An engineered Pseudomonas putida strain, its preparation method and applications

Abstract

The present invention discloses an engineered Pseudomonas putida bacterium, its preparation method and application. The engineered Pseudomonas putida bacterium includes Pseudomonas putida and a recombinant plasmid introduced into Pseudomonas putida; a key gene affecting ethylene glycol metabolism has been knocked out in the genome of Pseudomonas putida; the recombinant plasmid contains a terephthalic acid catabolism module shown in SEQ ID NO.2 or SEQ ID NO.6; or a terephthalic acid catabolism module shown in SEQ ID NO.4 and a terephthalic acid transport module shown in SEQ ID NO.3. The present invention uses Pseudomonas putida as the starting strain, knocks out the key endogenous gene affecting ethylene glycol metabolism, and on this basis, externally introduces a terephthalic acid metabolism module and a terephthalic acid transport module to obtain an engineered Pseudomonas putida bacterium; this engineered Pseudomonas putida bacterium can efficiently co-utilize terephthalic acid and ethylene glycol.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to an engineered Pseudomonas putida and its preparation method and application. Background Art

[0002] Polyethylene terephthalate (PET), which is polymerized from terephthalic acid (TPA) and ethylene glycol (EG), is one of the most widely used plastics in human life. Improper recycling of PET will cause serious environmental problems, and biological upgrading and recycling has become a hot issue of global concern in recent years.

[0003] The complete degradation of PET has been developed from the mode of artificial microbial consortia, that is, constructing a PET degradation module, an EG conversion module and a TPA conversion module to perform different functions, which can accelerate the degradation of PET and achieve its complete conversion. However, microbial consortia require a relatively complex mixing mode, which is not conducive to practical application. One of the bottlenecks in PET biodegradation is the efficient catabolism of TPA and EG by the same strain of bacteria.

[0004] Pseudomonas putida ATCC NO.47054 (i.e., Pseudomonas putida KT2440) is a new synthetic biology chassis that has attracted wide interest among scholars in recent years and has been used to metabolize recalcitrant substrates, including lignin and petroleum-based plastics, etc. However, this strain cannot grow with terephthalic acid as the sole carbon source, which greatly limits its application in the field of PET biodegradation.

[0005] The present invention aims to propose a new modification strategy to endow Pseudomonas putida with the ability to efficiently co-utilize terephthalic acid and ethylene glycol. Summary of the Invention

[0006] The present invention provides an engineered Pseudomonas putida and its preparation method and application. This engineered Pseudomonas putida can efficiently utilize ethylene glycol and terephthalic acid simultaneously to achieve efficient biodegradation of PET.

[0007] The specific technical solutions are as follows:

[0008] An engineered Pseudomonas putida, comprising Pseudomonas putida and a recombinant plasmid introduced into Pseudomonas putida; the key gene affecting ethylene glycol metabolism is knocked out in the genome of Pseudomonas putida;

[0009] The recombinant plasmid is one of the following:

[0010] (1) Comprising a terephthalic acid catabolism module as shown in SEQ ID NO.2;

[0011] (2) It contains the terephthalic acid catabolism module shown in SEQ ID NO. 6;

[0012] (3) It contains the terephthalic acid catabolism module shown in SEQ ID NO. 4 and the terephthalic acid transport module shown in SEQ ID NO. 3.

[0013] Among them, the terephthalic acid catabolism module shown in SEQ ID NO. 2 is derived from Pseudomonas umsongensis GO16 (GenBank: CP044409.1); the terephthalic acid catabolism module shown in SEQ ID NO. 6 is obtained by replacing the transport protein module (SEQ ID NO. 5 (protein_id = "BAE47084.1")) in the terephthalic acid catabolism module (SEQ ID NO. 4) derived from Comamonas sp. E6 with the transport protein module (SEQ ID NO. 3) derived from Pseudomonas umsongensis GO16; the terephthalic acid catabolism module shown in SEQ ID NO. 4 is derived from Comamonas sp. E6 (GenBank: AB238679.1); the terephthalic acid transport module shown in SEQ ID NO. 3 is derived from Pseudomonas umsongensis GO16 (protein_id = "QFG29484.1"). The above strains are publicly available strains, and both the terephthalic acid catabolism module and the terephthalic acid transport module can be obtained by biosynthesis.

[0014] The terephthalic acid catabolism module is a terephthalic acid catabolism gene cluster composed of multiple genes, including a transcription factor, a transport protein, a ring hydroxylase 1,2-dioxygenase, and a dihydrodiol dehydrogenase; the terephthalic acid transport module is a terephthalic acid transport protein.

[0015] The strain type of the Pseudomonas putida is Pseudomonas putida KT2440 ATCC NO. 47054; it can be purchased from the ATCC Biological Resource Center.

[0016] Preferably, the terephthalic acid catabolism module and the terephthalic acid transport module are respectively inserted into two different recombinant plasmids, and both recombinant plasmids can self-replicate and be compatible in Pseudomonas putida.

[0017] Preferably, the original expression vector of the recombinant plasmid is pSEVA64.

[0018] Preferably, the key gene for ethylene glycol metabolism is gclR, and its base sequence is shown in SEQ ID NO.1. gclR (PP_4283) is an endogenous transcriptional repressor of the Gcl pathway (NCBI Reference Sequence: NC_002947.4).

[0019] The present invention discloses a method for preparing the engineered Pseudomonas putida, comprising:

[0020] (1) Using gene knockout technology to knockout the key gene in the Pseudomonas putida genome that affects ethylene glycol metabolism. After knockout, laboratory adaptive evolution is carried out to obtain the evolved engineered bacterium;

[0021] (2) Inserting the terephthalic acid catabolism module into the original expression vector; or, inserting the terephthalic acid catabolism module and the terephthalic acid transport module into two original expression vectors respectively to obtain a recombinant plasmid;

[0022] (3) Transforming the recombinant plasmid into the engineered bacterium after gene knockout to obtain the engineered Pseudomonas putida.

[0023] In step (1), the gene knockout technology can be CRISPR / Cas 9. By knocking out the key gene gclR, Pseudomonas putida using EG as the sole carbon source can be obtained.

[0024] Furthermore, the method for laboratory adaptive evolution includes the following steps:

[0025] (A) Pick a single colony of Pseudomonas putida on the LB solid medium, inoculate it into the liquid LB medium for culture, place it on a shaker, and culture it at 30 °C with a rotation speed of 220 r / min for 12 - 16 h to obtain a seed liquid culture;

[0026] (B) Inoculate the seed liquid culture obtained in step (1) into the evolution medium for culture according to an inoculation amount of 2 v / v%, place it on a shaker, and culture it at 30 °C with a rotation speed of 220 r / min until the cell OD reaches 0.4 - 0.8 to obtain the first-generation cell culture;

[0027] (C) Transfer the first-generation cell culture obtained in step (2) to fresh evolution medium, place it on a shaker, and culture it at 30 °C with a rotation speed of 220 r / min until the cell OD reaches 0.4 - 0.8 to obtain the second-generation cell culture of the strain;

[0028] (D) Continuously repeat step (3) to obtain the 10th - 20th generation cell culture;

[0029] (E) When the cell growth cycle is stable, stop subculturing to obtain the evolved engineered bacteria.

[0030] Further, in step (A), the formula of the liquid LB medium is as follows: in 1 L of distilled water, there are 10 g of peptone, 5 g of yeast extract, 10 g of NaCl, and 50 mg of gentamicin; 15 - 20 g / L of agar powder needs to be additionally added to the LB liquid medium.

[0031] Further, in steps (B) and (C), the formula of the evolution medium (MSM medium) is as follows: in 1 L of distilled water, there are 3.88 g of K2HPO4, 2.12 g of NaH2PO4·2H2O, 2.00 g of (NH4)2SO4, 0.1 g of MgCl2·6H2O, 10 mg of EDTA, 2 mg of ZnSO4·7H2O, 1 mg of CaCl2·2H2O, 5 mg of FeSO4·7H2O, 0.2 mg of Na2MoO4·2H2O, 0.2 mg of CuSO4·5H2O, and 0.4 mg of CoCl2·6H2O.

[0032] Further, the number of cell passages in step (D) is 10 - 15.

[0033] The present invention also provides the application of the engineered Pseudomonas putida bacteria in the efficient co - utilization of ethylene glycol and terephthalic acid.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention uses Pseudomonas putida as the starting strain, knocks out the key endogenous genes affecting ethylene glycol metabolism, and on this basis, exogenously introduces the terephthalic acid metabolism module and the terephthalic acid transport module to obtain the engineered Pseudomonas putida bacteria; this engineered Pseudomonas putida bacteria can efficiently co - utilize terephthalic acid and ethylene glycol. Description of the Drawings

[0036] Figure 1 Growth curve and EG consumption curve of the engineered Pseudomonas putida KT1 constructed for Example 1 with different concentrations of ethylene glycol as the sole carbon source; among them, A represents the growth curve of KT1 in the medium with different concentrations of EG; B represents the EG consumption curve of KT1 in the medium with different concentrations of EG.

[0037] Figure 2Growth curve and terephthalic acid consumption rate curve of the engineered Pseudomonas putida KT1-G constructed for Example 2 with terephthalic acid as the sole carbon source; wherein, A represents the growth curve of KT1-G with terephthalic acid as the sole carbon source; B represents the terephthalic acid consumption rate curve.

[0038] Figure 3 Growth curve and terephthalic acid consumption rate curve of the engineered Pseudomonas putida KT1-EK and KT1-EK2 constructed for Example 4 and Example 5 with terephthalic acid as the sole carbon source; wherein, A represents the growth curve of KT1-EK and KT1-EK2 with terephthalic acid as the sole carbon source; B represents the terephthalic acid consumption rate curve.

[0039] Figure 4 Growth curve of the co-utilization of ethylene glycol and terephthalic acid by the engineered Pseudomonas putida KT1-G and KT1-EK2 constructed for Example 6 and the consumption rate curves of the two substrates; wherein, A represents the co-utilization of ethylene glycol by KT1-G and KT1-EK2; B represents the terephthalic acid consumption rate curve; C represents the ethylene glycol consumption curve.

[0040] Figure 5 Schematic diagram of the combination of terephthalic acid catabolic modules in different engineered bacteria. Detailed implementation mode

[0041] The present invention will be further described in detail below in conjunction with specific embodiments. The detailed implementation mode and specific operation process are given. The embodiments will help to understand the present invention, but the protection scope of the present invention is not limited to the following embodiments.

[0042] The sequence shown in SEQ ID NO.1 in the sequence listing of the present invention is the nucleotide sequence of the gclR gene, and the sequence shown in SEQ ID NO.2 is the nucleotide sequence of the terephthalic acid catabolic module (Artificial Sequence) from Pseudomonas citronellolis GO16 (including the terephthalic acid metabolic module and the terephthalic acid transport module); the sequence shown in SEQ ID NO.3 is the nucleotide sequence of the terephthalic acid transport module (Artificial Sequence) from Pseudomonas citronellolis GO16; the sequence shown in SEQ ID NO.4 is the nucleotide sequence of the terephthalic acid catabolic module (Artificial Sequence) from Comamonas sp. E6 (including the terephthalic acid metabolic module and the terephthalic acid transport module); the sequence shown in SEQ ID NO.5 is the nucleotide sequence of the terephthalic acid transport module (Artificial Sequence) from Comamonas sp. E6. The terephthalic acid catabolic module described in the present invention contains both the terephthalic acid metabolic module and the terephthalic acid transport module.

[0043] Example 1 Construction of Pseudomonas putida KT1 and Its Cultivation with Ethylene Glycol as the Sole Carbon Source

[0044] (1) Pseudomonas putida KT2440 (ATCC 47054) was stored in glycerol with a volume fraction of 25% and was long-term stored in a -80 °C ultra-low temperature refrigerator. When activating, it was streaked on an LB solid medium, and after overnight culture, a single colony was picked and inoculated into a shake tube containing 5 mL of sterilized LB liquid medium. Under the condition of 30 °C and a rotation speed of 220 r / min, it was cultured for 16 h to obtain a cell-activated culture.

[0045] (2) The gclR gene (the base sequence is as shown in SEQ ID NO.1) in KT2440 was knocked out by the CRISPR / Cas9 gene editing method. After obtaining a positive transformant, a single colony was picked and inoculated into a shake tube containing 5 mL of sterilized LB liquid medium. Under the condition of 30 °C and a rotation speed of 220 r / min, it was cultured for 16 h to obtain a seed solution;

[0046] (3) The seed solution was transferred to a 250 mL conical flask containing 50 mL of sterilized minimal medium without salts, and 30 mM ethylene glycol was added as the sole carbon source to the medium. The initial cell OD600 after inoculation was 0.01.

[0047] (4) Measure the cell turbidity every 6 hours until the OD600 reaches 0.6 - 0.8, and then transfer again to fresh minimal inorganic salt medium at 2 v / v%, where the medium contains 60 mM ethylene glycol.

[0048] (5) Repeat step (4), with an additional 30 mM ethylene glycol added to the medium for each transfer, and finally maintain the ethylene glycol concentration in the medium at 180 mM.

[0049] (6) When the cell growth cycle is stable, stop subculturing to obtain Pseudomonas putida KT1 that grows with ethylene glycol as the sole carbon source.

[0050] The formula of LB liquid medium is: in 1 L of distilled water, there are 10 g of tryptone, 5 g of yeast extract powder, and 10 g of sodium chloride.

[0051] The formula of minimal inorganic salt medium is: in 1 L of distilled water, there are 3.88 g of K2HPO4, 2.12 g of NaH2PO4·2H2O, 2.00 g of (NH4)2SO4, 0.1 g of MgCl2·6H2O, 10 mg of EDTA, 2 mg of ZnSO4·7H2O, 1 mg of CaCl2·2H2O, 5 mg of FeSO4·7H2O, 0.2 mg of Na2MoO4·2H2O, 0.2 mg of CuSO4·5H2O, and 0.4 mg of CoCl2·6H2O.

[0052] In this example, there was no obvious change in the cell turbidity of Pseudomonas putida ATCC 47054 in the medium, that is, Pseudomonas putida ATCC 47054 could not grow with ethylene glycol as the sole carbon source; while KT1 could efficiently utilize ethylene glycol and grow with ethylene glycol as the sole carbon source. Example 2 Construction of the engineered Pseudomonas putida KT1 - G and its cultivation with terephthalic acid as the sole carbon source

[0053] (1) Construction of the engineered strain: Synthesize the terephthalic acid catabolic module (SEQ ID NO.2) from Pseudomonas umsongensis GO16 through GenScript. This terephthalic acid catabolic module includes a terephthalic acid metabolic module and a terephthalic acid transport module shown in SEQ ID NO.3.

[0054] Ligate the target gene fragment with the PCR - linearized plasmid (pSEVA644) vector backbone seamlessly to obtain the recombinant plasmid pSEVA64 - tphG.

[0055] The recombinant plasmid pSEVA64-tphG was electrotransformed into competent cells of Pseudomonas putida KT1. Pseudomonas putida KT1 was cultured in LB medium until the mid-logarithmic phase, and the cells were collected by centrifugation (4°C, 6000 rpm, 10 min) and washed three times with electrotransformation buffer (3 mM HEPES). The obtained cells were competent cells.

[0056] 10 μL of the pSEVA64-tphG plasmid at a concentration of 100 ng / μL was mixed with 100 μL of competent cells and added to an electroporation cuvette (inner diameter 2 mm). Electroporation was carried out under the conditions of 2500 V, 25 μF, and 200 Ω, and then immediately 1 mL of LB culture medium was added. The bacterial suspension in the electroporation cuvette was transferred to a centrifuge tube and cultured on a shaker at 30°C for 2 h for cell recovery. After recovery, the cells were screened on an LB solid plate containing gentamicin resistance, and the growing colonies were picked and inoculated into an LB liquid medium containing gentamicin and cultured at 30°C for 16 h. The cells were collected and stored for later use. The obtained engineered strain was named Pseudomonas putida KT1-G.

[0057] (2) Culturing the engineered bacteria obtained in step (1) with terephthalic acid as the sole carbon source: Single colonies on the LB solid medium were picked and inoculated into a shake tube containing 5 mL of sterilized LB medium for culture. It was placed on a shaker and cultured at 30°C at a rotation speed of 220 r / min for 16 h to obtain the seed liquid culture of the strain; the LB medium contained 50 mg / L of gentamicin.

[0058] (3) The cell liquid culture in step (2) was inoculated into a 250 mL conical flask containing 50 mL of sterilized inorganic salt medium at 2 v / v%, and the medium contained 60 mM terephthalic acid as the sole carbon source.

[0059] (4) Repeat step three and measure the cell turbidity during the growth process until the cell growth cycle is stable, stop subculturing, and obtain Pseudomonas putida KT1-G that grows with terephthalic acid as the sole carbon source.

[0060] It should be noted that the obtained recombinant Pseudomonas putida engineered strain KT1-G can grow with terephthalic acid as the sole carbon source.

[0061] The formula of the inorganic salt medium is as follows: in 1 L of distilled water, it contains 3.88 g of K2HPO4, 2.12 g of NaH2PO4·2H2O, 2.00 g of (NH4)2SO4, 0.1 g of MgCl2·6H2O, 10 mg of EDTA, 2 mg of ZnSO4·7H2O, 1 mg of CaCl2·2H2O, 5 mg of FeSO4·7H2O, 0.2 mg of Na2MoO4·2H2O, 0.2 mg of CuSO4·5H2O, and 0.4 mg of CoCl2·6H2O.

[0062] Construction of the engineered Pseudomonas putida KT1-E and its cultivation using terephthalic acid as the sole carbon source in Example 3

[0063] (1) Construction of the engineered strain: Synthesize the terephthalic acid catabolic module (SEQ ID NO.4) derived from Comamonas sp. E6 by GenScript; this terephthalic acid catabolic module includes the terephthalic acid metabolic module and the terephthalic acid transport module shown in SEQ ID NO.5.

[0064] The target gene fragment was seamlessly ligated with the PCR-linearized plasmid (pSEVA644) vector backbone to obtain the recombinant plasmid pSEVA64-tphE.

[0065] The recombinant plasmid pSEVA64-tphE was electrotransformed into the competent cells of Pseudomonas putida KT1. Pseudomonas putida KT1 was cultured in LB medium until the mid-logarithmic phase, and the cells were collected by centrifugation (4°C, 6000 rpm, 10 min) and washed three times with electroporation buffer (3 mM HEPES). The obtained cells were competent cells. 10 μL of the pSEVA64-tphG plasmid with a concentration of 100 ng / μL was mixed with 100 μL of the competent cells and then added to an electroporation cuvette (inner diameter 2 mm). Electroporation was performed under the conditions of 2500 V, 25 μF, and 200 Ω, and then 1 mL of LB culture medium was immediately added. The bacterial solution in the electroporation cuvette was transferred to a centrifuge tube and cultured on a shaker at 30°C for 2 h for cell recovery. After recovery, the cells were screened on an LB solid plate containing gentamicin resistance, and the growing colonies were picked and inoculated into an LB liquid medium containing gentamicin and cultured at 30°C for 16 h. The cells were collected and stored for later use.

[0066] The obtained engineered strain was named Pseudomonas putida KT1-E.

[0067] (2) Cultivate the engineered bacteria obtained in step (1) using terephthalic acid as the sole carbon source: Pick a single colony from the LB solid medium, inoculate it into a shaking tube containing 5 mL of sterilized LB medium for cultivation, place it on a shaker, and cultivate at 30 °C at a rotation speed of 220 r / min for 16 h to obtain the seed liquid culture of the said strain; wherein the LB medium contains 50 mg / L gentamicin.

[0068] (3) Inoculate the cell liquid culture in step (2) into a 250 mL conical flask containing 50 mL of sterilized inorganic salt medium at 2 v / v%, and the medium contains 60 mM terephthalic acid as the sole carbon source.

[0069] (4) Measure the cell turbidity every 6 - 12 hours until 72 hours. In this example, there was no obvious change in the cell turbidity of Pseudomonas putida KT1-E in the medium with terephthalic acid as the sole carbon source, that is, the engineered bacteria Pseudomonas putida KT1-E could not grow using terephthalic acid as the sole carbon source.

[0070] The formula of the inorganic salt medium is: in 1 L of distilled water, it contains: 3.88 g of K2HPO4, 2.12 g of NaH2PO4·2H2O, 2.00 g of (NH4)2SO4, 0.1 g of MgCl2·6H2O, 10 mg of EDTA, 2 mg of ZnSO4·7H2O, 1 mg of CaCl2·2H2O, 5 mg of FeSO4·7H2O, 0.2 mg of Na2MoO4·2H2O, 0.2 mg of CuSO4·5H2O, 0.4 mg of CoCl2·6H2O.

[0071] Example 4 Construction of the engineered bacteria Pseudomonas putida KT1-EK and cultivation using terephthalic acid as the sole carbon source

[0072] (1) Construction of the engineered bacteria: Co-express the recombinant plasmid pSEVA25-tphK containing the transporter module (SEQ ID NO.3) in the terephthalic acid catabolic module of Pseudomonas guguanensis GO16 and the recombinant plasmid pSEVA64-tphE of the terephthalic acid catabolic module (SEQ ID NO.4) of Comamonas sp. E6 in KT1 to obtain the engineered bacteria KT1-EK.

[0073] Use PCR to amplify the transporter module (SEQ ID NO.3) in the terephthalic acid catabolic module of Pseudomonas guguanensis GO16. Recombine the gel recovery product of the transporter target fragment and the plasmid backbone, and obtain the expression vector pSEVA25-tphK after transformation and plasmid extraction.

[0074] The recombinant plasmid pSEVA25-tphK was electrotransformed into competent cells of Pseudomonas putida KT1-E. Among them, the method for preparing competent cells and the electrotransformation method are shown in Example 3. Colonies on the plate were picked and inoculated into LB liquid medium containing kanamycin and gentamicin, and cultured at 30 °C for 16 h, and the cells were collected and stored for later use. The obtained engineered strain was named Pseudomonas putida KT1-EK.

[0075] (2) Culturing the engineered bacterium obtained in step (1) with terephthalic acid as the sole carbon source: A single colony on the LB solid medium was picked and inoculated into a shake tube containing 5 mL of sterilized LB medium for culture. It was placed on a shaker and cultured at 30 °C at a rotation speed of 220 r / min for 16 h to obtain the seed liquid culture of the strain; the LB medium contained 50 mg / L kanamycin and 50 mg / L gentamicin.

[0076] (3) The cell liquid culture in step (2) was inoculated into a 250 mL conical flask containing 50 mL of sterilized inorganic salt medium at 2 v / v%, and the medium contained 5 g / L terephthalic acid as the sole carbon source.

[0077] (4) The cell turbidity was measured every 6 - 12 hours, and 1 mL of the bacterial liquid was taken for centrifugation to obtain the supernatant for measuring the concentration of terephthalic acid, and 1 mL of the bacterial liquid was taken for centrifugation to obtain the supernatant for measuring the concentration of terephthalic acid until 72 hours. In this example, the growth curve of Pseudomonas putida KT1-EK is as shown in the appendix Figure 3 and after 48 hours, the OD of the bacterial cells 600 exceeds 2.

[0078] The formula of the inorganic salt medium is as follows: in 1 L of distilled water, it contains: 3.88 g of K2HPO4, 2.12 g of NaH2PO4·2H2O, 2.00 g of (NH4)2SO4, 0.1 g of MgCl2·6H2O, 10 mg of EDTA, 2 mg of ZnSO4·7H2O, 1 mg of CaCl2·2H2O, 5 mg of FeSO4·7H2O, 0.2 mg of Na2MoO4·2H2O, 0.2 mg of CuSO4·5H2O, 0.4 mg of CoCl2·6H2O.

[0079] Example 5 Construction of the engineered strain Pseudomonas putida KT1-EK2 and its culture with terephthalic acid as the sole carbon source

[0080] (1) Construction of engineered bacteria: Replace the transporter module (SEQ ID NO. 5) in the terephthalic acid catabolic module (SEQ ID NO. 4) derived from Comamonas sp. E6 with the transporter module (SEQ ID NO. 3) derived from Pseudomonas guguanensis GO16; the catabolic module after replacement is shown in SEQ ID NO. 6. The obtained overexpression vector is pSEVA64-tphE-K.

[0081] Electrotransform the recombinant plasmid pSEVA64-tphE-K into competent cells of Pseudomonas putida KT1. Among them, the methods for preparing competent cells and electrotransformation are referred to in Example 3. Pick the colonies on the plate and inoculate them into an LB liquid medium containing kanamycin and gentamicin, and culture at 30 °C for 16 h, and collect the cells for standby. The obtained engineered strain is named Pseudomonas putida KT1-EK2.

[0082] (2) Cultivate the engineered bacteria obtained in step (1) with terephthalic acid as the sole carbon source: Pick a single colony from the LB solid medium and inoculate it into a shake tube containing 5 mL of sterilized LB medium for cultivation. Place it on a shaker and culture at 30 °C at a rotation speed of 220 r / min for 16 h to obtain the seed liquid culture of the strain; the LB medium contains 50 mg / L gentamicin.

[0083] (3) Inoculate the cell liquid culture in step (2) into a 250 mL conical flask containing 50 mL of sterilized inorganic salt medium at 2 v / v%, and the medium contains 60 mM terephthalic acid as the sole carbon source.

[0084] (4) Measure the cell turbidity every 6 - 12 hours, and aspirate 1 mL of the bacterial liquid, centrifuge it to take the supernatant and measure the concentration of terephthalic acid until 48 hours. In this example, the growth curve of Pseudomonas putida KT1-EK2 is as shown in the appendix Figure 3 and after 48 hours, the OD600 of the bacteria exceeds 5.

[0085] The formula of the inorganic salt medium is the same as that in Example 1.

[0086] Example 6 Cultivation of the engineered bacteria Pseudomonas putida KT1-G and KT1-EK2 using ethylene glycol and terephthalic acid as carbon sources

[0087] (1) Activation of engineered bacteria: The engineered bacteria Pseudomonas putida KT1-G and KT1-EK2 were streaked and activated on LB solid plates respectively, and single colonies were picked and transferred to LB liquid medium for overnight culture of the seed solution. KT1-G and KT1-EK2 are resistant to gentamicin.

[0088] (2) Culturing the engineered bacteria in step (1) using ethylene glycol and terephthalic acid as carbon sources. They were transferred to the minimal inorganic salt medium at 2 V / V% respectively for shake flask fermentation. The medium contained 60 mM terephthalic acid and 60 mM ethylene glycol.

[0089] (3) The cell turbidity was measured every 6 - 12 hours, and 1 mL of the bacterial solution was taken for centrifugation to obtain the supernatant to measure the concentrations of ethylene glycol and terephthalic acid until 72 hours. In this example, the growth curves of the engineered bacteria Pseudomonas putida KT1-G and KT1-EK2 and the consumption rates of the two substrates are as shown in the appendix Figure 4 shown.

[0090] Conclusion: KT1-EK2 can completely consume 60 mM terephthalic acid and 60 mM ethylene glycol within 56 h, and ethylene glycol is preferentially consumed completely within 48 h.

[0091] The present invention provides a new strategy for modifying Pseudomonas putida to efficiently co-utilize ethylene glycol and terephthalic acid. There are many methods and ways to specifically implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this example can be implemented by existing technologies.

Claims

1. An engineered Pseudomonas putida, characterized in that, It includes Pseudomonas putida and a recombinant plasmid introduced into Pseudomonas putida to satisfy self - replication; the key gene affecting ethylene glycol metabolism has been knocked out in the genome of Pseudomonas putida; The recombinant plasmid is one of the following: (1) It contains the terephthalic acid catabolism module shown in SEQ ID NO.2; (2) It contains the terephthalic acid catabolism module shown in SEQ ID NO.6; (3) It contains the terephthalic acid catabolism module shown in SEQ ID NO.4 and the terephthalic acid transport module shown in SEQ ID NO.3; The key gene for ethylene glycol metabolism is gclR, and its nucleotide sequence is shown in SEQ ID NO.

1.

2. The engineered Pseudomonas putida as claimed in claim 1, wherein, The strain type of Pseudomonas putida is Pseudomonas putida KT2440 ATCC NO.47054.

3. The engineered Pseudomonas putida as described in claim 1, wherein For (3), the terephthalic acid catabolism module and the terephthalic acid transport module are inserted into two different recombinant plasmids respectively, and both recombinant plasmids satisfy self - replication and compatibility in Pseudomonas putida.

4. The engineered Pseudomonas putida as claimed in claim 1, wherein The original expression vector of the recombinant plasmid is pSEVA64.

5. A method for preparing the engineered Pseudomonas putida as described in any one of claims 1 to 4, characterized in that, It includes: (1) Using gene knockout technology to knock out the key gene affecting ethylene glycol metabolism in the genome of Pseudomonas putida. After knockout, laboratory adaptive evolution is carried out to obtain the evolved engineered bacteria; The key gene for ethylene glycol metabolism is gclR, and its nucleotide sequence is shown in SEQ ID NO.1; (2) Inserting the terephthalic acid catabolism module with the nucleotide sequence shown in SEQ ID NO.2 or SEQ ID NO.6 into the original expression vector; or inserting the terephthalic acid catabolism module with the nucleotide sequence shown in SEQ ID NO.4 and the terephthalic acid transport module with the nucleotide sequence shown in SEQ ID NO.3 into two original expression vectors respectively to obtain recombinant plasmids; (3) Transforming the recombinant plasmid into the engineered bacteria after gene knockout to obtain Pseudomonas putida engineered bacteria; The method of the laboratory adaptive evolution includes the following steps: (A) Pick a single colony of Pseudomonas putida on the LB solid medium, inoculate it into the liquid LB medium for culture, place it on a shaker, and culture it at 30 °C with a rotation speed of 220 r / min for 12 - 16 h to obtain a seed liquid culture; (B) Inoculate the seed liquid culture obtained in step (A) into the evolution medium for culture according to an inoculation amount of 2 v / v%, place it on a shaker, and culture it at 30 °C with a rotation speed of 220 r / min until the cell OD reaches 0.4 - 0.8 to obtain the first - generation cell culture; (C) Transfer the first - generation cell culture obtained in step (B) to fresh evolution medium, place it on a shaker, and culture it at 30 °C with a rotation speed of 220 r / min until the cell OD reaches 0.4 - 0.8 to obtain the second - generation cell culture of the strain; (D) Continuously repeat step (C) to obtain the 10th - 20th generation cell culture; (E) When the cell growth cycle is stable, passage is stopped to obtain the evolved engineered bacterium.

6. The preparation method according to claim 5, characterized in that, The formula of the evolution medium is as follows: in 1 L of distilled water, there are 3.88 g of K2HPO4, 2.12 g of NaH2PO4·2H2O, 2.00 g of (NH4)2SO4, 0.1 g of MgCl2·6H2O, 10 mg of EDTA, 2 mg of ZnSO4·7H2O, 1 mg of CaCl2·2H2O, 5 mg of FeSO4·7H2O, 0.2 mg of Na2MoO4·2H2O, 0.2 mg of CuSO4·5H2O, and 0.4 mg of CoCl2·6H2O.

7. Use of the engineered bacterium of Pseudomonas putida according to any one of claims 1 to 4 or the engineered bacterium of Pseudomonas putida prepared by the preparation method according to any one of claims 5 to 6 in the co-utilization of ethylene glycol and terephthalic acid.

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