.03g；CoCl₂，0.2g；CuCl₂·2H₂O，0.01g；NiCl₂·6H₂O，0.02g；NaMoO₄·2H₂O，0.03g。

Example 2 transformation of D-galactose with Pseudomonas putida ATCC 47054 intact cells as catalyst.

(1) Preparing a biocatalyst: a loop of bacterial cells was inoculated from LB agar slant medium storing Pseudomonas putida ATCC 47054 into a shake tube containing 5mL of sterilized LB liquid medium, and cultured at 30 ℃ and 200rpm for 12 hours to activate the cells, thereby obtaining a cell-activating solution. Inoculating the cell activating solution into a 250mL conical flask filled with 50mL of sterilized LB liquid culture medium according to the volume percentage of 1%, and culturing for 12h under the conditions of 30 ℃ and 200rpm to obtain an amplification culture of the strain. And centrifuging at 10000r/min for 10min, collecting thallus cells, washing the thallus for 2 times by using a phosphate buffer solution, and separating to obtain the microbial intact cells, namely the biocatalyst.

(2) And (3) biotransformation: mixing the biocatalyst prepared in the step (1) with phosphate buffer solution containing 5g/L D-galactose, wherein the reaction system is 25mL, and the concentration of the biocatalyst is 1.6g dry weight/L. The reaction was carried out at 200rpm for 24h at 30 ℃.

(3) And (3) centrifuging the conversion solution obtained in the step (2) for 10min at 10000r/min, removing the biocatalyst added in the step (2), and carrying out HPLC detection on the obtained clear liquid.

(4) After 24h, no D-galactose is detected by HPLC, all D-galactose is oxidized into galactaric acid, and the obtained galactaric acid is not further metabolized.

The LB agar slant culture medium comprises the following components: 1L of distilled water contained: 10g of tryptone, 5g of yeast extract powder, 10g of sodium chloride and 15g of agar powder.

The formula of the phosphate buffer solution is as follows: na (Na)₂HPO₄ 7.59g/L，KH₂PO₄1.81g/L, and the solvent is distilled water.

Example 3 culture of Pseudomonas rhodosia CCTCC NO: M2021356 and Escherichia coli BL21, respectively, using galactonic acid as sole carbon source (DE 3).

(1) A loop of the bacterial cells was inoculated from an LB agar slant medium storing Pseudomonas rhododendrons CCTCC NO: M2021356 into a shake tube containing 5mL of a sterilized LB liquid medium, and cultured at 30 ℃ and 200rpm for 12 hours to activate the cells, thereby obtaining a cell activation solution. Meanwhile, a loop of bacterial cells was taken from LB agar slant medium storing Escherichia coli BL21(DE3), inoculated into a shake tube containing 5mL of sterilized LB liquid medium, and cultured at 30 ℃ and 200rpm for 12 hours to activate the cells, thereby obtaining a cell-activating solution.

(2) The two cell activation cultures were centrifuged at 8000r/min for 5 minutes, the supernatant was discarded, the cells were washed with physiological saline, and then they were inoculated into a 250mL conical flask containing 50mL of sterilized inorganic salt medium containing 5g/L of galactonic acid as a sole carbon source, and the OD of the initial cells after inoculation was 0.2.

(3) Cell turbidity during growth was measured. In this example, both Pseudomonas rhodosia CCTCC NO: M2021356 and Escherichia coli (DE3) were able to grow on galactaric acid, but the growth rate of Pseudomonas rhodosia CCTCC NO: M2021356 was 0.22h^-1The growth rate of Escherichia coli (DE3) was 0.18h^-1。

The inorganic salt medium formulation was the same as in example 1.

Example 4 construction of Pseudomonas putida Putida NL908, a P.putida engineered bacterium, and its culture using D-galactose as the sole carbon source.

(1) Construction of engineering bacteria: the genomic DNA of E.coli (Escherichia coli) BL21(DE3) was prepared by a conventional method, and reference was made to a method for minipreparation of bacterial genomes in "A. Nature molecular biology Manual" published by scientific Press. The synthetic primers dgoKADT-f1 and dgoKADT-r1 were used to obtain the galactosyl acid transport (SEQ ID No.1) and metabolic module (SEQ ID No.2) from the genomic DNA by PCR amplification.

Plasmid pSEVA2213(http:// seva. cnb. csic. es) was digested with XbaI and PstI, and the digested product was recovered by agarose gel and ligated with DNA ligase to obtain recombinant plasmid pSEVA 2213-EdgoKADT.

Wherein the primer sequence for amplifying the target fragment is as follows:

dgoKADT-f1：

(XbaI restriction sites underlined, ribosome binding sites in bold italics)

dgoKADT-r1：5’-atatCTGCAGttagccaacgcgcttcac-3' (PstI cleavage site underlined)

The recombinant plasmid pSEVA2213-EdgoKADT was electroporated into Pseudomonas putida ATCC 47054 competent cells. Pseudomonas putida ATCC 47054 was cultured in LB medium to the middle logarithmic phase, the cells were collected by centrifugation (4 ℃, 6000rpm, 10min) and washed twice with an electrotransfer buffer (10% glycerol, 1mM HEPES, 0.3M sucrose, pH 7.0), and the resulting cells were competent cells. mu.L of pSEVA 2213-dgokADAT plasmid at a concentration of 100 ng/. mu.L was mixed with 100. mu.L of competent cells, and the mixture was added to an electric cuvette (inner diameter: 2mm), and then electric shock was applied thereto under 2400V, 25. mu.F and 200. omega. conditions, followed immediately by addition of 500. mu.L of LB medium. Transferring the bacterial liquid in the electric rotating cup to a centrifuge tube, and culturing on a shaking table at 30 ℃ for 1h for cell recovery. After recovery, the cells are screened on an LB solid plate containing kanamycin resistance, grown colonies are selected and inoculated in an LB liquid culture medium containing kanamycin, the colonies are cultured for 12 hours at the temperature of 30 ℃, and the cells are collected and stored for later use. The resulting engineered strain was named Pseudomonas putida NL 908.

(2) Culturing the engineering bacteria obtained in the step (1) by taking D-galactose as a unique carbon source: selecting a ring 1 of pseudomonas putida engineering bacteria NL908 strain cultured by an LB culture medium solid inclined plane, inoculating the ring in a shake tube filled with 5mL of sterilized LB culture medium for culturing, tying a breathable sealing film, placing on a shaker, and culturing at the temperature of 30 ℃ at the rotating speed of 200r/min for 10h to obtain a seed liquid culture of the strain; wherein the LB culture medium formula is as follows: 1L of distilled water contained: 10g of peptone, 5g of yeast powder and 10g of NaCl; the LB solid culture medium is added with 15g/L agar powder on the basis of the formula; the LB medium contained 50mg/L kanamycin.

(3) The liquid cell culture of step (2) was centrifuged at 8000r/min for 5 minutes, the supernatant was removed, washed with physiological saline, and then introduced into a 250mL Erlenmeyer flask containing 50mL of sterilized inorganic salt medium with an initial OD of 0.2, the medium containing 2g/L D-galactose as the sole carbon source.

(4) Cell turbidity during growth was measured. After 48 hours, the OD of the cells reached a maximum of 1.0. The obtained recombinant Pseudomonas putida NL908 can grow by taking D-galactose as a unique carbon source.

The inorganic salt medium formulation was the same as in example 1.

Example 4 construction of Pseudomonas putida Putida NL910 and cultivation thereof with D-galactose as the sole carbon source.

(1) Construction of engineering bacteria: the genomic DNA of Pseudomonas rhododendrose CCTCC NO: M2021356 was prepared by a conventional method, and the procedure was referred to as a method for minipreparation of bacterial genome in "draft of molecular biology", published by scientific Press. The galactonic acid transporter (SEQ ID NO.3) and the metabolic module (SEQ ID NO.4) were obtained from the above genomic DNA by PCR amplification using synthetic primers dgoKADT-f2 and dgoKADT-r 2.

Plasmid pSEVA2213(http:// seva. cnb. csic. es) was digested with KpnI and XbaI, and the digested product was recovered by agarose gel and ligated with DNA ligase to obtain recombinant plasmid pSEVA 2213-PdgoKADT.

Wherein the primer sequence for amplifying the target fragment is as follows:

dgoKADT-f2：

(underlined KpnI restriction sites, bold italics ribosome binding sites)

dgoKADT-r2：5’-tcttTCTAGAttacagctcgatgcgctcgaccttgcc-3' (XbaI restriction site underlined)

The recombinant plasmid pSEVA2213-PdgoKADT was electroporated into Pseudomonas putida ATCC 47054 competent cells. Pseudomonas putida ATCC 47054 was cultured in LB medium to the middle logarithmic phase, the cells were collected by centrifugation (4 ℃, 6000rpm, 10min) and washed twice with an electrotransfer buffer (10% glycerol, 1mM HEPES, 0.3M sucrose, pH 7.0), and the resulting cells were competent cells. mu.L of pSEVA 2213-dgokADAT plasmid at a concentration of 100 ng/. mu.L was mixed with 100. mu.L of competent cells, and the mixture was added to an electric cuvette (inner diameter: 2mm), and then electric shock was applied thereto under 2400V, 25. mu.F and 200. omega. conditions, followed immediately by addition of 500. mu.L of LB medium. Transferring the bacterial liquid in the electric rotating cup to a centrifuge tube, and culturing on a shaking table at 30 ℃ for 1h for cell recovery. After recovery, the cells are screened on an LB solid plate containing kanamycin resistance, grown colonies are selected and inoculated in an LB liquid culture medium containing kanamycin, the colonies are cultured for 12 hours at the temperature of 30 ℃, and the cells are collected and stored for later use. The resulting engineered strain was designated Pseudomonas putida NL 910.

(2) Culturing the engineering bacteria obtained in the step (1) by taking D-galactose as a unique carbon source: selecting a ring 1 of pseudomonas putida engineering bacteria NL910 cultured by a solid slant of an LB culture medium, inoculating the ring in a shake tube filled with 5mL of sterilized LB culture medium for culturing, tying a breathable sealing film, placing the shake tube on a shaking table, and culturing at the temperature of 30 ℃ at the rotating speed of 200r/min for 10 hours to obtain a seed liquid culture of the strain; wherein the LB culture medium formula is as follows: 1L of distilled water contained: 10g of peptone, 5g of yeast powder and 10g of NaCl; the LB solid culture medium is added with 15g/L agar powder on the basis of the formula; the LB medium contained 50mg/L kanamycin.

(3) The liquid cell culture of step (2) was centrifuged at 8000r/min for 5 minutes, the supernatant was removed, washed with physiological saline, and then introduced into a 250mL Erlenmeyer flask containing 50mL of sterilized mineral salts medium with an initial OD of 0.2, the medium containing 2 g/LD-galactose as the sole carbon source.

(4) The cell turbidity during the growth was measured, and the growth curve was as shown in FIG. 1, and the OD of the cells was 1.2. The obtained recombinant Pseudomonas putida NL910 can grow by taking D-galactose as a unique carbon source.

The inorganic salt medium formulation was the same as in example 1.

Example 5 construction of Pseudomonas putida NL911 and cultivation thereof with D-galactose as the sole carbon source.

(1) Genomic DNAs of Pseudomonas putida ATCC 47054 and Pseudomonas rhodosia CCTCC NO: M2021356 were prepared by a conventional method, and reference is made to a method for the miniprep of bacterial genomes in "compendium molecular biology guidelines" published by scientific publishers. The upstream homology arm of PP _0545 (nucleic acid sequence: AAN66172.1) was PCR-amplified from Pseudomonas putida ATCC 47054 genomic DNA using synthetic primers up-f and up-r; the putative downstream homology arm of PP-0545 was PCR amplified from Pseudomonas putida ATCC 47054 genomic DNA using synthetic primers down-f and down-r. The galactaric acid transport and metabolism module was PCR amplified from Pseudomonas rhododendrose CCTCC NO: M2021356 genomic DNA using synthetic primers dgo-f and dgo-r.

The PCR product was purified with a purification kit, followed by

The operational manual of the Uni Seamless Cloning and Assembly Kit (Beijing Quanjin Biotechnology Co., Ltd.) links the galactose acid transport and metabolism modules to the upstream and downstream homology arms to obtain linear fragments. The obtained linear fragment was used as a template, and the fragment was amplified in large amounts using primers up-f and down-r.

The amplified fragment and the plasmid pK18mobSacB were digested with Xba I and Hind III, respectively, and the digested products were recovered by agarose gel and ligated with DNA ligase to obtain the recombinant plasmid pK18 mobSacB-dgo.

Wherein, the related primer sequences are as follows:

up-f：5’-atatTCTAGAgcggccagcggcacgccttc-3' (Xba I restriction site underlined)

up-r：5’-aatggctatcccacatagtgaccgcgtcgccttcttcgcg-3’

dgo-f：5’-gcggtcactatgtgggatagccatt-3’

dgo-r：5’-cgaccggcgcctctgaggct-3’

down-f：5’-cctcagaggcgccggtcgaagggttgggccagtgttgg-3’

down-r：5’-accgAAGCTTttggcagcgcaaatgcgcccatcgc-3' (HindIII restriction site underlined)

The recombinant plasmid pK18mobSacB-dgo was electrotransformed into Pseudomonas putida ATCC 47054 competent cells. For the methods of preparing competence and electrotransformation, see example 4. Colonies on the plates were picked and inoculated into LB liquid medium containing kanamycin and cultured at 30 ℃ to logarithmic numberAnd (5) middle stage. PCR verification of bacterial liquid is carried out by using the primers up-f and down-r, and the obtained bacterial strain which can simultaneously amplify long fragments and short fragments is the correct single-exchange target bacterium. The correct single-crossover objective strain was inoculated into LB liquid medium and cultured overnight at 30 ℃. The culture solution is transferred to an LB liquid culture medium containing 15% of sucrose to be cultured to the middle logarithmic phase, and is transferred to an LB liquid culture medium containing 15% of sucrose again to be cultured to the middle logarithmic phase. Diluted bacterial liquid (generally 10)^-6Or 10^-7) Spread onto LB solid plates containing 15% sucrose, and cultured at 30 ℃ until single colonies appear. Selecting single growing bacterium, and carrying out PCR verification on the bacterium liquid by using primers up-f and down-r to obtain a strain which can only amplify short segments, namely the correct double-exchange target bacterium, wherein a galactonic acid transport module and a metabolic module are integrated on the genome of the strain. The resulting engineered strain was named Pseudomonas putida NL 911.

(2) Culturing the engineering bacteria obtained in the step (1) by taking D-galactose as a unique carbon source: selecting a ring 1 of a pseudomonas putida engineering bacterium NL911 strain cultured by an LB culture medium solid inclined plane, inoculating the ring in a shake tube filled with 5mL of sterilized LB culture medium for culturing, tying a breathable sealing film, placing on a shaker, and culturing at the temperature of 30 ℃ at the rotating speed of 200r/min for 10 hours to obtain a seed liquid culture of the strain; wherein the LB culture medium formula is as follows: 1L of distilled water contained: 10g of peptone, 5g of yeast powder and 10g of NaCl; the LB solid culture medium is added with 15g/L agar powder on the basis of the formula; the LB medium contained 50mg/L kanamycin.

(3) The liquid cell culture of step (2) was centrifuged at 8000r/min for 5 minutes, the supernatant was removed, washed with physiological saline, and then introduced into a 250mL Erlenmeyer flask containing 50mL of sterilized mineral salts medium with an initial OD of 0.2, containing 2g/L D-galactose as the sole carbon source.

(4) The turbidity of the cells during the growth was measured, and the growth curve was as shown in FIG. 2. the OD of the cells after 48 hours was 1.3.

The inorganic salt medium formulation was the same as in example 1.

Example 6 laboratory adapted evolution of the engineered Pseudomonas putida NL910 Pseudomonas putida and cultivation of the evolved strain obtained with D-galactose as sole carbon source.

(1) A loop of bacterial cells was inoculated from LB agar slant medium storing Pseudomonas putida NL910 into a shake tube containing sterilized 5mLLB liquid medium, and cultured at 30 ℃ and 200rpm for 12 hours to activate the cells, thereby obtaining a cell-activating solution.

(2) Inoculating the liquid seed culture obtained in the step (1) into a culture bottle filled with a sterilized evolution culture medium according to the inoculation amount of 0.5-5 v/v% for culture, tying a breathable sealing film, placing on a shaking table, and culturing at the temperature of 30 ℃ at the rotating speed of 300r/min until the OD of cells reaches 0.5 to obtain a first generation cell culture of the strain;

(4) repeating the step (3) to obtain a 15 th generation cell culture of the strain;

(5) the cell culture obtained in step (4) was centrifuged at 8000r/min for 5 minutes, the supernatant was discarded, the cells were washed with physiological saline, and then they were inoculated into a 250mL conical flask containing 50mL of sterilized inorganic salt medium containing 2g/L D-galactose as a sole carbon source, and the OD of the initial cells after inoculation was 0.2. Cell turbidity during growth was measured. The growth curve is shown in FIG. 3, and the time for the cells to reach stationary phase is reduced from 48h to 22 h.

The evolution culture medium is as follows: 1L of distilled water contained: d-galactose, 5 g; na (Na)₂HPO₄ 3.5g；KH₂PO₄ 1.5g；NH₄Cl 0.5g；NaCl 0.25g；MgSO₄0.25 g; 2.5mL of trace element solution. The formula of the trace element solution is as follows: 1L of distilled water contained: h₃BO₃，0.3g；ZnCl₂，0.05g；MnCl₂·4H₂O，0.03g；CoCl₂，0.2g；CuCl₂·2H₂O，0.01g；NiCl₂·6H₂O，0.02g；NaMoO₄·2H₂O，0.03g。

The inorganic salt medium formulation was the same as in example 1.

Comparative example 1 construction of Pseudomonas putida Putida NL909 engineered Pseudomonas putida and cultivation thereof with D-galactose as the sole carbon source

(1) Construction of engineering bacteria: genomic DNA preparation of Pseudomonas rhodolesiae CCTCC NO: M2021356 referring to example 4, a galactic acid metabolic module was PCR-amplified from the above genomic DNA using synthetic primers dgokAD-f and dgokAD-r.

Wherein the primer sequence for amplifying the target fragment is as follows:

dgoKAD-f：

(the kpn I cleavage site is underlined, the ribosome binding site is in bold italics)

dgoKAD-r：5’-tattAAGCTTtcaccactcggcgaagctgc-3' (HindIII restriction site underlined)

The PCR amplified fragment and the plasmid pSEVA 644-DeltalacI (http:// seva. cnb. csic. es) were digested with Kpn I and Hind III, respectively, and the digested products were recovered by agarose gel and ligated with DNA ligase to obtain the recombinant plasmid pSEVA 644-DeltalacI-dgoKAD.

The recombinant plasmid pSEVA644- Δ lacI-dgokAD was electrotransformed into Pseudomonas putida ATCC 47054 competent cells. See, among others, example 4 for a competent preparation method and an electrotransformation method. After the electric transformation, the bacterial liquid in the electric rotating cup is transferred to a centrifuge tube, and cultured on a shaking table at 30 ℃ for 1h for cell recovery. After recovery, the cells are screened on an LB solid plate containing gentamicin resistance, and grown colonies are selected and inoculated in an LB liquid culture medium containing gentamicin and cultured to the middle logarithmic phase at the temperature of 30 ℃. The resulting engineered strain was named Pseudomonas putida NL 909.

(2) Culturing the engineering bacteria obtained in the step (1) by taking D-galactose as a unique carbon source: selecting a ring 1 of pseudomonas putida engineering bacteria NL909 strain cultured by an LB culture medium solid inclined plane, inoculating the ring in a shake tube filled with 5mL of sterilized LB culture medium for culturing, tying a breathable sealing film, placing on a shaker, and culturing at the temperature of 30 ℃ at the rotating speed of 200r/min for 10h to obtain a seed liquid culture of the strain; wherein the LB culture medium formula is as follows: 1L of distilled water contained: 10g of peptone, 5g of yeast powder and 10g of NaCl; the LB solid culture medium is added with 15g/L agar powder on the basis of the formula; the LB medium contained 50mg/L kanamycin.

(3) The liquid cell culture of step (2) was centrifuged at 8000r/min for 5 minutes, the supernatant was removed, washed with physiological saline, and then introduced into a 250mL Erlenmeyer flask containing 50mL of sterilized inorganic salt medium with an initial OD of 0.2, the medium containing 2 g/LD-galactose as the sole carbon source.

(4) Cell turbidity during growth was measured up to 48 h. In this example, the cell turbidity of the engineering bacterium NL909 in the culture medium using D-galactose as the sole carbon source has no significant change, that is, the engineering bacterium NL909 cannot grow using D-galactose as the sole carbon source.

The inorganic salt medium formulation was the same as in example 1.

The present invention provides a novel strategy for transforming Pseudomonas putida to assimilate D-galactose, and many ways to implement the technical scheme, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and embellishments can be made without departing from the principle of the present invention, and these modifications and embellishments should also be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.

Sequence listing

<110> Nanjing university of forestry

<120> method for transforming pseudomonas putida to assimilate D-galactose

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1338

<212> DNA

<213> nucleotide Sequence of galactosate transport Module (Artificial Sequence)

<400> 1

atggtgagcg gcttcgctat gcccaaaatc tggagaaagc tcgctatgga tattcccgtt 60

aatgcagcaa agccggggcg tcggcgttat ctgacgctgg tgatgatctt tattacggtg 120

gtcatttgtt atgttgaccg cgctaacctg gccgtggctt ccgcccatat tcaggaagag 180

ttcggcatta cgaaagcgga aatgggctat gtattttcgg ccttcgcctg gctttatacg 240

ctatgccaga tccccggcgg ttggttttta gatcgcgtag gttctcgcgt gacttatttt 300

attgcgatat ttggttggtc agtggcgact ttattccagg gctttgccac gggattaatg 360

tcattaattg gtctgcgtgc gataaccggt attttcgaag cgcccgcttt cccgaccaat 420

aaccggatgg tgaccagctg gttcccggaa catgaacgcg cttccgccgt tggtttttat 480

acgtctggtc agtttgtcgg tctggcgttt ttgactccgc tgctgatctg gattcaggag 540

atgttgagct ggcactgggt gttcattgtc actggtggta tcggcattat ctggtcgctg 600

atttggttta aggtttatca gccgccgcgc ctgaccaaag gtatcagcaa agctgaactg 660

gattacattc gtgatggcgg cggtctggtg gatggtgatg cgccggtgaa gaaagaggcg 720

cgtcagccgt taacagccaa agactggaaa ctggtgttcc atcgtaaact gatcggcgtt 780

tatcttgggc aatttgcggt ggcttctaca ctgtggtttt tcttaacctg gttcccgaac 840

tatttaaccc aggaaaaagg aatcacggcg ctgaaagcag gctttatgac cacggtgcca 900

ttcctcgcgg cgtttgtcgg cgtcctgctc tctggctggg tcgcggatct gctggtacgt 960

aagggctttt cactgggctt tgcgcgtaaa acgccgatta tctgcggctt gctgatctcc 1020

acctgcatta tgggcgctaa ctacaccaac gatccgatga tgattatgtg cctgatggcg 1080

ctggcattct tcggcaacgg ttttgcttcg attacctggt cgctggtctc ttctctggca 1140

ccgatgcgcc tgattggttt aaccggcggc gtgtttaact tcgccggtgg tctgggcggc 1200

atcaccgttc cgctggtggt ggggtacctg gcgcagggtt acggtttcgc acctgcactg 1260

gtttatatct ccgccgtcgc gttgattggc gcgctctctt acatcctgct ggtggacgat 1320

gtgaagcgcg ttggctaa 1338

<210> 2

<211> 2625

<212> DNA

<213> nucleotide Sequence of galactose acid metabolism Module (Artificial Sequence)

<400> 2

atgacagctc gctacatcgc aattgactgg ggatcgacca atctgcgcgc ctggctttat 60

cagggcgacc actgcctgga gagcaggcaa tcagaagcag gcgtcacgcg cctgaacgga 120

aaatctccgg ctgcggtgtt agcagaagtc acgaccgact ggcgtgaaga gaatacgcca 180

gtggtaatgg caggaatggt cggcagcaat gtcggctgga aagttgctcc gtatttatct 240

gttcctgccc gtttttcgtc tattggcgaa caattaacgt ctgttggcga caatatctgg 300

attattcccg gattatgtgt ctctcatgac gataaccaca atgtgatgcg cggcgaagaa 360

acacaattga tcggcgcgcg agctctggct ccttcctctc tttatgtcat gcccggaacc 420

cattgcaaat gggtgcaggc cgatagccag caaatcaacg attttcgcac cgtgatgacc 480

ggtgaattac atcatttact gttaaatcac tcattgattg gcgcaggttt gccgccgcag 540

gaaaactctg ccgatgcctt cgcggctggt cttgagcgtg gtcttaatac gcccgccata 600

ttgccgcagc tttttgaagt tcgcgcctcg catgtgctgg gaacacttcc ccgcgaacag 660

gtcagcgaat ttctctctgg tttgttgatt ggtgcagagg tcgccagtat gcgcgactat 720

gtggcccatc aacacgccat cacccttgtc gccggaacat cgctgaccgc gcgctaccag 780

caagcctttc aggcgatggg ttgcgacgtg acggcggtgg cgggcgacac ggcatttcag 840

gctggtataa ggagcatcgc tcatgcagtg gcaaactaaa ctcccgctga tcgccatttt 900

gcgcggtatt acgcccgacg aggcgctggc gcatgttggc gcggtgattg acgccgggtt 960

cgacgcggtt gaaatcccgc tgaattcccc acaatgggag caaagcattc ccgccatcgt 1020

tgatgcgtac ggcgacaagg cgttgattgg cgcaggtacg gtactgaaac ctgaacaggt 1080

cgatgcgctc gccaggatgg gctgtcagct catcgttacg cccaatatcc atagtgaagt 1140

gatccgccgt gcggtgggct acggcatgac cgtctgcccc ggctgcgcga cggcgaccga 1200

agcctttacc gcgctcgaag cgggcgcgca ggcgctgaaa atatttccgt catcggcttt 1260

tggtccgcaa tacatcaaag cgttaaaagc ggtattgcca tcggacatcg cagtctttgc 1320

cgttggcggc gtgacgccag aaaacctggc gcagtggata gacgcaggtt gtgcaggggc 1380

gggcttaggc agcgatctct atcgcgccgg gcaatccgta gagcgcaccg cgcagcaggc 1440

agcagcattt gttaaggcgt atcgagaggc agtgcaatga aaatcaccaa aattaccacg 1500

tatcgtttac ctccccgctg gatgttcctg aaaattgaaa ccgatgaagg cgtggtcggt 1560

tggggcgagc ccgtgatcga aggccgcgcc cgtacggtgg aagcggcagt tcacgagctg 1620

ggtgactatt tgattggtca ggatccatcg cgcatcaatg acttatggca agtgatgtat 1680

cgcgccggat tctatcgcgg cggtccgatc ctgatgagcg ccatcgccgg gattgaccag 1740

gcattatggg atatcaaagg taaagtgctg aatgcgccgg tctggcaact gatgggcggc 1800

ctggttcgcg acaaaattaa agcctacagt tgggttggcg gcgatcgtcc ggcggatgtt 1860

atcgacggca ttaaaacgct acgcgaaatc ggcttcgata ccttcaaact gaacggttgt 1920

gaagaactgg ggctaattga taactcccgc gcggtagatg cggcggttaa caccgtggca 1980

caaattcgtg aagcttttgg caatcagatt gagtttggtc ttgatttcca cggtcgcgtc 2040

agcgcgccga tggcgaaagt gctgattaaa gaactggagc cgtatcgccc gctgtttatt 2100

gaggagccgg tgctggcgga acaagccgaa tactacccga aactggcggc acaaacgcat 2160

attccactgg cggcgggtga acgcatgttc tcacgcttcg attttaaacg cgtgctggag 2220

gcaggcggta tttcgattct gcaaccggat ctctcccacg cgggcggtat taccgaatgc 2280

tacaaaatcg ccggaatggc agaagcctat gacgtgaccc ttgcgccgca ctgtccgctc 2340

ggaccgattg cactggcggc ttgcctgcat atcgactttg tttcctataa cgccgtactt 2400

caggaacaaa gtatgggaat tcattacaac aaaggcgcgg agttactcga ctttgtgaaa 2460

aacaaagaag acttcagcat ggtcggcggc ttctttaaac cgttaacgaa accgggctta 2520

ggcgtggaaa tcgacgaagc taaagtgatt gagttcagta aaaatgcccc ggactggcgt 2580

aatccgctct ggcgtcatga agataacagc gtagcagagt ggtaa 2625

<210> 3

<211> 1311

<212> DNA

<213> nucleotide Sequence of galactosate transport Module (Artificial Sequence)

<400> 3

atgcaacctg aatccttgac cgggcaggct cctttagtca cgcctagccg aaagcgtttc 60

ttcatcatgg tgctgctgtt tatcaccgtg gtgatcaact acctggaccg cagcaatctg 120

tcgattgcag cgccggccct gaccagtgac ctgggtatcg acccggtgca cgtcggcctg 180

attttctcgg ccttcggctg gacctacgcg gccatgcaaa tccccggcgg ctggctggtg 240

gaccgggtgc cgccgcgcat tctctacacc gctgccctgc tgctgtggtc catcgccacg 300

gtgatgctcg ggtttgccgc cagctttatt gcgctgtttg tgctgcgcat ggcggtcggt 360

gcgttggaag cgccggccta tccgatcaac agccgcgtgg tcaccagttg gtttcccgag 420

cgtgaacgcg ccacggcgat tggcttttac acctccgggc agttcgtcgg gctggcgttc 480

ctgacaccgg tcttggcttg gctgcagcat cactatggct ggcacatggt gtttgtggtg 540

accggtggcg tcggcattct gtgggcggcg atctggtacg cggtgtaccg cgagccgcgt 600

gacttcaaag gtatcaacca ggccgaaatc gacctgatcc gcgacggtgg tgggctggtg 660

gacctcagca cccaagcggc caagaccaag acgccgttca gctgggtcga cctgggtatt 720

gtcctgagca agcgcaaact gtgggggatc tacctcgggc agttctgcct gaactccacg 780

ctgtggttct tcctgacgtg gttcccgacg tatctggtga agtaccgcgg catggacttc 840

atcaagtccg gcatgctcgc gtcgctgcca ttccttgcgg cattcgttgg ggtgctgtgt 900

tccgggctgt tttccgactg gctgatccgg cgcggcgcga gcgtggggtt tgcgcgcaag 960

ttgccgatca tcagcggcct gctgatctcc acggcgatca tcggcgccaa cttcgtcgaa 1020

tcgactccgt tggtcatcgc cttcctggcc gtggcgttct tcggcaacgg gctggcttcg 1080

atcacttggt cgctggtctc gaccctcgcc ccggcgcgct tgttgggcct gaccggcggg 1140

gtgttcaact tcatcggcaa cctggcggcg attaccacgc caatcgtcat cggtttcctg 1200

gccagcggcg attcgtttgc gccggcgatc acctacatcg cgctgctggc gttgctgggg 1260

gcgttgtcct acgtgctgct ggtaggcaag gtcgagcgca tcgagctgta a 1311

<210> 4

<211> 2784

<212> DNA

<400> 4

atgcaggcgc aattgatcgc gctcgactgg gggaccagct cccttcgtgc ttacaaactc 60

ggccccgcag gcgtggtgct ggaacaacgc tcgctggcgt cgggcatcat gcatctgccc 120

agcgaacccc gcgacattgc cggcgtgcgc tgcagcgatg gttttgagtt ggcgttcgac 180

gcagcgtgtg gagattggct cgacgccgag ccacacctgc cggtgatcgc ctgcggcatg 240

gtgggcagcg ctcagggctg gagcgaagcg gcataccgca atacgccggt ggacgtcgcc 300

agtctcggca aagccttgca caccgtacgc agcctgcgtg gcgtggcggt acatatcgtg 360

cccggcgtga tcgagcaagg ccgcttgccc aacgtaatgc gcggcgaaga aacccaggtg 420

ctcggcgtgc tgcacaaccg ggcggccggc ggtgaagtct tgatcggcct gccgggcagc 480

cactccaagt gggtcgaggt ggtcggtggc tgcatcaccc atttcgacac ctttatgacc 540

ggtgaagtgt tcgcgatcct cagcaagcac agcattctgg ggcgtacgca acagccttgc 600

gaacatttcc aggccgaagc ctttgatcgc ggcgtgcaag tggcgcgctc tcaggatggc 660

cagcgcggcg tgctgtcgac gctgttcagc gcccgcactc tgggcctgac cgccgaactc 720

gcgcccgacc agcaagcgga ctacctgtcc ggtctgatga tcggtcatga gctggccggt 780

ctgcctgagc gcgctcggca caccccgatc atcctggtcg gcgccggccc tttatgcgcg 840

cgctaccaac gcgccctcgc cctctgcggc tttgcccatg tcagcctggc cgaagaagcc 900

accgagcgcg gcctgtggca attggcgctg gcggccgggc tcactcaacc tgcaacggag 960

gcctgacatg ctcaagcaag cactcgctca aaacggtttg atcgccatcc tgcgtggcct 1020

gcgcacggag gaagcccagg cggtcggcca ggtgctgtac caggccggct ttcgcgtgat 1080

cgaagtaccc cttaattcac cggaccctta caccagcatc cgcatcctgc gtgacagctt 1140

gccagccgat tgcctgatcg gtgccggcac ggtgttgacg cccgagcagg tcgagcaggt 1200

gaaagccgcc ggtggccaag tgatcgtcat gccccacagt gacgccaagg tattgcacgc 1260

tgccaaagcg gcgggcctgt acctgtcgcc gggtgtggct acgcccaccg aggcctttgc 1320

cgcactggcc gaaggcgccg acgtgttgaa gctgttcccg gccgagcaaa tgggcccggc 1380

ggtgatcaag gcgtggttgg cggtattgcc ggcgggcacg ttgttgctgc cggtgggcgg 1440

cattaccccg gacaacatgc aggtgtttac cgacgccggc gccaaggggt tcgggctggg 1500

ttccgggttg ttcaaaccgg gtatgacagt ggatcaggta gcgagccgcg cccaggctta 1560

tgtcgccgcc tggaaagccc tgagctgaat tcaagttctg cgcccctggc gctgcatcta 1620

caagagagat aacagatgaa aatcaccaaa ctgacgacct ttatcgtccc gccgcgctgg 1680

tgcttcctga aggtcgaaac cgaccagggc gtgaccggct ggggtgagcc tgtggtcgaa 1740

ggccgcgccc acaccgtggc tgctgccgtt gaagaactgt ccgactacct gatcggtaaa 1800

gacccacgca acatcgaaga catctggacc gtgctctacc gcggcggttt ctaccgtggc 1860

ggcgcggtgc acatgagcgc cctcgccggt atcgaccagg cgctatggga catcaagggc 1920

aaggccctcg gcgtttccgt cagcgatctg ctgggcggcc aggtgcgcga caaaatccgc 1980

gtgtattcat ggatcggcgg cgaccgcccc gctgatactg cccgcgcggc taaagacgcc 2040

gtggcgcgtg gtttcactgc ggtgaaaatg aacggcaccg aagagctgca attcctcgac 2100

agcttcgaaa aagtcgacct ggccctggcc aacgtcgccg ccgtgcgtga tgcggtcggc 2160

cccaacgtcg gcatcggcgt cgacttccac ggccgcgtgc acaagccgat ggccaaagtg 2220

ctgatgaaag agctggaccc gtacaagctg atgtttatcg aagagcccgt actcagcgaa 2280

aactacgaag cgctcaagga actggcgccg ttgaccagca ccccgatcgc cctgggcgag 2340

cgcctgttct cgcgttggga tttcaagcgc gtgctcagcg aaggttatgt cgacatcatc 2400

cagccggacg cctcccacgc cggcggcatc accgaaaccc gcaagatcgc taacatggcc 2460

gaagcctacg acgtagcgct ggccctgcac tgcccgctgg gcccgattgc gctggcggcg 2520

tgcctgcaac tggatgcggc ctgctacaac gcgtttatcc aggagcaaag cctggggatc 2580

cactacaacg agagcaacga cttgctcgac tacgtgcgtg accccggcgt gttcgattat 2640

gagcagggct tcgtcaagat ccctaacggc ccgggcctgg gcatcgagat caacgaggaa 2700

tacgtgatcg agcgcgctgc catcggccac cgctggcgca acccgatctg gcgccacgcc 2760

gacggcagct tcgccgagtg gtga 2784

<210> 5

<211> 46

<212> DNA

<213> dgoKADT-f1(dgoKADT-r2(Artificial Sequence))

<400> 5

cgcgtctaga tagcaagagg aatataccat gacagctcgc tacatc 46

<210> 6

<211> 28

<212> DNA

<213> dgoKADT-r1(dgoKADT-r2(Artificial Sequence))

<400> 6

atatctgcag ttagccaacg cgcttcac 28

<210> 7

<211> 49

<212> DNA

<213> dgoKADT-f2(Artificial Sequence)

<400> 7

agagggtacc ctttaagaag gagatataca tatgcaggcg caattgatc 49

<210> 8

<211> 37

<212> DNA

<213> dgoKADT-r2(Artificial Sequence)

<400> 8

tctttctaga ttacagctcg atgcgctcga ccttgcc 37

<210> 9

<211> 30

<212> DNA

<213> up-f(Artificial Sequence)

<400> 9

atattctaga gcggccagcg gcacgccttc 30

<210> 10

<211> 40

<212> DNA

<213> up-r(Artificial Sequence)

<400> 10

aatggctatc ccacatagtg accgcgtcgc cttcttcgcg 40

<210> 11

<211> 25

<212> DNA

<213> dgo-f(Artificial Sequence)

<400> 11

gcggtcacta tgtgggatag ccatt 25

<210> 12

<211> 20

<212> DNA

<213> dgo-r(Artificial Sequence)

<400> 12

cgaccggcgc ctctgaggct 20

<210> 13

<211> 35

<212> DNA

<213> down-r(Artificial Sequence)

<400> 13

accgaagctt ttggcagcgc aaatgcgccc atcgc 35

<210> 14

<211> 49

<212> DNA

<213> dgoKAD-f(Artificial Sequence)

<400> 14

aattggtacc ctttaagaag gagatataca tatgcaggcg caattgatc 49

<210> 15

<211> 30

<212> DNA

<213> dgoKAD-r(Artificial Sequence)

<400> 15

tattaagctt tcaccactcg gcgaagctgc 30

Claims

1. A method for engineering Pseudomonas putida to assimilate D-galactose, wherein a recombinant Pseudomonas putida is constructed by introducing an exogenous galactic acid transport module and metabolic module into Pseudomonas putida, wherein the galactic acid transport module is a sequence encoding a protein that transports galactic acid from the periplasm of the cell to the cytoplasm, and the galactic acid metabolic module is a sequence encoding a protein that converts galactic acid in the cytoplasm into pyruvate and glyceraldehyde-3-phosphate in the central metabolic pathway.

2. The method of claim 1, wherein the source strain of exogenous galactonic acid transport and metabolic modules is Pseudomonas Hoffii (Pseudomonas aeruginosa)Pseudomonas rhodesiae) Escherichia coli (E.coli)Escherichia coli) Azotobacter vinelandii (A)Azotobacter vinelandii) Pseudomonas fluorescens (A)Pseudomonas fluorescens) And Bacillus crescentus: (Caulobacter crescentus) Any one of them.

3. The method according to claim 2, wherein the pseudomonas hopcalis is pseudomonas hopcalis with the preservation number of CCTCC NO: M2021356, wherein the sequence of the galactaric acid transport module of the pseudomonas hopcalis is shown as SEQ ID NO.3, and the sequence of the galactaric acid metabolism module of the pseudomonas hopcalis is shown as SEQ ID NO. 4; the Escherichia coli is Escherichia coli (A)Escherichia coli) BL21(DE3) with the sequence of the galactaric acid transport module being SEQ ID No.1 and the sequence of the galactaric acid metabolism module being SEQ ID No. 2.

4. The method of claim 1, wherein the means for introducing the exogenous galactonic acid transport and metabolism module is any one of the following methods:

(1) respectively placing the exogenous galactonic acid transport module and the exogenous metabolic module on two different plasmids, and then transforming the plasmids into the pseudomonas putida, wherein the two recombinant plasmids meet the self-replication and compatibility in the pseudomonas putida;

(3) the exogenous galactoside acid transport module and the exogenous metabolic module are all integrated into the genome of the pseudomonas putida and are synchronously replicated with the genome.

5. The method of claim 1, further comprising the step of subjecting the obtained Pseudomonas putida race to laboratory adaptive evolution.

6. The method of claim 4, wherein the laboratory adaptive evolution comprises the steps of:

(5) when the cell growth cycle was stable, passage was stopped.

7. The method of claim 6, wherein in step (2), the evolution medium formula is: 1L of distilled water contained: d-galactose 1-10 g、Na₂HPO₄ 1~5 g、KH₂PO₄ 1~3 g、NH₄Cl 0.1~1g、NaCl 0.1~1 g、MgSO₄0.1-0.4 g, 2-3 mL of trace element solution and 50mg of kanamycin; the formula of the trace element solution is as follows: 1L of distilled water contained: h₃BO₃ 0.1~0.5 g、ZnCl₂ 0.01~0.1 g、MnCl₂·4H₂O 0.01~0.05 g、CoCl₂ 0.1~0.5 g、CuCl₂·2H₂O 0.01~0.5 g、NiCl₂·6H₂O 0.01~0.1g、NaMoO₄·2H₂O 0.01~0.05 g。

8. The method according to claim 6, wherein the number of passages of the cells in the step (4) is 15 to 20.

9. A recombinant pseudomonas putida is characterized in that the recombinant pseudomonas putida is prepared by using pseudomonas hopcalis (A) with a preservation number of CCTCC NO: M2021356Pseudomonas rhodesiae) The galactose acid transport module and the metabolic module of the pseudomonas putida are connected to a plasmid pSEVA2213 to obtain a recombinant plasmid, then the recombinant plasmid is electrically transformed into pseudomonas putida ATCC 47054 competent cells, and an LB solid plate containing kanamycin resistance is used for screening to obtain the recombinant pseudomonas putida, wherein the sequence of the galactose acid transport module of the pseudomonas hopohii is shown in SEQ ID No.3, and the sequence of the galactose acid metabolic module of the pseudomonas hopohii is shown in SEQ ID No. 4.

10. Recombinant pseudomonas putidaPseudomonas putida) ZF21, characterized in that it is obtained by laboratory adapted evolution of recombinant Pseudomonas putida according to claim 9 (ZF: (ZF))Pseudomonas putida) ZF21 was deposited at the chinese type culture collection at 2021, 09/10 under the following accession numbers: CCTCC NO: M20211155.