CN113073072B

CN113073072B - Pseudomonas putida engineering bacterium and application thereof

Info

Publication number: CN113073072B
Application number: CN202110361120.4A
Authority: CN
Inventors: 郑兆娟; 王静; 谭黄虹; 欧阳嘉; 刘青
Original assignee: Nanjing Forestry University
Current assignee: Nanjing Forestry University
Priority date: 2021-04-02
Filing date: 2021-04-02
Publication date: 2022-08-26
Anticipated expiration: 2041-04-02
Also published as: CN113073072A

Abstract

The invention discloses a pseudomonas putida strainPseudomonas putida) T05, which has been deposited at the China center for type culture Collection at 03-15.2021 under the following deposition numbers: CCTCC NO: M2021227. The invention discloses a method for preparing galactaric acid by using galactose and a biological catalysis method, wherein galactaric acid is an attractive biological-based platform compound, the method uses D-galacturonic acid rich in pectin biomass as a substrate to construct a pseudomonas putida engineering bacterium for efficiently oxidizing D-galacturonic acid to produce galactaric acid, the efficient synthesis of the galactaric acid is realized, and the production capacity of the galactaric acid is obviously superior to that of the reported biological catalysis method.

Description

Pseudomonas putida engineering bacterium and application thereof

Technical Field

The invention relates to the field of biocatalytic conversion, and particularly relates to a pseudomonas putida engineering bacterium and application thereof.

Background

With the increasing scarcity of fossil resources and the exacerbation of environmental problems, the production of energy and chemicals based on renewable resources is receiving increasing attention from researchers. Biomass is the only renewable carbon resource, and the conversion of biomass into platform compounds and bulk chemicals is an important development direction for the high-value utilization of biomass. Galactaric acid is an attractive bio-based platform compound, has a structure similar to glucaric acid, belongs to the same class of hexose diacid, and has a potential application field similar to glucaric acid. Glucaric acid is typically prepared chemically or biologically from glucose in lignocellulosic biomass as a precursor, while galactaric acid is oxidized from D-galacturonic acid in pectin biomass. Researchers are interested in galactose diacid relatively late compared to glucaric acid, and their preparation methods and application fields are under continuous development.

The reaction condition for synthesizing the galactaric acid by biological catalysis is mild, the selectivity is high, the process is simple, and the method is green and environment-friendly. The field is currently in the beginning. Among the strains that have been used for galactaric acid production are Aspergillus niger (Aspergillus niger), Trichoderma reesei (Trichoderma reesei), Coniochaeta sp, Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Escherichia coli (Escherichia coli). The galactose diacid produced by the strains has a common characteristic: firstly, the original reduction or isomerization pathway of D-galacturonic acid is damaged, then the metabolic pathway of galactaric acid is further damaged, and finally, the corresponding D-galacturonic acid oxidizing capability is endowed to the strain. The productivity of these catalysts is not satisfactory in terms of the yield and productivity of galactaric acid. At present, no report exists on the efficient production of galactaric acid by using Pseudomonas putida (Pseudomonas putida) as a catalyst.

Disclosure of Invention

The invention aims to: aiming at the defects of the prior art, the invention provides a pseudomonas putida engineering bacterium and a method for synthesizing galactaric acid by using the pseudomonas putida engineering bacterium through biocatalysis, so that the production process is simplified, and the yield, the yield and the productivity of the galactaric acid are improved.

The engineering bacteria are Pseudomonas putida engineering bacteria which are named as Pseudomonas putida (Pseudomonas putida) T05, the strains are preserved in China Center for Type Culture Collection (CCTCC) in 03-15 months in 2021, and the preservation numbers are as follows: CCTCC NO: M2021227, address: china, wuhan university.

The above-mentioned Pseudomonas putida engineered bacterium (Pseudomonas putida) T05 was constructed by the following method.

Pseudomonas putida ATCC47054 (Pseudomonas putida KT2440, accession number ATCC No.47054, purchased from ATCC) is used as an initial strain, and the growth rate and D-galacturonic acid metabolism rate of the strain in an inorganic salt medium using D-galacturonic acid as a unique carbon source are improved after ion beam mutagenesis. And (3) deleting possible galactaric acid metabolism genes in the mutant bacteria by utilizing a homologous recombination technology until the strain cannot metabolize the galactaric acid. And further overexpressing glucose dehydrogenase and uronate dehydrogenase in the strain, and finally completing the construction of the pseudomonas putida engineering bacterium T05.

Wherein the glucose dehydrogenase is preferably derived from Pseudomonas putida (Pseudomonas putida) ATCC47054, and the gene nucleotide sequence is shown as SEQ ID NO. 1; the uronate dehydrogenase is preferably derived from radiorhizobium radiobacter (Agrobacterium tumefaciens), ATCC 33970, and has a gene nucleotide sequence shown in SEQ ID NO. 2.

The application of the pseudomonas putida engineering bacteria and the new pseudomonas putida engineering bacteria prepared by taking the pseudomonas putida engineering bacteria as the starting bacteria in preparing the galactaric acid is also within the protection scope of the invention.

Wherein, the application comprises the following steps:

(1) selecting 1-2 rings of a Pseudomonas putida (Pseudomonas putida) T05 strain cultured by an LB (lysogenum Bromide) culture medium solid slant, inoculating the rings into a container filled with a sterilized LB culture medium for culturing, tying a breathable sealing film, placing the container on a shaking table, and culturing at the temperature of 30 ℃ for 12 +/-2 hours at the rotating speed of 200 +/-10 r/min 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 GCY culture medium according to the inoculation amount of 0.5-10 v/v% for culture, tying a breathable sealing film, placing on a shaking table, and culturing at the temperature of 25-40 ℃ at the rotating speed of 50-300 r/min for 8-14 h to obtain an amplification culture of the strain;

(3) centrifuging the amplified culture obtained in the step (2), collecting bacterial cells, washing the bacterial cells for 3 times by using physiological saline, and separating to obtain the bacterial cells;

(4) catalyzing D-galacturonic acid conversion by using the bacterial cells obtained in the step (3) to obtain a conversion solution containing a target product galactaric acid;

(5) and (5) adjusting the pH value of the conversion solution in the step (4) to 1.5, wherein the obtained precipitate is galactaric acid crystals.

Wherein in the step (4), the D-galacturonic acid is a buffer solution containing the D-galacturonic acid; the pH value of the buffer solution is 5.0-9.0; the content of D-galacturonic acid in the buffer solution is 10-150 mM; the mass volume ratio of the somatic cells to the buffer solution is 3-50 g of stem cells/L; the reaction conditions are 15-50 ℃, 100-800 r/min and 10-12 h of reaction.

In the step (5), the pH of the conversion solution is adjusted by acid, wherein the acid is sulfuric acid or hydrochloric acid.

Preferably, the LB medium formula is as follows: 1L of distilled water contained: 5-15 g of peptone, 3-7 g of yeast powder and 5-15 g of NaCl, wherein the LB culture medium contains 50mg/L of gentamicin and 50mg/L of kanamycin; the LB solid culture medium is added with 20g/L agar powder on the basis of the formula. Preferably, the LB medium formula is as follows: 1L of distilled water contained: 10g of peptone, 5g of yeast powder and 10g of NaCl, wherein the LB culture medium contains 50mg/L of gentamicin and 50mg/L of kanamycin; the LB solid culture medium is added with 20g/L agar powder on the basis of the formula.

The GCY culture medium comprises the following components in percentage by weight: 1L of distilled water contained: 3-10 g of glycerol, 5-15 g of corn steep liquor dry powder, 1-3 g of yeast powder and 1-3 g of NH ₄ Cl、1～2g KH ₂ PO ₄ ·3H ₂ O、0.5～1.5g K ₂ HPO ₄ 、0.2～0.8g MgSO ₄ ·7H ₂ O, 50mg gentamicin and 50mg kanamycin. Preferably, the GCY medium formula is: 1L of distilled water contained: 5g of glycerol, 10g of corn steep liquor dry powder, 2g of yeast powder and 2g of NH ₄ Cl、1.1g KH ₂ PO ₄ ·3H ₂ O、0.9g K ₂ HPO ₄ 、0.5g MgSO ₄ ·7H ₂ O, 50mg gentamicin and 50mg kanamycin.

Has the advantages that: the method can oxidize high-concentration D-galacturonic acid into galactaric acid, and has the advantages of high product yield, high reaction rate, simple and controllable catalytic process, good practicability and easy industrialization.

Drawings

FIG. 1 is a graph showing the change in the composition of various substances during the synthesis of galactaric acid by Pseudomonas putida T5 catalyzing 130mM D-galacturonic acid in the catalysis procedure described in example 9 (xxx, D-galacturonic acid;. tangle-solidup, galactaric acid).

Detailed Description

The present invention will be described in further detail with reference to specific examples. Given the detailed embodiment and the specific operation process, the examples will help to understand the present invention, but the scope of the present invention is not limited to the following examples.

Wherein, the conversion rate of D-galactonic acid and the yield of galactaric acid are calculated by the following formulas:

example 1 preparation of Pseudomonas putida (Pseudomonas putida) T05.

Ion beam mutagenesis to obtain Pseudomonas putida (Pseudomonas putida) T01.

Pseudomonas putida (Pseudomonas putida KT2440, accession number ATCC No.47054) cultured on a slant with GM medium was inoculated into a 500mL Erlenmeyer flask containing 50mL of sterilized GM medium, and cultured on a shaker at 30 ℃ for 12 hours at a rotation speed of 200r/min to prepare a liquid culture of Pseudomonas putida cells.

The prepared cell liquid culture is centrifuged at 800r/min for 5 minutes, the supernatant is removed, and the cell liquid culture is washed with physiological saline and suspended to prepare a bacterial suspension (about OD 1). Sucking 100 μ L of the culture medium, spreading on a culture dish containing solid GM culture medium, blowing to dry with sterile air to obtain bacterial membrane, and performing ion implantation with ion energy of 30keV and implantation dosage of 1 × 10 ¹⁴ ions/cm ² ，20×10 ¹⁴ ions/cm ² ，50×10 ¹⁴ ions/cm ² ，80×10 ¹⁴ ions/cm ² ，110×10 ¹⁴ ions/cm ² . The plates after ion implantation were washed with 1mL of physiological saline. Respectively sucking 100 mu L of the injection with different injection doses, diluting 1000-10000 times, respectively coating the injection in a culture dish filled with a solid GM culture medium, standing and culturing for 12-24 h at 30 ℃, selecting a single colony with high growth speed, carrying out amplification culture, detecting the metabolic speed of D-galacturonic acid by HPLC (high performance liquid chromatography), and selecting the strain with the highest metabolic speed. The strain having the highest D-galacturonic acid metabolism rate was named as Pseudomonas putida (Pseudomonas putida) T01.

The GM culture formula comprises the following components: 1L of distilled water contained: 5g D-galacturonic acid, 4.25g Na ₂ HPO ₄ ·2H ₂ O，1.5g KH ₂ PO ₄ ，0.25g NaCl，0.5g NH ₄ Cl and 0.24g MgSO ₄ And 2.5mL of trace element solution. The formula of the trace element solution is (mg/L): h ₃ BO ₃ ：300，ZnCl ₂ ：50，MnCl ₂ ·4H ₂ O：30，CoCl ₂ ：200，CuCl ₂ ·2H ₂ O 10，NiCl ₂ ·6H ₂ O：20，NaMoO ₄ ·2H ₂ O: 30. the GM solid culture medium is added with 20g/L agar powder on the basis of the formula.

And (II) genetically modifying to obtain Pseudomonas putida (Pseudomonas putida) T02.

The genomic DNA of Pseudomonas putida T01 was prepared by a conventional method, and the procedure was described in "molecular biology Manual edition" published by scientific Press for miniprep of bacterial genome. PCR-amplifying the putative GARD1 upstream homology arm from the above genomic DNA using synthetic primers GARD1a-f and GARD1 a-r; the downstream homology arm of the putative galactaric acid metabolism gene 1 was PCR amplified from the above genomic DNA using the synthetic primers GARD1b-f and GARD1 b-r. Recombinant PCR was performed using the obtained upstream and downstream homology arms as templates, and then the recombinant fragments were amplified by PCR using primers GARD1a-f and GARD1 b-r.

The recombinant fragment and the suicide plasmid pK18mobSacB are respectively digested by EcoR I and Hind III, and the digested products are recovered by nucleic acid gel and then connected by DNA ligase to obtain the knock-out plasmid pK18mobSacB-GARD 1.

Wherein the primer sequences for amplifying the recombinant fragments are as follows:

GARD1a-f：5’-GCACGAATTCGGCCAGCTCCCGTCACTGAA-3’(EcoR I)

GARD1a-r：5’-CCGCTGCCCGACTTGACGATATGCCGAGGATATTGCGGGT-3’

GARD1b-f：5’-ACCCGCAATATCCTCGGCATATCGTCAAGTCGGGCAGCGG-3’

GARD1b-r：5’-GACAAAGCTTTGAAGTTATGGCCGAGGATC-3’(Hind III)

the knock-out plasmid pK18mobSacB-GARD1 was electroporated into Pseudomonas putida T01 competent cells. Pseudomonas putida T01 was cultured in LB medium to the middle logarithmic phase, the cells were harvested by centrifugation (4 ℃, 6000rpm, 10min) and washed twice with an electrotransfer buffer (10% glycerol, 1mM HEPES, 0.3M sucrose, pH 7.0), and the cells were competent cells. mu.L of pK18mobSacB-GARD1 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 electric shock was applied under 2400V, 25. mu.F, 200. omega. conditions, followed immediately by 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 picked and inoculated in an LB liquid culture medium containing kanamycin, and the colonies are cultured to the middle logarithmic phase at the temperature of 30 ℃. PCR verification of bacteria liquid is carried out by using primers GARD1a-f and GARD1b-r, and the obtained bacterial strain capable of simultaneously amplifying long fragments and short fragments is the correct single-exchange target bacteria. The correct single-crossover objective strain was inoculated into LB liquid medium and cultured overnight at 30 ℃. The culture solution is transferred to LB liquid culture medium containing 10% of sucrose to be cultured to the middle logarithmic phase, and is transferred to LB liquid culture medium containing 10% of sucrose again to be cultured to the middle logarithmic phase. Diluted bacterial liquid (generally 10) ^-6 Or 10 ^-7 ) Spread on LB solid plates containing 12% sucrose, and cultured at 30 ℃ until single colonies appear. Selecting single growing bacteria, and carrying out bacteria liquid PCR verification by using primers GARD1a-f and GARD1b-r to obtain a strain which can only amplify short segments, namely a correct double-exchange target bacterium, namely a supposed engineering bacterium with the inactivated galactaric acid metabolism gene 1. The resulting engineered strain was named Pseudomonas putida (Pseudomonas putida) T02.

And (III) genetically modifying to obtain Pseudomonas putida (Pseudomonas putida) T03.

The genomic DNA of Pseudomonas putida T02 was prepared by a conventional method, and the procedure was described in "molecular biology Manual edition" published by scientific Press for miniprep of bacterial genome. PCR-amplifying the putative GARD2a-f and GARD2a-r upstream homology arms of galactaric acid metabolism gene 1 from the genomic DNA as described above using synthetic primers GARD2a-f and GARD2 a-r; the downstream homology arm of the putative mucic acid metabolizing gene 2 was PCR amplified from the above genomic DNA using the synthetic primers GARD2b-f and GARD2 b-r. Recombinant PCR was performed using the obtained upstream and downstream homology arms as templates, and then the recombinant fragments were amplified by PCR using primers GARD2a-f and GARD2 b-r.

The recombinant fragment and the suicide plasmid pK18mobSacB are respectively digested by EcoR I and Hind III, and the digested products are recovered by nucleic acid gel and then connected by DNA ligase to obtain the knock-out plasmid pK18mobSacB-GARD 2.

Wherein the primer sequences of the amplified recombinant fragments are as follows:

GARD2a-f：5’-GCACGAATTCGCTCGATCGGATCATCTCAT-3’(EcoR I)

GARD2a-r：5’-CACTGCGCCCAGGGATTTCTTTCACCGAGGTGAGAATGCC-3’

GARD2b-f：5’-GGCATTCTCACCTCGGTGAAAGAAATCCCTGGGCGCAGTG-3’

GARD2b-r：5’-GACAAAGCTTGCGCTTGCACGTTGTACAGT-3’(Hind III)

the knock-out plasmid pK18mobSacB-GARD2 was electroporated into Pseudomonas putida T02 competent cells. Among these, the methods for preparing competence, for electrotransformation, for screening of single-and double-crossover target bacteria are described in example 2. Selecting single bacteria growing after double exchange, and carrying out bacteria liquid PCR verification by using primers GARD2a-f and GARD2b-r to obtain a strain which can only amplify short segments, namely correct double-exchange target bacteria, namely presumed engineering bacteria with the galactaric acid metabolic genes 1 and 2 inactivated together. The resulting engineered strain was named Pseudomonas putida (Pseudomonas putida) T03.

And (IV) genetically modifying to obtain the Pseudomonas putida (Pseudomonas putida) T04.

The genomic DNA of Pseudomonas putida ATCC47054 was prepared by a conventional method, and the procedure was described in "guidelines for molecular biology", published by scientific Press for miniprep of bacterial genomes. The synthesized primers GDH-f and GDH-r are used for obtaining the glucose dehydrogenase gene from the genomic DNA through PCR amplification, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

Wherein the primer sequences are as follows:

GDH-f：5’-TCGGTACCTCGTCTCCAAGACGATCCCGACGAGGAAAGAAGTTATGAGCACTGAAGGTGCG-3’(Kpn I)

GDH-r：5’-GCCCCCTGCAGTCATTACTCGGCTAATTTGTAAGCAATGATG-3’(Pst I)

the PCR amplified fragment and the plasmid pSEVA 644-. DELTA.lacI (derived from SEVA) were digested with Kpn I and Pst I, respectively, and the digested products were recovered on a nucleic acid gel and ligated with a DNA ligase to obtain a recombinant plasmid pSEVA 644-. DELTA.lacI-GDH.

The recombinant plasmid pSEVA644- Δ lacI-GDH was electroporated into P.putida T03 competent cells. For the methods of preparation and electrotransformation, see example 2. 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 (Pseudomonas putida) T04.

And (V) genetically modifying to obtain Pseudomonas putida (Pseudomonas putida) T05.

Genomic DNA of Rhizobium radiobacter (Agrobacterium tumefaciens, ATCC 33970, purchased from ATCC) was prepared by a conventional method, and reference was made to a method for minipreparation of bacterial genome in "eds molecular biology guidelines" published by scientific Press. The uronic acid dehydrogenase gene is obtained by PCR amplification from the genomic DNA by using the synthesized primers UDH-f and UDH-r, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2. According to conventional molecular biological methods, using

Cloning of the PCR amplified uronate dehydrogenase gene into the pBBR1MCS-2 plasmid (from Addgene) with the Uni Seamless Cloning and Assembly Kit (from all-type gold). The specific operation is as follows:

1) two pairs of primers were designed for amplification of the target gene and plasmid, respectively.

2) The primers for amplifying the target gene are as follows:

an upstream primer: 5'-ATTTCACACAGGAAACAGCTATGAAACGGCTTCTTGTTA-3'

A downstream primer: 5'-TTAACAAAATATTAACGCTCAGCTCTGTTTGAAGATCGGGT-3'

3) The primers for amplifying the plasmid are as follows:

an upstream primer: 5'-GCGTTAATATTTTGTTAAAATTC-3'

A downstream primer: 5'-AGCTGTTTCCTGTGTGAAATTGTTA-3'

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

The Uni Seamless Cloning and Assembly Kit Manual of operations ligates the fragment of interest and the plasmid.

5) The resulting recombinant plasmid was electrically transformed into pseudomonas putida T04 competent cells. Wherein, the preparation method of competence and the electric transformation method are seen in the preparation steps of Pseudomonas putida (Pseudomonas putida) T02. 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. And (3) screening the recovered cells on an LB solid plate containing double resistance of gentamicin and kanamycin, selecting growing bacterial colonies, inoculating the bacterial colonies into an LB liquid culture medium containing double resistance of gentamicin and kanamycin, culturing at 30 ℃ to logarithmic phase, collecting bacteria, and storing in a low-temperature refrigerator for later use. The obtained engineering strain is named as Pseudomonas putida (Pseudomonas putida) T05 and is preserved in China center for type culture Collection at 03 and 15 days 2021 with the preservation numbers as follows: CCTCC NO of M2021227, hereinafter directly abbreviated as Pseudomonas putida engineering bacteria T05.

Example 2 use of pseudomonas putida T05 for galactaric acid production.

(1) Selecting a ring 1 of pseudomonas putida engineering bacteria T05 strain cultured by an LB medium solid slant, inoculating the ring in a 1L conical flask filled with 100mL of sterilized LB medium for culture, 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 20g/L agar powder on the basis of the formula; the LB medium contained 50mg/L gentamicin and 50mg/L kanamycin.

(2) Inoculating the seed solution prepared from pseudomonas putida T05 into a 3L conical flask filled with sterilized 500mL GCY culture medium according to the inoculation amount of 1 v/v%, tying a breathable sealing film, placing on a shaking table, and culturing at 30 ℃ at the rotating speed of 200r/min for 10h to obtain the amplification culture of the strain. Wherein the formula of the GCY culture medium is as follows: 1L of distilled water contained: 5g of glycerol, 10g of corn steep liquor dry powder, 2g of yeast powder and 2g of NH ₄ Cl，1.1g KH ₂ PO ₄ ·3H ₂ O，0.9g K ₂ HPO ₄ ，0.5g MgSO ₄ ·7H ₂ O; 50mg gentamicin, 50mg kanamycin.

(3) Centrifugally collecting thallus cells, washing thallus for 3 times by using normal saline, and separating to obtain complete microbial cells;

(4) mixing the bacterial cells obtained in the step (3) with a Tris-HCl buffer solution (100mM, pH 7.0) containing D-galacturonic acid, enabling the concentration of the D-galacturonic acid in the mixture to be 30mM and the concentration of a biocatalyst to be 10g dry cells/L, and reacting for 5 hours at 35 ℃ and 300r/min to obtain a conversion solution;

(5) and (4) centrifuging the conversion solution obtained in the step (4) for 10min at 10000r/min, removing the biocatalyst added in the step (4), diluting the obtained clear solution by 10 times, and then carrying out HPLC detection.

(6) HPLC detection shows that the conversion solution contains no D-galacturonic acid, the concentration of the product galactaric acid is 30mM, the product is not degraded, and the conversion rate and the yield are both 100%.

Example 3 use of pseudomonas putida T05 for galactaric acid production.

(1) Selecting 2 rings of pseudomonas putida engineering bacteria T05 strain cultured by LB culture medium solid slant, inoculating in a 1L conical flask filled with 100mL sterilized LB culture medium for culturing, tying a breathable sealing film with a breathable film, placing on a shaker, and culturing at 30 ℃ for 12h at a rotating speed of 210r/min to obtain a seed liquid culture of the strain; wherein the formulation of the LB medium is the same as in example 2.

(2) Inoculating the seed solution prepared from pseudomonas putida T05 into a 3L conical flask filled with sterilized 500mL GCY culture medium according to the inoculation amount of 3 v/v%, tying a breathable sealing film, placing on a shaker, and culturing at 25 ℃ at the rotating speed of 350r/min for 14h to obtain the amplification culture of the strain. Wherein the GCY medium formulation is the same as in example 2.

(4) mixing the bacterial cells obtained in the step (3) with a Tris-HCl buffer solution (150mM, pH 7.5) containing D-galacturonic acid, enabling the concentration of the D-galacturonic acid in the mixture to be 50mM and the concentration of a biocatalyst to be 15g dry cells/L, and reacting for 3h at 30 ℃ and 200r/min to obtain a conversion solution;

(5) and (4) centrifuging the conversion solution obtained in the step (4) for 10min at 10000r/min, removing the biocatalyst added in the step (4), diluting the obtained clear solution by 15 times, and carrying out HPLC detection.

(6) HPLC detection shows that the conversion solution contains no D-galacturonic acid, the concentration of the product galactaric acid is 50mM, the product is not degraded, and the conversion rate and the yield are both 100%.

Example 4 application of Pseudomonas putida T05 to galactaric acid production.

(1) Selecting a ring 1 of pseudomonas putida engineering bacteria T05 strain cultured by an LB culture medium solid slant, inoculating the ring in a 1L conical flask filled with 100mL of sterilized LB culture medium for culture, tying a breathable sealing film, placing the conical flask on a shaking table, and culturing at the temperature of 30 ℃ for 11h at the rotating speed of 190r/min to obtain a seed liquid culture of the strain; wherein the formulation of the LB medium is the same as in example 2.

(2) Inoculating the seed solution prepared from pseudomonas putida T05 into a 3L conical flask filled with sterilized 500mL GCY culture medium according to the inoculation amount of 5 v/v%, tying a breathable sealing film, placing on a shaker, and culturing at 35 ℃ at the rotating speed of 300r/min for 11h to obtain the amplification culture of the strain. Wherein the GCY medium formulation is the same as in example 2.

(4) mixing the bacterial cells obtained in the step (3) with a Tris-HCl buffer solution (200mM, pH 8.0) containing D-galacturonic acid, enabling the concentration of the D-galacturonic acid in the mixture to be 100mM and the concentration of a biocatalyst to be 20g dry cells/L, and reacting for 6h at 35 ℃ and 600r/min to obtain a conversion solution;

(5) and (5) centrifuging the conversion solution obtained in the step (4) at 10000r/min for 10min, removing the biocatalyst added in the step (4), diluting the obtained clear solution by 25 times, and carrying out HPLC detection.

(6) HPLC detection shows that the conversion solution contains no D-galacturonic acid, the concentration of the product galactaric acid is 100mM, the product is not degraded, and the conversion rate and the yield are both 100%.

Example 5 application of Pseudomonas putida T05 to galactaric acid production.

(1) Selecting a ring 1 of pseudomonas putida engineering bacteria T05 strain cultured by an LB medium solid slant, inoculating the ring in a 1L conical flask filled with 100mL of sterilized LB medium for culture, tying a breathable sealing film, placing the conical flask on a shaker, and culturing for 11h at the rotating speed of 210r/min at the temperature of 30 ℃ to obtain a seed liquid culture of the strain; wherein the formulation of the LB medium is the same as in example 2.

(2) Inoculating the seed solution prepared from pseudomonas putida T05 into a 3L conical flask filled with sterilized 500mL GCY culture medium according to the inoculation amount of 1.5 v/v%, sealing with a breathable sealing film, placing on a shaking table, and culturing at 32 ℃ at the rotating speed of 200r/min for 12h to obtain the amplified culture of the strain. Wherein the GCY medium formulation is the same as in example 2.

(4) mixing the bacterial cells obtained in the step (3) with a Tris-HCl buffer solution (200mM, pH 7.0) containing D-galacturonic acid, enabling the concentration of the D-galacturonic acid in the mixture to be 130mM and the concentration of the biocatalyst to be 18g dry cells/L, and reacting for 12h at 35 ℃ and 300r/min to obtain a conversion solution;

(5) and (5) centrifuging the conversion solution obtained in the step (4) at 10000r/min for 10min, removing the biocatalyst added in the step (4), diluting the obtained clear solution by 30 times, and carrying out HPLC detection.

(6) HPLC detection shows that the conversion solution contains no D-galacturonic acid, the concentration of the product galactaric acid is 130mM, the product is not degraded, and the conversion rate and the yield are both 100%.

Comparative example 1:

(1) selecting a ring 1 of pseudomonas putida engineering bacteria ATCC47054 strain cultured by an LB culture medium solid slant, inoculating the ring in a 1L conical flask filled with 100mL of sterilized LB culture medium for culture, tying a breathable sealing film, placing the conical flask on a shaker, and culturing at the temperature of 30 ℃ for 11h at the rotating speed of 200r/min to obtain a seed liquid culture of the strain; wherein the LB medium formulation is the same as example 6, but without antibiotics.

(2) Inoculating the seed solution prepared from the pseudomonas putida ATCC47054 into a 3L conical flask filled with a sterilized 500mL GCY culture medium according to the inoculation amount of 1 v/v%, sealing by a breathable sealing film, placing on a shaking table, and culturing at the temperature of 30 ℃ at the rotating speed of 200r/min for 12h to obtain the amplification culture of the strain. Wherein the GCY medium formulation is the same as example 2, but does not contain antibiotics.

(4) mixing the bacterial cells obtained in the step (3) with a Tris-HCl buffer solution (100mM, pH 7.0) containing D-galacturonic acid, enabling the concentration of the D-galacturonic acid in the mixture to be 10mM and the concentration of a biocatalyst to be 10g of dry cells/L, and reacting for 10 hours at 35 ℃ and 200r/min to obtain a conversion solution;

(5) and (4) centrifuging the conversion solution obtained in the step (4) at 10000r/min for 10min, removing the biocatalyst added in the step (4), and carrying out HPLC detection on the obtained clear liquid.

(6) HPLC detection shows that no D-galacturonic acid remains in the conversion solution, the concentration of the product galactaric acid is basically not detected, the product is completely degraded, the conversion rate is 100%, and the yield is 0%.

Comparative example 2

(1) Selecting a ring 1 of pseudomonas putida engineering bacteria T03 strain cultured by an LB culture medium solid slant, inoculating the ring in a 1L conical flask filled with 100mL of sterilized LB culture medium for culture, tying a breathable sealing film, placing the conical flask on a shaking table, and culturing for 12h at the rotating speed of 200r/min at the temperature of 30 ℃ to obtain a seed liquid culture of the strain; wherein the LB medium formulation is the same as example 2, but without antibiotics.

(2) Inoculating the seed solution prepared from pseudomonas putida T03 into a 3L conical flask filled with sterilized 500mL GCY culture medium according to the inoculation amount of 1 v/v%, tying a breathable sealing film, placing on a shaking table, and culturing at 30 ℃ for 11h at the rotating speed of 200r/min to obtain the amplification culture of the strain. Wherein the GCY medium formulation is the same as example 2, but does not contain antibiotics.

(4) mixing the bacterial cells obtained in the step (3) with Tris-HCl buffer solution (100mM, pH 7.0) containing galactaric acid, enabling the concentration of the galactaric acid in the mixture to be 50mM and the concentration of a biocatalyst to be 10g dry cells/L, and reacting for 24 hours at the temperature of 30 ℃ and at the speed of 200r/min to obtain a conversion solution;

(6) HPLC detection shows that the concentration of the galactaric acid in the transformation solution is not changed, which indicates that the galactaric acid metabolic capability of the wild bacteria is completely inactivated.

The invention provides a thought and a method for constructing engineering bacteria of pseudomonas putida for producing galactaric acid, and a method and a way for realizing the technical scheme are many, the above description is only a preferred embodiment of the invention, and it should be noted that, for a person skilled in the art, a plurality of improvements and decorations can be made without departing from the principle of the invention, and the improvements and decorations are also regarded as the protection scope of the 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> Pseudomonas putida engineering bacterium and application thereof

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2412

<212> DNA

<213> nucleotide Sequence of preferred Gene for glucose dehydrogenase (Artificial Sequence)

<400> 1

atgagcactg aaggtgcgaa ccaaggaagc cgctggctac cgcgcctgat tggcgcgctg 60

ctgttgctga tgggcctggc cctgctggcc ggcggtatca agctgagcca gctgggcgga 120

tcgctgtact acctgatcgc cggtattggc tttgcccttt cgggcgtcct gctgctggcc 180

caacgccaga tcgccctggg cctgtatggc ctggtgctgc tgggcagcac cgtgtgggcc 240

ctgttcgaag taggcctgga ctggtggcaa ctggtgccac ggctggctat ctggttcgcc 300

atcggcgtgg tgttgctgct gccgtgggca cgtcgcccgc tgatcggccc agccagcaaa 360

gccaacactg cactgctcgg cgtggcagtg gtcgcttcgg gcgcttgcgc gctggccagc 420

cagttcaccc atcccggtga agtgttcggc gaactgggcc gcgacagcag cgaaatggcc 480

agcgccgccc cggccatgcc cgacggcgaa tggcaggcct acggccgtac cgagcatggc 540

gaccgctact cgccgctgcg ccagatcacc ccgcagaacg cctaccgtct ggaagaagcc 600

tggcgcattc gcaccggtga tctgccaacc gaaaacgacc cggtggagct gaccaaccag 660

aacaccccgc tgaaggtcaa cggcatgctt tacgcctgca ccgcgcacag tcgcctgctg 720

gcgctggacc cggacactgg cgcagaaatc tggcgctacg acccgcaggt caagagcccg 780

accggcacct tcaagggctt tgcccacatg acctgccgtg gcgtctcgta ctatgacgaa 840

aaccgctacg tcagccgcga cggcagcccg gcgccgaaaa ttaccgatgc aggccaggcc 900

gtggcccaag cctgcccgcg tcgcctctat ctgcctaccg cggatgcccg cctgatcgcc 960

atcaacgccg acaacggcaa ggtctgcgaa ggcttcgcca accagggcgt gatcgacctc 1020

accaccggca ttggcccatt caccgccggc ggctattact ccacctcgcc tgctgcgatt 1080

acccgtgacc tggtgatcat cggtggccac gtcaccgaca acgagtcgac caatgagccg 1140

tccggggtga tccgcgccta cgacgtgcac gacggccacc tggtgtggaa ctgggacagt 1200

aacaacccgg acgacaccaa gccattggct gccggcaaaa tgtacagccg caactcggcc 1260

aacatgtggt cgatcgccag cgtcgacgaa gaccttggca tgatctacct gccgctgggc 1320

aaccagaccc cggaccagtg gggcgccgac cgcaccccgg gcgccgagaa gtacagcgcc 1380

ggcgtggtcg cccttgacct ggccaccggc aaggcacgct ggaactatca gttcacccac 1440

cacgacctgt gggacatgga cgtcggcagc cagccgaccc tggtacacct gaaaaccgac 1500

gatggtgtga aaccggcgat catcgtaccg accaagcaag gcagcctgta cgtgctcgac 1560

cgccgcgacg gtacgccaat cgtgccgatc cgcgagatcc ccaccccaca aggcgcagtg 1620

gaaggcgacc acacctcgcc cacccaggcc cgctccgacc tcaacctgct cggcccagag 1680

ctgaccgaac aggccatgtg gggcgccacg cctttcgacc agatgctgtg ccgcatccag 1740

ttccgcgaac tgcgctacga aggccagtac accccgccat ccgaacaagg ttcgttggtc 1800

taccccggca acgtcggtgt attcaactgg ggcagcgtgt cggtcgaccc ggtgcgccag 1860

ctgctgttca cttcgcccaa ctacatggcg ttcgtgtcga agatggtccc gcgtgagcag 1920

gttgccgaag gcagcaagcg cgaaagcgag accagcggcg tgcagccgaa caccggcgca 1980

ccgtatgcag tgatcatgca cccgttcatg tcgccgctcg gtgtaccgtg ccaggcaccc 2040

gcctggggct acgtcgccgc catcgacctg ttcaccaaca aggtggtgtg gaaacacaag 2100

aacggcacca cccgcgacag caccccgcta ccgatcggcc tgccggttgg cgtgccgagc 2160

atgggtggct cgatcgtcac cgccggtggc gtcggcttcc tcagcggcac gctcgaccag 2220

tacctgcgcg cctatgacgt gaacaacggc aaggagttgt ggaaagcacg cctgccagcg 2280

ggtggccagg ctacgccgat gagctacacc ggcaaggacg gcaagcagta cgtgctggtg 2340

actgccggcg gccatggctc gctgggcacc aagatgggcg attacatcat tgcttacaaa 2400

ttagccgagt aa 2412

<210> 2

<211> 798

<212> DNA

<213> nucleotide Sequence of preferred Gene for uronate dehydrogenase (Artificial Sequence)

<400> 2

atgaaacggc ttcttgttac cggtgcggcg ggccagcttg gccgcgtcat gcgcgagcgt 60

ctcgcaccga tggcggagat actgcgcctt gccgatctct ccccgctcga cccggcaggg 120

ccgaacgaag aatgcgtgca atgcgacctt gccgatgcca atgccgtgaa tgccatggtc 180

gccggttgcg acggtattgt tcatctcggc ggcatctcgg tggagaagcc cttcgaacaa 240

atccttcagg gcaatatcat cgggctttat aatctctacg aggccgcccg cgcccatgga 300

cagccacgca tcgtctttgc cagctccaac cacacgatcg gctattatcc gcagaccgaa 360

cggctcggtc cggatgttcc ggcgcggccg gacggtcttt acggcgtctc caaatgtttc 420

ggcgaaaacc tcgcccgcat gtatttcgat aaattcgggc aggagacggc gctggtgcgc 480

atcggctcct gtacgccgga acccaacaat taccgcatgc tgtccacctg gttttcgcac 540

gatgatttcg tgtcgctgat cgaggcggtg tttcgcgcgc cggtgctcgg ctgcccggtc 600

gtctgggggg catcggccaa tgatgcgggc tggtgggaca attcgcatct tggctttctg 660

ggctggaaac cgaaggataa tgccgaggcc ttccggcggc atataaccga gacgacaccg 720

ccaccggacc cgaatgacgc gttggtgcgg ttccagggcg gtacgtttgt cgacaacccg 780

atcttcaaac agagctga 798

<210> 3

<211> 30

<212> DNA

<213> GARD1a-f(Artificial Sequence)

<400> 3

gcacgaattc ggccagctcc cgtcactgaa 30

<210> 4

<211> 39

<212> DNA

<213> GARD1a-r(Artificial Sequence)

<400> 4

ccgctgcccg acttgacgat atgccgagga tattgcggg 39

<210> 5

<211> 40

<212> DNA

<213> GARD1b-f(Artificial Sequence)

<400> 5

acccgcaata tcctcggcat atcgtcaagt cgggcagcgg 40

<210> 6

<211> 30

<212> DNA

<213> GARD1b-r(Artificial Sequence)

<400> 6

gacaaagctt tgaagttatg gccgaggatc 30

<210> 7

<211> 30

<212> DNA

<213> GARD2a-f(Artificial Sequence)

<400> 7

gcacgaattc gctcgatcgg atcatctcat 30

<210> 8

<211> 40

<212> DNA

<213> GARD2a-r(Artificial Sequence)

<400> 8

cactgcgccc agggatttct ttcaccgagg tgagaatgcc 40

<210> 9

<211> 40

<212> DNA

<213> GARD2b-f(Artificial Sequence)

<400> 9

ggcattctca cctcggtgaa agaaatccct gggcgcagtg 40

<210> 10

<211> 30

<212> DNA

<213> GARD2b-r(Artificial Sequence)

<400> 10

gacaaagctt gcgcttgcac gttgtacagt 30

<210> 11

<211> 61

<212> DNA

<213> GDH-f(Artificial Sequence)

<400> 11

tcggtacctc gtctccaaga cgatcccgac gaggaaagaa gttatgagca ctgaaggtgc 60

g 61

<210> 12

<211> 42

<212> DNA

<213> GDH-r(Artificial Sequence)

<400> 12

gccccctgca gtcattactc ggctaatttg taagcaatga tg 42

<210> 13

<211> 39

<212> DNA

<213> upstream primer for amplifying target Gene (Artificial Sequence)

<400> 13

atttcacaca ggaaacagct atgaaacggc ttcttgtta 39

<210> 14

<211> 41

<212> DNA

<213> downstream primer for amplifying target Gene (Artificial Sequence)

<400> 14

ttaacaaaat attaacgctc agctctgttt gaagatcggg t 41

<210> 15

<211> 23

<212> DNA

<213> upstream primer for amplifying plasmid (Artificial Sequence)

<400> 15

gcgttaatat tttgttaaaa ttc 23

<210> 16

<211> 25

<212> DNA

<213> downstream primer for amplifying plasmid (Artificial Sequence)

<400> 16

agctgtttcc tgtgtgaaat tgtta 25

Claims

1. A pseudomonas putida engineering bacterium is named as pseudomonas putida (A)Pseudomonas putida) T05, which has been deposited at the China center for type culture Collection at 03-15.2021 under the following deposition numbers: CCTCC NO: M2021227.

2. The use of the engineered pseudomonas putida as set forth in claim 1 in the preparation of galactaric acid.

3. Use according to claim 2, characterized in that it comprises the following steps:

(1) selecting Pseudomonas putida cultured on LB culture medium solid slant (Pseudomonas putida) Inoculating 1-2 rings of T05 strain, culturing in a container containing sterilized LB culture medium, sealing with air-permeable sealing film, placing on a shaking table, and culturing at 30 deg.C at rotation speed of 200 + -10 r/minCulturing for 12 +/-2 h to obtain a seed liquid culture of the strain;

(5) and (4) adjusting the pH value of the conversion solution in the step (4) to 1.5, wherein the obtained precipitate is the galactaric acid crystal.

4. The use according to claim 3, wherein in step (4), the D-galacturonic acid is in a buffer solution containing D-galacturonic acid; the pH value of the buffer solution is 5.0-9.0; the content of D-galacturonic acid in the buffer solution is 10-150 mM; the mass volume ratio of the somatic cells to the buffer solution is 3-50 g of stem cells/L; the reaction conditions are 15-50 ℃, 100-800 r/min and 10 min-12 h.

5. Use according to claim 3, characterized in that in step (5) the pH of the conversion solution is adjusted with an acid, said acid being sulfuric acid or hydrochloric acid.

6. The use according to claim 3, wherein the LB medium formulation is: 1L of distilled water contained: 5-15 g of peptone, 3-7 g of yeast powder and 5-15 g of NaCl, wherein the LB culture medium contains 50mg/L of gentamicin and 50mg/L of kanamycin; the LB solid culture medium is added with 20g/L agar powder on the basis of the formula.

7. Use according to claim 3, characterized in thatCharacterized in that the formula of the GCY culture medium is as follows: 1L of distilled water contains: 3-10 g of glycerol, 5-15 g of corn steep liquor dry powder, 1-3 g of yeast powder and 1-3 g of NH ₄ Cl、1~2 g KH ₂ PO ₄ ·3H ₂ O、0.5~1.5 g K ₂ HPO ₄ 、0.2~0.8 g MgSO ₄ ·7H ₂ O, 50mg gentamicin and 50mg kanamycin.