Application of a broad-spectrum type III esterase derived from rhodococcus in phthalate-contaminated bioremediation

Abstract

The application discloses application of a broad-spectrum type III esterase derived from Rhodococcus in phthalate ester pollution bioremediation, and overcomes the limitation of low degradation efficiency of a natural strain; through genetic engineering technology, the esterase 5359 gene is cloned into a Rhodococcus-E.coli shuttle vector pNV18 to construct a recombinant Rhodococcus engineering bacterium WT-pNV18-5359 overexpressing the esterase. Experimental results show that the engineering bacterium significantly improves the degradation performance: in single PAEs degradation test, the degradation rates of long-chain PAEs such as DEHP, DnOP and DiNP are respectively increased by 15.71%, 13.99% and 12.82% compared with the wild type; in a mixed PAEs system, the degradation of long-chain PAEs is obviously superior, and the engineering bacterium has good genetic stability and does not affect growth. Through genetic engineering to strengthen the application of known enzymes, efficient and stable PAEs pollution bioremediation is realized.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology and genetic engineering technology, specifically relating to the application of a broad-spectrum type III esterase derived from Rhodococcus in the bioremediation of phthalic acid ester pollution. Background Technology

[0002] Phthalate esters (PAEs), as plasticizers, are widely present in the global environment through volatilization and leaching. Long-chain PAEs (such as DEHP and DnOP) are difficult to degrade naturally due to their stable structure and strong hydrophobicity, posing a challenge to environmental remediation. Microbial degradation is a green and environmentally friendly remediation pathway, with esterases being the key catalytic enzymes. However, the degradation efficiency of natural bacterial strains is often limited by the expression level of their endogenous enzymes, making it difficult to meet the requirements for efficient remediation. Microbial degradation is an effective and environmentally friendly way to remove PAE pollution from the environment. In this process, esterases play a central catalytic role, responsible for hydrolyzing the ester bonds of PAEs.

[0003] Genetic engineering technology provides an effective means to enhance the degradation capabilities of microorganisms. By introducing key degradation enzyme genes into host bacteria and achieving efficient expression, the degradation rate and scope of target pollutants can be significantly improved. However, this technical approach still faces several challenges: for example, the expression efficiency of exogenous genes in the host, the maintenance of protein activity, the environmental adaptability of engineered bacteria, and genetic stability all require further exploration and optimization. Currently, there are no reports in the literature on enhancing the degradation capabilities of engineered bacteria for PAEs. Therefore, constructing efficient and stable engineered degradation bacteria has significant application value.

[0004] The early stage of this invention was derived from Rhodococcus Rhodococcus A novel type III esterase, 5359, was identified in sp. AH-ZY2, which exhibits broad-spectrum PAE degradation activity. Based on this, the present invention aims to construct an engineered Rhodococcus strain overexpressing this esterase using genetic engineering techniques, thereby enhancing the degradation performance of the strain. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide an application of a broad-spectrum type III esterase derived from Rhodococcus in the bioremediation of phthalic acid ester pollution. This invention provides a genetically enhanced Rhodococcus engineered strain, which, through the introduction and overexpression of a broad-spectrum type III esterase derived from [a specific organism, likely Rhodococcus], [is described]. Rhodococcus The type III esterase 5359 gene of sp. AH-ZY2 significantly enhances the degradation ability of various PAEs, especially long-chain PAEs.

[0006] In a first aspect, the present invention provides a recombinant Rhodococcus engineered bacterium, the engineered bacterium comprising a recombinant expression vector containing a nucleotide sequence encoding esterase 5359 (as shown in SEQ ID NO: 2) and its expression regulatory elements, the recombinant expression vector being pNV18-5359, which is introduced into the Rhodococcus AH-ZY2 host via electroporation; the recombinant Rhodococcus engineered bacterium is inoculated in a PAE-contaminated environment, and the overexpressed esterase 5359 directly hydrolyzes the PAEs into phthalic acid.

[0007] Secondly, the present invention provides the application of the above-mentioned engineered bacteria in the degradation of phthalate compounds.

[0008] The PAEs include, but are not limited to, one or more of the following: dimethyl phthalate (DMP), diethyl phthalate (DEP), dipropyl phthalate (DPrP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), di(2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DnOP), and diisononyl phthalate (DiNP).

[0009] The beneficial effects of this invention are: 1) Enhanced degradation performance: Compared with wild-type strains, the engineered strains overexpressing esterase 5359 significantly improved the degradation rate of various PAEs, especially showing stronger degradation ability for long-chain PAEs that are difficult to degrade (such as DEHP and DnOP).

[0010] 2) Highly targeted application: This engineered bacterium is directly constructed based on environmentally adaptable Rhodococcus strains, making it more suitable for bioremediation of actual PAE-contaminated environments (such as soil).

[0011] 3) Good genetic stability: The use of a Rhodococcus-Escherichia coli shuttle vector ensures the stable inheritance and expression of exogenous genes in engineered bacteria. Attached Figure Description

[0012] Figure 1 Phylogenetic tree of esterase 5359; Figure 2 Image of the expression vector plasmid for esterase 5359 in Rhodococcus; Figure 3 This is a gel image showing the overexpression of esterase 5359 in Rhodococcus. Figure 4 For WT-pNV18- 5359 Degradation test of engineered bacteria on single PAEs; Figure 5 For WT-pNV18- 5359 Degradation test of engineered bacteria on mixed PAEs; Figure 6 For WT-pNV18- 5359 Growth test of engineered bacteria in LB; Figure 7 The growth of colony transformants on antibiotic plates under different electric shock conditions. Detailed Implementation

[0013] The present invention will be further described below with reference to embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto. Example 1: Discovery, cloning, and construction of recombinant expression vector for esterase 5359 gene Based on currently reported literature and genome annotations, esterase 5359 (amino acid sequence shown in SEQ ID NO: 1) was sequence-compared with currently reported esterases, and a phylogenetic tree was constructed, revealing that esterase 5359 belongs to the carboxylesterase family. Figure 1 Literature review predicts that the active sites of esterase 5359 are Ser199 (serine), Thr377 (threonine), and His416 (histidine). The surface area of ​​the active site of esterase 5359 is 802.52 Ų, the depth of the active pocket is 18.28 Å, and the radius of the active site is 22.5 Å. Furthermore, esterase 5359 shows a maximum similarity of only 37.77% to the currently reported type III esterase CarEW ((AIZ00845)). Previous experiments have reported that esterase 5359 can degrade phthalic acid esters (PAEs) to phthalic acid (PA) in one step (Hou Zhengyu, Pan Hejuan, GuMengjie, et al. Simultaneously degradation of various phthalate esters by Rhodococcus sp. AH-ZY2: Strain, omics and enzymatic study[J]. Journal of Hazardous Materials, 2024, 474: 134776.), which will not be elaborated further here.

[0014] Example 2: Construction of engineered bacteria and PAE degradation experiment Functional synergistic analysis of esterase 5359 with Rhodococcus host Esterase 5359 originates from Rhodococcus, therefore the intracellular environment of Rhodococcus (such as the molecular chaperone system and redox state) is more conducive to the proper folding and maintenance of activity of this esterase. The gene sequence of esterase 5359 is consistent with the codon usage preferences of Rhodococcus. Figure 2As shown, the pNV18 plasmid is a Rhodococcus-Escherichia coli shuttle plasmid, using a replicon that can replicate stably in both Rhodococcus and Escherichia coli. The pNV18 plasmid is used to ensure that the esterase gene can be expressed and function efficiently, stably, and specifically in Rhodococcus.

[0015] 1. Recombinant expression plasmid pNV18- 5359 Construction: Using the Rhodococcus-Escherichia coli shuttle vector pNV18 as the backbone, suitable restriction endonuclease sites (such as...) were selected from its multiple cloning sites. BamH I and Hind III). Based on the coding gene sequence of esterase 5359 (SEQ ID NO: 2), a specific primer pNV18- with a homologous arm was designed and synthesized. 5359 -F / R (Table 1). Using plasmids containing this gene or AH-ZY2 genomic DNA as templates, the 5359 gene fragment was amplified by high-fidelity PCR. The purified PCR product was ligated to the pNV18 vector linearized with the same restriction endonuclease using a one-step cloning kit. Figure 2 The recombinant plasmid pNV18- was transformed into E. coli DH5α. After antibiotic selection, colony PCR, and plasmid sequencing verification, the correct recombinant plasmid pNV18- was obtained. 5359 .

[0016] Table 1 Primer sequences involved in this invention

[0017] Note: Homologous arms of plasmids are represented by uppercase letters, and complementary parts of the target gene sequence are represented by lowercase letters.

[0018] 2. Construction of Rhodococcus AH-ZY2 engineered strain by electroconversion: Optimization of electrostatic conditions To achieve the use of exogenous plasmids in Rhodococcus ( Rhodococcus To achieve efficient transfection of plasmid DNA in *Rhodococcus aureus* sp. AH-ZY2, this study systematically optimized the electroporation conditions. *Rhodococcus aureus* is a Gram-positive bacterium, and its thick peptidoglycan cell wall structure poses a significant barrier to traditional transformation methods. Therefore, high-voltage electroporation is necessary to mediate the transmembrane transport of plasmid DNA. Key parameters for electroporation include voltage, capacitance, and resistance, with voltage being particularly sensitive: too low a voltage fails to form effective reversible micropores on the cell membrane, preventing plasmid entry; too high a voltage leads to irreversible damage to the cell membrane, causing cell death.

[0019] Based on the above principles, this study conducted gradient tests on voltage parameters (1.0 kV, 1.5 kV, 2.0 kV, 2.5 kV, 3.0 kV) under fixed capacitance (25 μF) and resistance (200 Ω). Competent cells in mid-logarithmic growth, thoroughly washed with ice-cold glycerol buffer, were mixed with an equal volume of plasmid DNA and pulsedly discharged in a pre-cooled electroporation cuvette. Immediately after electroporation, LB resuscitation medium was added, and the cells were cultured with shaking at a suitable temperature, followed by plating onto selective plates containing the appropriate antibiotics.

[0020] Transformation efficiency was evaluated by the number of resistant colonies obtained. Results are as follows: Figure 7 As shown, voltage and transformation efficiency exhibit a significant correlation: within the range of 1.0–2.0 kV, the number of colonies gradually increases with increasing voltage; it reaches a peak at 2.5 kV, at which point the highest number of transformants can be obtained per microgram of plasmid DNA; when the voltage rises to 3.0 kV, the number of colonies drops sharply, indicating that excessively high voltage has a serious adverse effect on cell viability.

[0021] Wild-type Rhodococcus aureus AH-ZY2 was inoculated into LB liquid medium (10.0 g / L NaCl, 10.0 g / L peptone, 5.0 g / L yeast extract) and cultured to mid-logarithmic growth phase (OD). 600 =0.8), to prepare competent cells. Take an appropriate amount of purified recombinant plasmid pNV18- 5359 (Or use empty pNV18 as a control), gently mix with competent cells and place in a pre-chilled electroporation cuvette. Electroporate using an electroporator at optimized voltage (2.5 kV, 25 μF, 200 Ω). Immediately after electroporation, add LB recovery medium (10.0 g / L NaCl, 10.0 g / L peptone, 5.0 g / L yeast extract) and incubate at 37°C for 12 h. Spread the revived culture onto LB agar plates containing kanamycin and incubate at 37°C until single colonies appear.

[0022] Competent cell preparation method: Pick a single colony of Rhodococcus from a fresh plate and inoculate it into a test tube containing 20 mL of LB medium. Take 1.5 mL of the overnight culture and transfer it to 100 mL of LB medium for expansion culture. Culture until OD... 600 =0.6-0.8. Centrifuge the bacterial culture at 6,000 rpm for 5 min at 4°C, discard the supernatant, and collect the bacterial cells. Resuspend the bacterial cells in 10 mL of pre-cooled 10% glycerol solution, centrifuge at 6,000 rpm for 5 min, discard the supernatant, and collect the bacterial cells. Resuspend the washed bacterial cells in 5 mL of 10% glycerol solution, aliquot into 100 μL tubes, and store at -80°C.

[0023] 3. Screening and Validation of Engineered Microorganisms: Single colonies from resistance plates were picked and expanded. Primers pNV18- from Table 1 were used. 5359 -F / R was used for PCR verification to ensure that the plasmid had been transferred ( Figure 3 In the diagram, M represents Mark, and 1, 2, and 3 represent WT-pNV18- 5359 The transformant, 4 is pNV18- 5359 Plasmid, 5 represents water, band size, 1759 bp.

[0024] 4. Evaluation of the degradation performance of engineered bacteria: For WT-pNV18- 5359 The WT strain was cultured in 200 mL LB (using 50 mg / L Kan as a transformant) until OD. 600 = 1.2, centrifuge at 8000 rpm, wash with 1×PBS, and adjust to OD. 600 = 10. For the single PAEs degradation experiment, the reaction system was 1 mL (920 μL bacterial suspension, 80 μL PAEs stock solution) (10 g / L). After reacting at 37℃ for 1 h, the sample was extracted with 3 times its volume of methanol, vortexed for 10 min, and centrifuged for 5 min. The supernatant was used for HPLC analysis of PAEs residues. For the degradation experiment of 8 mixed PAEs, the reaction system was 1 mL (920 μL bacterial suspension, 80 μL PAEs stock solution) (10 g / L), and the control group used PBS and PAEs stock solution (control group). The sample was cultured at 37℃ and 220 rpm, and samples were taken every day, extracted with 3 times its volume of methanol, vortexed for 10 min, and centrifuged for 5 min. The supernatant was used for HPLC analysis of PAEs residues.

[0025] 5. Growth curve determination: The OD values ​​of wild-type and engineered strains were measured every 12 hours in LB medium using a UV spectrophotometer. 600 All experiments were performed in triplicate, and the data were plotted and analyzed using GraphPad Prism 9 software.

[0026] The results showed that in the presence of eight individual PAEs, overexpression of WT-pNV18 significantly improved the degradation of multiple PAEs. Compared with wild-type WT, WT-pNV18- 5359 The degradation rates of DEHP, DnOP, and DiNP increased by 15.71%, 13.99%, and 12.82% respectively within 1 hour. Figure 4 When 8 mixed PAEs are present, WT-pNV18- 5359 It improved the degradation efficiency of long-chain PAEs. The degradation effect on short-chain PAEs showed no significant change compared to WT. Figure 5 pNV18-5359 Plasmid transformation had no significant effect on the growth of WT in LB medium. Figure 6 ).

Claims

1. The application of a broad-spectrum type III esterase derived from Rhodococcus in the bioremediation of phthalic acid ester contamination, characterized in that, A recombinant Rhodococcus engineered bacterium overexpressing a broad-spectrum type III esterase was constructed. This recombinant Rhodococcus engineered bacterium used Rhodococcus AH-ZY2 as the host and contained the recombinant expression vector pNV18-5359. This vector contained the nucleotide sequence encoding the type III esterase 5359 shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO:

2. By overexpressing the type III esterase 5359 gene, the degradation ability of phthalate compounds was enhanced.

2. The application as described in claim 1, characterized in that, Recombinant Rhodococcus engineered bacteria were inoculated into a PAE-contaminated environment, and the PAEs were directly hydrolyzed into phthalic acid by the overexpressed esterase 5359.

3. The application as described in claim 2, characterized in that, PAEs include one or more of DEHP, DnOP, DiNP, DMP, DEP, DPRP, DBP, and BBP.

4. The application as described in claim 1, characterized in that, The recombinant expression vector was introduced into the Rhodococcus AH-ZY2 host via electroporation. The electroporation parameters were 2.5 kV, 25 μF, and 200 Ω.

5. The application as described in claim 3, characterized in that, PAEs include one or more of DEHP, DnOP, and DiNP.

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