Application of recombinant bacteria and plant culture method

Abstract

The invention relates to application of recombinant bacteria and a plant culture method. According to the application of the recombinant bacterium in preparation of indoleacetic acid, a PETase gene, an MHETase gene, a tphA1 gene, a tphA2 gene, a tphA3 gene, a tphB gene and a tpaK gene are integrated in a genome of the bacterium. A PETase gene, an MHETase gene, a tphA1 gene, a tphA2 gene, an A3 gene, a tphB gene and a tpaK gene are integrated in a genome of the recombinant bacterium, PET and PBAT plastics can be degraded under natural conditions, and the degradation efficiency is high. According to the recombinant bacteria, plastics such as PET plastics and PBAT plastics are used as carbon sources, plant growth hormone indoleacetic acid is synthesized to be used for preparing indoleacetic acid, degradation of waste plastics in the environment is accelerated, degradation products are converted into indoleacetic acid to promote growth of plants, and secondary pollution is relieved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of recombinant bacteria and plant culture methods. Background Technology

[0002] Petroleum-based plastics are polymeric materials made from chemical monomer raw materials through addition polymerization or condensation polymerization. They are diverse and widely used, including polyethylene terephthalate (PET), polybutylene terephthalate (PBAT), polyethylene (PE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), and polyurethane (PUR). PET and PBAT plastics are polyester plastics. Due to their excellent physicochemical properties, they are widely used as packaging, containers, and clothing. Currently, the conventional treatment methods for PET plastics mainly include physical methods, chemical methods, incineration, and landfill [3]. Physical methods decompose plastics by sorting, grinding, heating, and other processes. This method cannot completely degrade plastics and easily generates new environmental pollutants (microplastics). Chemical methods degrade PET plastics through processes such as glycolysis, alcoholysis, hydrolysis, and ammonolysis. These methods usually require high temperature conditions and the addition of catalysts, resulting in high costs and easy secondary pollution [5]. Most PET plastics in the environment (approximately 72%) are difficult to recycle effectively and are mainly disposed of through incineration and landfill. Incineration releases toxic and harmful substances, polluting water and air. Landfilling has a long processing time, can cause soil compaction, and hinders crop growth. While PBAT plastics are biodegradable, their degradation process requires high-temperature composting, and their degradation products are difficult for microorganisms in the environment to utilize, potentially leading to secondary pollution.

[0003] Microbial resources are abundant, diverse in type and function, and can be developed into fertilizers with different functions and uses. Microbial fertilizers mainly play the role of indirect nutrition and can be divided into five categories according to the type of microorganism: (1) bacterial fertilizers (such as rhizobium fertilizer, nitrogen-fixing bacteria fertilizer, phosphorus-solubilizing bacteria fertilizer, potassium-solubilizing bacteria fertilizer, and photosynthetic bacteria fertilizer); (2) actinomycete fertilizers (such as antibiotic fertilizer); (3) fungal fertilizers (mycorrhizal fungal fertilizers: including ectomycorrhizal fungi and endomycorrhizal fungi); (4) algal fertilizers (such as nitrogen-fixing cyanobacteria fertilizer); (5) compound microbial fertilizers. Currently reported microorganisms cannot achieve the degradation of waste PET and PBAT plastics in the environment or the metabolic function of degradation products (terephthalic acid, ethylene glycol, butanediol, adipic acid). Therefore, it is necessary to develop a method that can effectively degrade waste PET and PBAT plastics in the environment and utilize the degradation products to generate plant growth hormones to promote plant growth, thereby controlling environmental pollution while allowing the degradation products to enter the geochemical cycle.

[0004] Waste PET and PBAT plastics in the environment are difficult to degrade. Moreover, waste plastics in the natural environment are usually "degraded" into small microplastics (<5μm) rather than being completely degraded into single-molecule compounds. These single-molecule compounds, including terephthalic acid, ethylene glycol, butanediol, adipic acid, etc., cannot be further utilized by microorganisms in the environment, thus causing secondary pollution to the environment. Summary of the Invention

[0005] Therefore, this application requires providing an application of the recombinant bacteria. This recombinant bacteria can degrade PET and PBAT plastics under natural conditions, and can convert the degraded single-molecule compounds into indoleacetic acid, which can be used for the preparation of indoleacetic acid.

[0006] In addition, it is necessary to provide a plant cultivation method.

[0007] The application of a recombinant bacterium in the preparation of indoleacetic acid, wherein the genome of the recombinant bacterium integrates the PETase gene, MHETase gene, tphA1 gene, tphA2 gene, tphA3 gene, tphB gene and tpaK gene.

[0008] In the application of the above-mentioned recombinant bacteria in the preparation of indoleacetic acid, the genome of this recombinant bacteria integrates the PETase gene, MHETase gene, tphA1 gene, tphA2 gene, A3 gene, tphB gene, and tpaK gene. It can degrade PET and PBAT plastics under natural conditions and convert the degraded single-molecule compounds into indoleacetic acid, which can be used for the preparation of indoleacetic acid. Experimental verification showed that the degradation rate of PBAT by the above-mentioned recombinant bacteria under natural conditions reached 35%, and it had a significant effect on promoting the root growth of Arabidopsis thaliana.

[0009] In one embodiment, the recombinant bacteria uses *Pseudomonas putida*, a bacterium with ethylene glycol metabolism capabilities, as chassis cells.

[0010] Furthermore, the recombinant bacteria uses Pseudomonas putidaPP1 strain with accession number CCTCC NO:M 20231659 as the chassis cells.

[0011] In one embodiment, the PETase and MHETase genes are derived from Ideonella sakaiensis.

[0012] In one embodiment, the tphA1, tphA2, tphA3, and tphB genes are derived from Commonas testosterone.

[0013] In one embodiment, the tpaK gene is derived from Rhodococcus jostii.

[0014] In one embodiment, the amino acid sequence of PETase is shown in SEQ ID No. 1;

[0015] And / or, the amino acid sequence of MHETase is shown in SEQ ID No. 2;

[0016] And / or, the amino acid sequence of tpaK is shown in SEQ ID No. 3;

[0017] And / or, the amino acid sequence of tphA1 is shown in SEQ ID No. 4;

[0018] And / or, the amino acid sequence of tphA2 is shown in SEQ ID No. 5;

[0019] And / or, the amino acid sequence of tphA3 is shown in SEQ ID No. 6;

[0020] And / or, the amino acid sequence of tphB is shown in SEQ ID No. 7.

[0021] In one embodiment, the PETase gene, MHETase gene, and tphA1 gene are all linked to the Pgro4 promoter and the T0 terminator.

[0022] And / or, both the tphA2 and tphA3 genes are linked to the Pem7-2 promoter and the T0 terminator;

[0023] And / or, the tphB gene is linked to the Pem7-5 promoter and the T0 terminator;

[0024] And / or, the tpaK gene is linked to the Pem7-9 promoter and the T0 terminator.

[0025] In one embodiment, the base sequence of the Pgro4 promoter is shown in SEQ ID No. 8;

[0026] And / or, the base sequence of the Pem7-2 promoter is shown in SEQ ID No. 9;

[0027] And / or, the base sequence of the Pem7-5 promoter is shown in SEQ ID No. 10;

[0028] And / or, the base sequence of the Pem7-9 promoter is shown in SEQ ID No. 11;

[0029] And / or, the base sequence of the T0 terminator is shown in SEQ ID No. 12.

[0030] In one embodiment, the recombinant bacteria are transformed with a mutant plasmid, the mutant plasmid carrying a mutant DNA polymerase gene and a sacB gene, wherein the mutant DNA polymerase gene is either the dnaQ926 DNA polymerase gene or the mutD5 DNA polymerase gene.

[0031] Furthermore, the amino acid sequence of dnaQ926 DNA polymerase is shown in SEQ ID No. 13; the amino acid sequence of mutD5 DNA polymerase is shown in SEQ ID No. 14.

[0032] In one embodiment, the recombinant bacteria were obtained by carbon source pressure screening, wherein the carbon source is phthalic acid (TPA), 1,4-butanediol (BDO), adipic acid (AA), or bis(2-hydroxyethyl) terephthalate (BHET).

[0033] In one embodiment, the recombinant bacteria overexpress the PETase gene and the MHETase gene.

[0034] A plant culture comprising a recombinant bacterium and a plant culture medium, wherein the genome of the recombinant bacterium integrates the PETase gene, MHETase gene, tphA1 gene, tphA2 gene, tphA3 gene, tphB gene, and tpaK gene.

[0035] A plant cultivation method includes the following steps:

[0036] The recombinant bacteria were inoculated into a plant culture medium containing plastic and then transplanted into plants to be cultured. The genome of the recombinant bacteria integrated the PETase gene, MHETase gene, tphA1 gene, tphA2 gene, tphA3 gene, tphB gene and tpaK gene.

[0037] In one embodiment, the plastic article contains at least one of polyethylene terephthalate, polybutylene terephthalate, phthalic acid, 1,4-butanediol, adipic acid, and bis(2-hydroxyethyl) terephthalate.

[0038] In one embodiment, the plant to be cultivated is Arabidopsis thaliana or Nicotiana benthamiana. Attached Figure Description

[0039] Figure 1 The flowchart shows the synthetic pathway of indoleacetic acid in strain PP1.

[0040] Figure 2 HPLC detection results for indoleacetic acid produced by the metabolism of strain PP1;

[0041] Figure 3 This is a comparison chart showing the ability of different strains to degrade PBAT in plant culture medium in Example 3. Detailed Implementation

[0042] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples and accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0043] One embodiment of this application provides the application of a recombinant bacterium in the preparation of indoleacetic acid. The genome of this recombinant bacterium integrates the PETase gene, MHETase gene, tphA1 gene, tphA2 gene, tphA3 gene, tphB gene, and tpaK gene.

[0044] In the application of the above-mentioned recombinant bacteria in the preparation of indoleacetic acid, the genome of this recombinant bacteria integrates the PETase gene, MHETase gene, tphA1 gene, tphA2 gene, A3 gene, tphB gene, and tpaK gene. It can degrade PET and PBAT plastics under natural conditions and convert the degraded single-molecule compounds into indoleacetic acid, which can be used for the preparation of indoleacetic acid. Experimental verification showed that the degradation rate of PBAT by the above-mentioned recombinant bacteria under natural conditions reached 35%, and it had a significant effect on promoting the root growth of Arabidopsis thaliana.

[0045] In one embodiment, since ethylene glycol is a monomeric compound of PET plastic, the terephthalic acid and ethylene glycol produced during the degradation of PET are difficult for other microorganisms to utilize, thus causing secondary pollution problems. Therefore, the recombinant bacteria use Pseudomonas putidae, which has the ability to metabolize ethylene glycol, as chassis cells.

[0046] Microorganisms, as natural decomposers, inhabit diverse and rich environments, possessing highly evolved and adaptable abilities. Microbial degradation of plastics is an environmentally friendly and energy-efficient method, representing a safe and ecological approach. Among these microorganisms, *Pseudomonas putida* is widely distributed in the environment, exhibiting strong environmental adaptability, natural multifunctionality, and significant intrinsic oxidative stress and solvent tolerance. Therefore, *Pseudomonas putida* is a promising microbial host for research on the depolymerization and degradation product transformation of PBAT and PET plastics.

[0047] Furthermore, the recombinant bacteria used *Pseudomonas putida* PP1 strain with accession number CCTCC NO:M 20231659 as the chassis cells. *Pseudomonas putida* PP1 strain was deposited at the China Center for Type Culture Collection (CCTCC) on September 11, 2023, with accession number CCTCC NO:M 20231659.

[0048] It should be noted that the recombinant bacteria are not limited to the chassis cells mentioned above, but can also be other chassis cells, such as Escherichia coli and yeast.

[0049] In one embodiment, the PETase and MHETase genes are derived from Ideonella sakaiensis.

[0050] In one embodiment, the tphA1, tphA2, tphA3, and tphB genes are derived from Commonas testosterone.

[0051] In one embodiment, the tpaK gene is derived from Rhodococcus jostii.

[0052] In one embodiment, the amino acid sequence of PETase is shown in SEQ ID No. 1.

[0053] In one embodiment, the amino acid sequence of MHETase is shown in SEQ ID No. 2.

[0054] In one embodiment, the amino acid sequence of tpaK is shown in SEQ ID No. 3.

[0055] In one embodiment, the amino acid sequence of tphA1 is shown in SEQ ID No. 4.

[0056] In one embodiment, the amino acid sequence of tphA2 is shown in SEQ ID No. 5.

[0057] In one embodiment, the amino acid sequence of tphA3 is shown in SEQ ID No. 6.

[0058] In one embodiment, the amino acid sequence of tphB is shown in SEQ ID No. 7.

[0059] In one embodiment, the PETase gene, MHETase gene, and tphA1 gene are all linked to the Pgro4 promoter and the T0 terminator.

[0060] In one embodiment, both the tphA2 and tphA3 genes are linked to the Pem7-2 promoter and the T0 terminator.

[0061] In one embodiment, the tphB gene is linked to the Pem7-5 promoter and the T0 terminator.

[0062] In one embodiment, the tpaK gene is linked to the Pem7-9 promoter and the T0 terminator.

[0063] In one embodiment, the base sequence of the Pgro4 promoter is shown in SEQ ID No. 8.

[0064] In one embodiment, the base sequence of the Pem7-2 promoter is shown in SEQ ID No. 9.

[0065] In one embodiment, the base sequence of the Pem7-5 promoter is shown in SEQ ID No. 10.

[0066] In one embodiment, the base sequence of the Pem7-9 promoter is shown in SEQ ID No. 11.

[0067] In one embodiment, the base sequence of the T0 terminator is shown in SEQ ID No. 12.

[0068] In one embodiment, the recombinant bacteria are transformed with a mutant plasmid carrying a mutant DNA polymerase gene and a sacB gene (amino acid sequence as shown in SEQ ID No. 16). The mutant DNA polymerase gene is either the dnaQ926 gene (amino acid sequence as shown in SEQ ID No. 14) or the mutD5 gene (amino acid sequence as shown in SEQ ID No. 15). This application utilizes DNA polymerase mutant genome mutation technology to evolve the recombinant bacteria.

[0069] In one embodiment, the recombinant bacteria were obtained through carbon source pressure screening. The carbon source included one of phthalic acid (TPA), 1,4-butanediol (BDO), adipic acid (AA), and bis(2-hydroxyethyl) terephthalate (BHET). To improve the efficiency of screening beneficial mutant strains, high-throughput screening models were constructed using phthalic acid, 1,4-butanediol, adipic acid, and bis(2-hydroxyethyl) terephthalate as the sole carbon source for screening pressure. It should be noted that the carbon source is not limited to phthalic acid, 1,4-butanediol, adipic acid, and bis(2-hydroxyethyl) terephthalate; other carbon sources can also be used as the sole carbon source for pressure screening to obtain strains capable of metabolizing that carbon source.

[0070] The culture for 1,4-butanediol (BDO) pressure screening included (g / L): 1.8g BDO, 3g KH2PO4, 7g Na2HPO4, 1.4g urea, 0.5g NaCl, 0.25g MgSO4, and 1mL of trace elements solution (perliter) (2g ZnSO4·7H2O, 1g MnCl2·2H2O, 0.3g Na2B4O7, 0.2g Na2MoO4·2H2O, 0.2g CuSO4·7H2O, 0.4g CoCl2·6H2O, and 5g FeSO4·7H2O).

[0071] The culture for phthalic acid (TPA) stress screening included (g / L): 4g TPA, 3g KH2PO4, 7g Na2HPO4, 1.4g urea, 0.5g NaCl, 0.25g MgSO4 and 1mL of trace elements solution (per liter) (2g ZnSO4·7H2O, 1g MnCl2·2H2O, 0.3g Na2B4O7, 0.2g Na2MoO4·2H2O, 0.2g CuSO4·7H2O, 0.4g CoCl2·6H2O and 5g FeSO4·7H2O).

[0072] The culture for adipic acid (AA) stress screening included (g / L): 3g AA, 3g KH2PO4, 7g Na2HPO4, 1.4g gurea, 0.5g NaCl, 0.25g MgSO4, and 1mL of trace element solution (per liter) (2g ZnSO4·7H2O, 1g MnCl2·2H2O, 0.3g Na2B4O7, 0.2g Na2MoO4·2H2O, 0.2g CuSO4·7H2O, 0.4g CoCl2·6H2O, and 5g FeSO4·7H2O).

[0073] The culture for pressure screening of bis(2-hydroxyethyl) terephthalate (BHET) included (g / L): 3g BHET, 3g KH2PO4, 7g Na2HPO4, 1.4g urea, 0.5g NaCl, 0.25g MgSO4 and 1mL of trace elements solution (per liter) (2g ZnSO4·7H2O, 1g MnCl2·2H2O, 0.3g Na2B4O7, 0.2g Na2MoO4·2H2O, 0.2g CuSO4·7H2O, 0.4g CoCl2·6H2O and 5g FeSO4·7H2O).

[0074] In one embodiment, the recombinant bacteria overexpressed the PETase gene and the MHETase gene.

[0075] The genome of the aforementioned recombinant bacteria integrates the PETase, MHETase, tphA1, tphA2, tphA3, tphB, and tpaK genes, enabling it to degrade PET and PBAT plastics under natural conditions with high efficiency. This allows it to be applied to the degradation of plastic products. Experimental results show that, under natural conditions, shake-flask fermentation of the aforementioned recombinant bacteria achieves a degradation rate of 9.5 mg PET / day for PET and 17.8 mg PBAT / day for PBAT, demonstrating high degradation efficiency.

[0076] Compared to enzyme-based degradation, the recombinant bacteria of this application can degrade waste PET and PBAT plastics in the environment without requiring prior recycling and degradation treatment.

[0077] Existing enzymes require high temperatures to degrade PET and PBAT plastics, while the recombinant bacteria of this application can degrade PET and PBAT plastics under natural conditions.

[0078] Furthermore, the recombinant bacteria of this application can not only degrade PET and PBAT plastics, but also further metabolize their degradation products EG, TPA, BDO and AA, achieving complete degradation and reducing the problem of secondary pollution of plastics to the environment.

[0079] One embodiment of this application also provides a method for constructing the above-mentioned recombinant bacteria, comprising the following steps: integrating the PETase gene, MHETase gene, tphA1 gene, tphA2 gene, tphA3 gene, tphB gene and tpaK gene into the genome of a chassis cell to obtain recombinant bacteria.

[0080] The detailed description of the recombinant bacteria mentioned above is provided above and will not be repeated here.

[0081] In one embodiment, after the step of integrating the PETase gene, MHETase gene, tphA1 gene, tphA2 gene, tphA3 gene, tphB gene, and tpaK gene into the genome of the chassis cell, the method further includes the following step: transforming a mutant plasmid into the gene-integrated chassis cell, the mutant plasmid carrying a mutant DNA polymerase gene and a sacB gene (amino acid sequence as shown in SEQ ID No. 15), the mutant DNA polymerase gene being either the dnaQ926 gene (amino acid sequence as shown in SEQ ID No. 13) or the mutD5 gene (amino acid sequence as shown in SEQ ID No. 14).

[0082] In one embodiment, after the step of transforming the mutant plasmid into the gene-integrated chassis cells, the method further includes the following step: performing carbon source stress screening on the cells transformed with the mutant plasmid, the carbon source including at least one of phthalic acid, 1,4-butanediol, adipic acid and bis(2-hydroxyethyl) terephthalate.

[0083] In one embodiment, after the step of performing carbon source stress screening on cells transformed with mutant plasmids, the method further includes the step of overexpressing the PETase gene and the MHETase gene in the stress-screened cells.

[0084] In one embodiment, the step of overexpressing the PETase gene and the MHETase gene in cells obtained through stress screening includes:

[0085] The signal peptide sequence of the PETase gene was replaced with the SPpelB sequence to obtain the SPpelB-PETase gene.

[0086] The SPpelB-PETase gene and the MHETase gene were ligated into the expression plasmid to obtain the recombinant expression plasmid.

[0087] The recombinant expression plasmid was transformed into cells obtained through stress selection.

[0088] In one embodiment, the SPpelB sequence is shown in SEQ ID No. 25.

[0089] The application of the above-mentioned recombinant bacteria, or the recombinant bacteria obtained by the above-mentioned recombinant bacteria construction method, in degrading plastic products.

[0090] In one embodiment, the plastic article contains at least one of polyethylene terephthalate, polybutylene terephthalate, phthalic acid, 1,4-butanediol (BDO), adipic acid, and bis(2-hydroxyethyl) terephthalate.

[0091] This application constructs an engineered *Pseudomonas malodorans* strain capable of degrading PBAT and PET plastics and further metabolizing their degradation products. Simultaneously, a laboratory adaptive evolution strategy is employed to enhance the degradation efficiency of this strain for PBAT and PET plastics. Using this method, the *Pseudomonas malodorans* strain achieved a degradation rate of 9.5 mg PET / day for PET plastic and 17.8 mg PBAT / day for PBAT plastic in shake-flask fermentation.

[0092] One embodiment of this application provides a plant culture comprising the above-mentioned recombinant bacteria and a plant culture medium, wherein the genome of the recombinant bacteria integrates the PETase gene, MHETase gene, tphA1 gene, tphA2 gene, tphA3 gene, tphB gene and tpaK gene.

[0093] The detailed description of the recombinant bacteria mentioned above is provided above and will not be repeated here.

[0094] The plant culture medium formula (g / L) is as follows: Murashige & Skoog (MS) 2.17 g / L, glucose 2 g / L, KH₂PO₄ 1.5 g / L, Na₂HPO₄ 1.5 g / L, Agar 12 g / L (pH adjusted to 6.5 with 2M HCl solution). It should be noted that the plant culture medium is not limited to the above formula and can be adjusted according to the type of plant being cultured.

[0095] The aforementioned plant cultures can significantly promote plant growth.

[0096] One embodiment of this application provides a plant cultivation method, comprising the following steps:

[0097] The recombinant bacteria were inoculated into a plant culture medium containing plastic and then transplanted into the plants to be cultured. The genome of the recombinant bacteria integrated the PETase gene, MHETase gene, tphA1 gene, tphA2 gene, tphA3 gene, tphB gene and tpaK gene.

[0098] The detailed description of the recombinant bacteria mentioned above is provided above and will not be repeated here.

[0099] The plant culture medium formula (g / L) is as follows: Murashige & Skoog (MS) 2.17 g / L, glucose 2 g / L, KH₂PO₄ 1.5 g / L, Na₂HPO₄ 1.5 g / L, Agar 12 g / L (pH adjusted to 6.5 with 2M HCl solution). It should be noted that the plant culture medium is not limited to the above formula and can be adjusted according to the type of plant being cultured.

[0100] The plastic products contain at least one of polyethylene terephthalate, polybutylene terephthalate, phthalic acid, 1,4-butanediol, adipic acid, and bis(2-hydroxyethyl) terephthalate.

[0101] The plants to be cultivated are Arabidopsis thaliana or Nicotiana benthamiana.

[0102] The above-mentioned plant culture method uses recombinant bacteria as a carbon source to synthesize the plant growth hormone indoleacetic acid. This accelerates the degradation of waste plastics in the environment and converts the degradation products into indoleacetic acid to promote plant growth, thereby reducing secondary pollution.

[0103] The following are specific examples.

[0104] Unless otherwise specified, the drugs and instruments used in the examples are conventional choices in the art. Experimental methods not specifying particular conditions in the examples are typically performed under standard conditions, such as those described in literature, books, or methods recommended by the reagent kit manufacturer.

[0105] In the following embodiments, the sequences involved are shown in Tables 1 to 3.

[0106] Table 1: Required primer sequences

[0107]

[0108] Table 2: Required gene base sequences

[0109]

[0110]

[0111]

[0112]

[0113] Table 3: Amino acid sequences of the required enzymes

[0114]

[0115]

[0116]

[0117]

[0118] Example 1:

[0119] Bioinformatics methods were used to mine tryptophan decarboxylase (Tdc), monoamine oxidase (AofH), and aldehyde dehydrogenase (AldH) from the genome of the original strain *Pseudomonas putida* PP1. Figure 1 Using genomic DNA from *Pseudomonas putidaPP1* strain as a template, PCR reactions were performed using tdc-F / R, aofH-F / R, and aldH-F / R primers, respectively, to obtain the tdc, aofH, and aldH gene fragments. These three genes were then ligated into the pBBR1MCS plasmid, and the Pgro4 promoter sequence was added upstream of each gene to obtain the pMCS-tdc, pMCS-aofH, and pMCS-aldH plasmids. The plasmid was transformed into the PET and PBAT plastic degrading strain *Pseudomonas putida* PP1 (deposited at the China Center for Type Culture Collection (CCTCC) on September 11, 2023, accession number CCTCC NO: M 20231659) to obtain strains PP1 / pMCS-tdc, PP1 / pMCS-aofH, and PP1 / pMCS-aldH, respectively. These strains were fermented in LB medium in shake flasks for 24 h. The cells were then collected and sonicated with PBS to obtain the supernatant of each strain. 200 μL of the supernatant was transferred to a 2 mL EP tube, and 0.1 g of L-tryptophan was added. The mixture was incubated at 30 °C and 400 rpm for 6 h. After the reaction, the sample was extracted with ethyl acetate at a volume ratio of 1:1. The ethyl acetate layer was collected, dried under nitrogen, redissolved in methanol, and analyzed by HPLC. Figure 2 The test results showed that the above three enzymes can catalyze the final synthesis of indoleacetic acid from tryptophan, meaning that the Pseudomonas putida PP1 strain has the ability to synthesize indoleacetic acid using L-tryptophan as a precursor.

[0120] Example 2:

[0121] Laboratory adaptive evolution (ALE) was employed to enable strain PP2 to degrade the products of PET and PBAT plastics, aiming to achieve complete degradation of these plastics. Specifically, ALE involved evolving strain PP2 based on DNA polymerase mutant genomic mutation technology. To improve the efficiency of screening beneficial mutant strains, a high-throughput screening model using TPA as the sole carbon source was constructed. The DNA polymerase gene was cloned from *E. coli*, and an amino acid mutation was performed at a specific site to obtain the dnaQ926 mutant. The dnaQ926 gene was ligated into the pBBR1MCS plasmid, and an arabinose promoter (Para) was added upstream of the gene to obtain the pMCS-Para-dnaQ926 mutant plasmid. To eliminate the mutant plasmid in subsequent screening, the sacB gene was added downstream of the dnaQ926 gene to obtain the pMCS-Para-dnaQ926-sacB mutant plasmid. The pMCS-Para-dnaQ926-sacB mutant plasmid was transformed into strain PP2 to obtain strain PP2 / pMCS-Para-dnaQ926-sacB.

[0122] Pick single clones of PP2 / pMCS-Para-dnaQ926-sacB and add 0.8M arabinose to LB medium. Incubate at 30℃ and 200rpm for 12h. Take 100μL of culture, centrifuge at 13000xg for 1min, discard the supernatant, resuspend the bacterial cells in PBS buffer, and spread them on MM_TPA plates with TPA as the sole carbon source. The MM_TPA medium formulation (g / L) is: 4g TPA, 3g KH2PO4, 7g Na2HPO4, 1.4g urea, 0.5g NaCl, 0.25g MgSO4, and 1mL of trace elements solution (per liter) (2g ZnSO4·7H2O, 1g MnCl2·2H2O, 0.3g Na2B4O7, 0.2g Na2MoO4·2H2O, 0.2g CuSO4·7H2O, 0.4g CoCl2·6H2O, 5g FeSO4·7H2O). MM_TPA plates coated with the bacterial solution were incubated at 30℃ for 3 days. Larger single-clone strains were picked from the plates and subjected to TPA degradation metabolism verification in MM_TPA liquid medium. The optimal growth monoclonal strain ALE_TPA / pMCS-Para-dnaQ926-sacB was inoculated into LB medium and cultured for 12 h. Then, it was plated on 20% sucrose-LB plates to eliminate the corresponding plasmids, thus obtaining strain ALE_TPA.

[0123] Example 3:

[0124] The rapid degradation of PET and PBAT plastics was achieved by overexpressing PETase and MHETase using plasmids. First, the signal peptide sequence of the PETase gene was replaced with the SPpelB sequence to obtain the SPpelB-PETase gene. This gene, along with the MHETase gene, was then ligated into the pBBR1MCS plasmid. A Pgro4 promoter sequence was added upstream of the SPpelB-PETase gene, and a T0 terminator sequence was added downstream of the MHETase gene to obtain the pMCS-Pgro4-PMHETase plasmid. The empty pBBR1MCS vector was transformed into PP1 chassis cells to obtain the PP1 / pMCS strain. Simultaneously, the pMCS-Pgro4-PMHETase plasmid was transformed into the PP1 and ALE_TPA strains, respectively, to obtain the PP2 / pMCS-Pgro4-PMHETase and ALE_TPA / pMCS-Pgro4-PMHETase strains.

[0125] Strains PP1 / pMCS, PP2 / pMCS-Pgro4-PMHETase, and ALE_TPA / pMCS-Pgro4-PMHETase were inoculated into plant solid culture medium, and then Arabidopsis thaliana was transplanted onto the medium and placed in PBAT plastic for degradation for 30 days. The plant culture medium formula was (g / L): Murashige & Skoog (MS) 2.17 g / L, glucose 2 g / L, KH2PO4 1.5 g / L, Na2HPO4 1.5 g / L, Agar 12 g / L (pH adjusted to 6.5 with 2M HCl solution). The results showed that the degradation rate of PBAT by strain ALE_TPA / pMCS-Pgro4-PMHETase reached 35%. Figure 3 Furthermore, compared with the control, Arabidopsis thaliana grew better in plates inoculated with strains PP1 and ALE_TPA, and strain ALE_TPA had a significant effect on promoting the root growth of Arabidopsis thaliana.

[0126] This application utilizes recombinant bacteria to synthesize the plant growth hormone indoleacetic acid (IAA) using plastics such as PET and PBAT as carbon sources. This accelerates the degradation of waste plastics in the environment while converting the degradation products into IAA to promote plant growth and reduce secondary pollution.

[0127] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0128] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Use of a recombinant bacterium for the preparation of indoleacetic acid, characterized in that, The genome of the recombinant bacteria integrates PETase gene, MHETase gene, tphA1 gene, tphA2 gene, tphA3 gene, tphB gene and tpaK gene.

2. Use according to claim 1, characterized in that, The recombinant bacteria take Pseudomonas putida with ethylene glycol metabolic ability as the chassis cell. Further, the recombinant bacteria take Pseudomonas putida PP1 bacteria with the preservation number of CCTCC NO: M 20231659 as the chassis cell.

3. Use according to claim 1, characterized in that, The PETase gene and the MHETase gene are from Ideonella sakaiensis; And / or, the tphA1 gene, the tphA2 gene, the tphA3 gene and the tphB gene are from Comamonas testosterones; And / or, the tpaK gene is from Rhodococcus jostii.

4. Use according to claim 3, characterized in that, The amino acid sequence of the PETase is shown in SEQ ID No. 1; And / or, the amino acid sequence of the MHETase is shown in SEQ ID No. 2; And / or, the amino acid sequence of the tpaK is shown in SEQ ID No. 3; And / or, the amino acid sequence of the tphA1 is shown in SEQ ID No. 4; And / or, the amino acid sequence of the tphA2 is shown in SEQ ID No. 5; And / or, the amino acid sequence of the tphA3 is shown in SEQ ID No. 6; And / or, the amino acid sequence of the tphB is shown in SEQ ID No.

7.

5. The use according to claim 1, characterized in that, The PETase gene, the MHETase gene and the tphA1 gene are all connected with the Pgro4 promoter and the T0 terminator; And / or, the tphA2 gene and the tphA3 gene are both connected with the Pem7-2 promoter and the T0 terminator; And / or, the tphB gene is connected with the Pem7-5 promoter and the T0 terminator; And / or, the tpaK gene is connected with the Pem7-9 promoter and the T0 terminator.

6. Use according to claim 5, characterized in that, The base sequence of the Pgro4 promoter is shown in SEQ ID No. 8; And / or, the base sequence of the Pem7-2 promoter is shown in SEQ ID No. 9; And / or, the base sequence of the Pem7-5 promoter is shown in SEQ ID No. 10; And / or, the base sequence of the Pem7-9 promoter is shown in SEQ ID No. 11; And / or, the base sequence of the T0 terminator is shown in SEQ ID No.

12.

7. The use according to any one of claims 1 to 6, characterized in that, The recombinant bacteria are transformed with a mutant plasmid, and the mutant plasmid carries a mutant DNA polymerase gene and a sacB gene, wherein the mutant DNA polymerase gene is a dnaQ926 DNA polymerase gene or a mutD5 DNA polymerase gene. Further, the amino acid sequence of the dnaQ926 DNA polymerase is shown in SEQ ID No. 13; and the amino acid sequence of the mutD5 DNA polymerase is shown in SEQ ID No.

14.

8. Use according to claim 7, characterized in that, The recombinant bacteria are screened by carbon source pressure, and the carbon source is phthalic acid, 1,4-butanediol, adipic acid or bis(2-hydroxyethyl) terephthalate.

9. Use according to claim 8, characterized in that, The PETase gene and the MHETase gene are overexpressed in the recombinant bacteria.

10. A plant culture, characterized by, The recombinant bacteria and the plant culture medium are provided, and the PETase gene, the MHETase gene, the tphA1 gene, the tphA2 gene, the tphA3 gene, the tphB gene and the tpaK gene are integrated into the genome of the recombinant bacteria.

11. A plant culturing method characterized by, The method comprises the following steps: The recombinant bacteria are inoculated into the plant culture medium containing the plastic, and then transplanted into the plant culture to be cultivated, and the PETase gene, the MHETase gene, the tphA1 gene, the tphA2 gene, the tphA3 gene, the tphB gene and the tpaK gene are integrated into the genome of the recombinant bacteria.

12. The plant culturing method according to claim 11, wherein, The plastic product contains at least one of polyethylene terephthalate, polybutylene terephthalate, phthalic acid, 1,4-butanediol, adipic acid and bis(2-hydroxyethyl) terephthalate; And / or, the plant to be cultivated is Arabidopsis thaliana or Nicotiana benthamiana.

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