Redoccus engineering bacteria for enhanced degradation of phthalate esters by heterologous expression of est16 and application thereof

Abstract

This invention discloses a Rhodococcus engineered bacterium enhanced by heterologous expression of Est16 for phthalate degradation and its applications. This invention utilizes genetic engineering technology to transform Est16 derived from... Glutamicibacter The type III esterase Est16 gene of sp. ZJUTW (amino acid sequence shown in SEQ ID NO:1) was heterologously expressed in Rhodococcus ( Rhodococcus The recombinant engineered bacterium WT-pNV18-Est16 was constructed from sp. AH-ZY2. RT-qPCR confirmed that the Est16 gene expression level was significantly upregulated in this engineered bacterium. Degradation experiments showed that the engineered bacterium exhibited excellent performance: after culturing for 5 days in a high-concentration (1600 mg / L) mixed PAE environment, the degradation rates of recalcitrant long-chain PAEs such as DnOP and DiNP were significantly increased by 55.04% and 59.15%, respectively, compared to the wild-type bacterium. This invention provides a stable and promising microbial resource for the efficient bioremediation of PAE contamination, especially long-chain PAE contamination.

Description

Technical Field

[0001] This invention belongs to the fields of environmental microbiology and genetic engineering, specifically relating to a Rhodococcus engineered bacterium that enhances the degradation of phthalates through heterologous expression of Est16 and its application. Background Technology

[0002] Phthalate esters (PAEs), common plasticizers, are a class of widespread endocrine disruptors. Microbial degradation is an effective means of removing PAE pollution from the environment. Rhodococcus aureus AH-ZY2 is a strain capable of degrading a variety of PAEs, but its degradation efficiency is limited by the substrate profile and activity of its own esterases.

[0003] Research on phthalic acid esterases mainly includes type I, type II, and type III enzymes. Type I enzymes can degrade PAEs to phthalic acid monoesters (MAPs). Type II enzymes can degrade MAPs to PAs. Type III enzymes can directly degrade PAEs to PAs in a one-step reaction. The types and research targets of type III enzymes are still very limited. Hou et al. reported in 2023 that type III esterase 5359 can efficiently degrade eight types of PAEs. Song et al. reported in 2022... Rhodococcus sp. LW-XY12, the article proposed that the KXC42_04905 esterase has a 38.7% similarity to Carboxylesterase, predicting it to be a type III enzyme, but no heterologous expression verification was performed. Ding et al. mentioned in their 2015 article... Bacillus The CarEW esterase in sp. K91 can degrade DiBP into MiBP and PTH, and has the function of a type III enzyme. This esterase belongs to the serine esterase family. Sphingobium The sp. SM42 genome contains EstB and EstG esterases. EstG esterases can degrade not only DBP but also MBP. Currently, research on type III esterases is limited, and further investigation is needed. Therefore, the search for new type III esterases is imperative.

[0004] Currently, most reported PAE-degrading esterases are derived from bacteria and fungi, but most esterases have a narrow substrate spectrum, especially exhibiting low degradation efficiency for long-chain PAEs (such as DEHP, DnOP, and DiNP). Furthermore, existing research largely focuses on the discovery and modification of endogenous esterases in single bacterial strains, with few reports on enhancing the degradation efficiency of host bacteria through heterologous expression of highly efficient esterases. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide a *Rhodococcus* engineered bacterium enhanced by heterologous expression of Est16 for phthalate degradation and its applications. This bacterium is constructed by heterologously introducing the gene encoding the esterase Est16 into a *Rhodococcus* host.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of esterase Est16 in the degradation of phthalates.

[0007] The present invention also provides a Rhodococcus engineered bacterium enhanced by heterologous expression of Est16 to degrade phthalates. The Rhodococcus engineered bacterium is constructed by heterologously introducing the gene encoding the esterase Est16 into the Rhodococcus host. The amino acid sequence of the esterase Est16 is shown in SEQ ID NO: 1.

[0008] Furthermore, the nucleotide sequence of the gene encoding the esterase Est16 is shown in SEQ ID NO: 2.

[0009] Furthermore, the gene for the esterase Est16 was introduced into the Rhodococcus host via the recombinant expression vector pNV18-Est16. The pNV18-Est16 vector was constructed by inserting the nucleotide sequence shown in SEQ ID NO: 2 into the Rhodococcus-Escherichia coli shuttle vector pNV18 as the backbone.

[0010] Furthermore, phthalic acid ester contaminants include one or more of 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).

[0011] Preferably, the phthalate contaminants include one or more of DEHP, DnOP, and DiNP.

[0012] The beneficial effects of this invention are: 1) Novel esterase function: Est16 is a type III esterase that can catalyze the hydrolysis of various PAEs to PA in one step, and it maintains high activity, especially for long-chain PAEs.

[0013] 2) Significantly enhanced degradation ability: After expressing Est16 in Rhodococcus AH-ZY2, the engineered bacteria significantly improved the degradation ability of mixed PAEs, especially the degradation efficiency of DnOP and DiNP.

[0014] 3) Good biocompatibility: The expression of Est16 does not significantly inhibit the growth of host bacteria, making it suitable for constructing stable and efficient PAE-degrading engineered bacteria.

[0015] 4) Broad application prospects: This invention provides new enzyme resources and engineered bacteria construction strategies for the bioremediation of PAEs pollution. Attached Figure Description

[0016] Figure 1 Phylogenetic tree of esterase Est16; Figure 2 pET-28a (+)- Est16 Plasmid mapping; Figure 3 Heterologous expression of esterase Est16 in BL21(DE3) and SDS-PAGE; Figure 4 Substrate profile assay for PAE degradation by esterase Est16; Figure 5 pNV18- Est16 Plasmid map; Figure 6 The recombinant strain WT-pNV18- Est16 Quantitative real-time PCR of esterase Est16 in China; Figure 7 The growth curve of esterase Est16 after expression in Rhodococcus AH-ZY2; Figure 8 For wild-type bacteria WT and recombinant bacteria WT-pNV18- Est16 Degradation efficiency for single PAEs; Figure 9 The recombinant strain WT-pNV18- Est16 Degradation efficiency of mixed PAEs; Figure 10 For wild-type fungi WT and WT-pNV18- Est16 Degradation efficiency for high concentrations of PAEs. Detailed Implementation

[0017] The present invention will be further described below with reference to the embodiments and the accompanying drawings, but the scope of protection of the present invention is not limited thereto.

[0018] Example 1: Sequence analysis of Est16 esterase and construction of a phylogenetic tree First, the whole-genome sequencing annotation results were analyzed, and literature was reviewed to identify currently reported esterase genes. Then, the NCBI database was used to compare the currently reported esterase genes with... GlutamicibacterComparative analysis was performed on esterase genes in the ZJUTW genome. Potentially functional esterase genes were identified (esterase sequences with greater than 30% sequence identity were selected). The NCBI database was used to analyze whether the esterase possessed the known conserved "GXSXG" domain. Finally, the Est16 esterase was selected for subsequent heterologous expression and functional analysis. The amino acid sequence of esterase Est16 is shown in SEQ ID NO: 1, and the nucleotide sequence of the gene encoding esterase Est16 is shown in SEQ ID NO: 2.

[0019] Literature review, sequence alignment analysis of currently reported esterases with Est16, and phylogenetic tree construction using MEGAX software confirmed that Est16 belongs to the carboxylesterase family. Figure 1 According to the NCBI comparative analysis, the esterase Est16 had the highest similarity to carboxylesterase (NCBI: QHH21706), at only 36.77%. Therefore, esterase Est16 was selected for heterologous expression.

[0020] Example 2: Gene cloning of Est16 esterase and... E. coli BL21(DE3) heterologous expression Gene cloning and recombinant bacterial construction: Glutamicibacter Using sp. ZJUTW genomic DNA as a template, pET28a(+)- Est16- F / R is the primer used to amplify the target esterase gene (Table 1). Bam HI and Hind III was used as the restriction enzyme site for the plasmid. Subsequently, the amplified gene was ligated into the pET-28(a)+ plasmid using a one-step cloning kit, and the recombinant plasmid was transformed into DH5α for plasmid amplification. The plasmid extracted from DH5α was then transformed into pET-28(a)+ using heat shock. E. coli BL21(DE3) Figure 2 ).

[0021] Table 1 Primer sequences involved in this invention

[0022] Note: Homologous arms of plasmids are represented by uppercase letters, and complementary parts of the target gene sequence are represented by lowercase letters.

[0023] Protein expression induction: After the recombinant bacteria were cultured at 37°C to mid-log phase, they were induced at 20°C with 0.5 mM IPTG for 16 hours to improve the expression of soluble proteins.

[0024] Protein purification: After the bacterial cells were sonicated, they were purified by affinity chromatography using a Ni-NTA gravity column. The purified product was dialyzed in Tris-HCl buffer (pH 7.5) to remove impurities.

[0025] Protein identification and quantification: The molecular weight and purity of the protein were finally determined by SDS-PAGE, and the protein concentration was determined by the BCA method. SDS-PAGE showed that its molecular weight was approximately 53.3 kDa. Figure 3 ), Figure 3 Cloning of the Est16 fragment, 1, 2, Est16 fragment (A); Verification of positive transformants: After introducing the pET28a (+)-Est16 recombinant plasmid into BL21, positive transformants 1, 2, 3, 4, and Est16 were obtained (B); Purification of Est16 esterase protein (1), 1st elution buffer was 250 mM imidazole; 2, 2nd elution buffer was 250 mM imidazole; 3, 3rd elution buffer was 250 mM imidazole; 4, 4th elution buffer was 250 mM imidazole (C). Liquid chromatography chromatograms of Est16 esterase degradation of DMP (D), DEP (E), DPRP (F), DBP (G), BBP (H), DEHP (I), DnOP (J) and DiNP (K), The detection conditions for DMP, DEP, DPRP, and DBP were: methanol:water = 90:10; the detection conditions for BBP, DEHP, DnOP, and DiNP were 100% methanol. For mixed PAEs, the detection conditions were: mobile phase A (water) and mobile phase B (methanol), using the following gradient elution program: 0.00–7.00 min, 75% B; 7.00–9.00 min, 75%–100% B; 9.00–16.00 min, 100% B. The injection volume was 10 μL, the column temperature was 30°C, the flow rate was 1.0 mL / min, the column was C18, and the detection wavelength was 235 nm.

[0026] from Figure 3 The DK data shows that Est16 can degrade DMP, DEP, DPRP, DBP, BBP, DEHP, DnOP, and DiNP. The degradation rates for DMP, DEP, DPRP, DBP, and BBP are higher than those for DEHP, DnOP, and DiNP.

[0027] Example 3: Determination of Enzymatic Properties of Est16 The degradation activity of purified enzyme Est16 was determined using eight PAEs as substrates. The reaction system contained 0.1 mg / mL Est16 and 800 mg / L PAEs, and was reacted at 37℃ for 4 h. After the reaction was completed, the reaction was stopped by heating in a 100℃ water bath for 15 min. After the reaction system cooled, twice the volume of HPLC-grade methanol was added to the reaction system, and the mixture was shaken thoroughly for 5 min. After centrifugation at 12,000 rpm for 5 min, the supernatant was collected and filtered through a 0.22 μL organic filter membrane. The residual amount of each PAE was determined by liquid chromatography. HPLC results showed that Est16 had a near 100% degradation rate for DMP, DEP, DPRP, DBP, and BBP, and a degradation rate of 72.84%, 56.65%, and 74.19% for DEHP, DnOP, and DiNP, respectively. Figure 4 ).

[0028] Example 4: Esterase Est16 gene in Rhodococcus Heterologous expression in sp. AH-ZY2 Building pNV18- Est16 Expression vector: using specific primer pNV18- Est16 -F / R (Table 1), obtained from the Rhodococcus AH-ZY2 genome by PCR amplification. Est16 Gene fragment. The purified fragment was ligated into the Rhodococcus-Escherichia coli shuttle vector pNV18, which had undergone the same enzyme digestion treatment, to construct the recombinant expression plasmid pNV18- Est16 ( Figure 5 The recombinant plasmid was introduced into wild-type competent Rhodococcus AH-ZY2 cells using electroporation at 2.5 kV. Positive transformants were obtained after resistance selection and named the engineered strain WT-pNV18-. Est16 Further analysis was performed using RT-qPCR, with the 16S rRNA gene as an internal control, and specific primers were used for detection. Est16 The transcriptional level of genes. The results showed that in engineered bacteria... Est16 The mRNA expression level of the gene was significantly upregulated compared to the wild-type strain WT (Rhodococcus aureus AH-ZY2), confirming that the gene was successfully transcribed and expressed in the AH-ZY2 host. Figure 6 ).

[0029] Growth impact assessment: WT-pNV18- Est16 It has no effect on the growth of the host bacteria in LB, indicating that the esterase Est16 does not affect the growth of the host bacteria. Rhodococcus Normal metabolism of sp. AH-ZY2 ( Figure 7 ).

[0030] Example 5: Evaluation of the degradation ability of engineered bacteria on PAEs Whole-cell degradation assay: Resting cells were used to degrade 800 mg / L of single PAEs. Recombinant strain WT-pNV18- Est16 Degradation efficiency for 8 types of PAEs. WT-pNV18- Est16 Wild-type AH-ZY2 was inoculated into PBS medium containing 800 mg / L of single or mixed PAEs, and cultured at 37℃ and 220 rpm. Samples were taken at different time points to detect PAE residues. The results showed that, in the presence of single PAEs, the engineered strain WT-pNV18-Est16 significantly improved the degradation rate of long-chain PAEs (including DEHP, DnOP, and DiNP) compared to the wild-type strain. Figure 8 In the presence of mixed PAEs, WT-pNV18- compared to WT Est16 Significantly reduced DMP accumulation, WT-pNV18- Est16 Improved the degradation of DnOP and DiNP ( Figure 9 The control group indicates no bacteria were added. Note: P < 0.001, **** . 0.001 <P<0.01,* * . 0.01<P<0.05,* . P> 0. 1, NS.).

[0031] Recombinant strain WT-pNV18- Est16 To assess the degradation efficiency of high-concentration PAEs under mixed PAEs conditions, the wild-type strain WT was used as a control group: the reaction system (840 μL bacterial culture + 160 μL mother liquor) was incubated at 37℃ and 220 rpm for 5 days at a final concentration of 1,600 mg / L. All samples were extracted with 3 volumes of methanol, vortexed for 1 hour, and then centrifuged at 8,000 rpm for 10 minutes. The PAEs degradation rate was detected by HPLC. The results showed that the residual amounts of DEHP, DnOP, and DiNP decreased on days 3 and 5. At day 3, the degradation rates of DEHP, DnOP, and DiNP increased by 13.49%, 41.09%, and 44.32%, respectively. At day 5, the degradation rates of DEHP, DnOP, and DiNP increased by 40.90%, 55.04%, and 59.15%, respectively. Figure 10 , Figure 10 In the table, A and B represent the residual concentrations at 3 days and 5 days, respectively, while C and D represent the degradation rates at 3 days and 5 days. Note: P < 0.001, ***. 0.001 <P<0.01,** . 0.01<P<0.05,* . P> 0.1, NS.).

Claims

1. A Rhodococcus engineered bacterium enhanced by heterologous expression of Est16 for phthalate degradation, characterized in that, The engineered Rhodococcus strain was constructed by heterologously introducing the gene encoding esterase Est16 into the Rhodococcus host. The amino acid sequence of esterase Est16 is shown in SEQ ID NO:

1.

2. The engineered Rhodococcus bacterium as described in claim 1, characterized in that, The nucleotide sequence of the gene encoding the esterase Est16 is shown in SEQ ID NO:

2.

3. The engineered Rhodococcus bacterium as described in claim 2, characterized in that, The gene for esterase Est16 was introduced into a Rhodococcus host via the recombinant expression vector pNV18-Est16. The pNV18-Est16 vector was constructed using the Rhodococcus-Escherichia coli shuttle vector pNV18 as its backbone and inserting the nucleotide sequence shown in SEQ ID NO:

2.

4. The application of the engineered Rhodococcus bacterium as described in any one of claims 1-3 in the enhanced degradation of phthalate pollutants, characterized in that, Phthalate contaminants include one or more of DMP, DEP, DPrP, DBP, BBP, DEHP, DnOP, and DiNP.

5. The application as described in claim 4, characterized in that, Phthalate contaminants include one or more of DEHP, DnOP, and DiNP.

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