Furandicarboxylic acid decarboxylase based on ancestral sequence reconstruction and use thereof

Abstract

The application discloses a furan dicarboxylic acid decarboxylase based on ancestor sequence reconstruction and application thereof, and belongs to the technical field of enzyme engineering and genetic engineering. The furan dicarboxylic acid decarboxylase is named as HmfF-N10, is obtained by reconstructing an ancestor sequence from multiple homologous enzyme sequences of a Prenylated flavin mononucleotide (prFMN) dependent furan dicarboxylic acid decarboxylase HmfF extracted from a database, and the amino acid sequence is shown as SEQ ID NO:1. Experiments show that HmfF-N10 has excellent thermal stability, the melting temperature is 86.83 DEG C, and HmfF-N10 still retains 60% of catalytic activity at 80 DEG C. In addition, HmfF-N10 has the activity of catalyzing terephthalic acid to generate benzoic acid by decarboxylation, and can be used for biodegradation of terephthalic acid plastic waste.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering and genetic engineering technology, specifically relating to a furanyldicarboxylase based on ancestral sequence reconstruction and its application in the decarboxylation of terephthalic acid. Background Technology

[0002] Terephthalic acid (TPA) is an important aromatic dicarboxylic acid widely used in the production of polyester materials such as polyethylene terephthalate (PET). However, PET degrades slowly in the natural environment, and its microplastic fragments can accumulate in ecosystems and be transferred through the food chain, posing a potential threat to environmental safety and human health. To achieve the green recycling of plastic waste, more and more research focuses on the biodegradation and transformation of terephthalic acid. Decarboxylases are enzymes that catalyze the decarboxylation of carboxyl compounds to generate corresponding products, and they have important applications in organic synthesis and green chemistry. However, the sources of enzymes with terephthalic acid decarboxylation activity are currently limited, and the development of efficient and stable terephthalic acid decarboxylases has important application prospects. In recent years, ancestral sequence reconstruction (ASR) has been widely used in enzyme functional modification and new enzyme design, enabling the discovery of ancestral protein sequences with high stability and excellent enzyme activity.

[0003] 2,5-Furandicarboxylic acid decarboxylase (HmfF) is an enzyme that catalyzes the decarboxylation of the biomass-derived platform compound 2,5-furandicarboxylic acid to 2-furancarboxylic acid. Belonging to the UbiD enzyme family, this enzyme relies on prenylated flavin mononucleotide (prFMN) as a cofactor and achieves efficient removal of the carboxyl group through a unique catalytic mechanism, showing significant potential for applications in green catalysis. Studies have shown that HmfF has a broad substrate spectrum, capable of catalyzing the decarboxylation of various 2,5-furandicarboxylic acid analogs and exhibiting good substrate heterogeneity. However, no natural HmfF has yet been found to catalyze the decarboxylation of terephthalic acid. Summary of the Invention

[0004] To expand the catalytic properties of furanyl dicarboxylases and enhance their stability and substrate adaptability, such as for terephthalic acid substrates, this invention aims to provide a furanyl dicarboxylase based on ancestor sequence reconstruction and its application in the decarboxylation of terephthalic acid. This invention utilizes ancestor sequence reconstruction (ASR) technology, and through computational biology analysis and protein engineering optimization, successfully constructed a novel furanyl dicarboxylase sequence, HmfF-N10, with catalytic activity. HmfF-N10 not only possesses excellent enzymatic stability but also achieves the decarboxylation of terephthalic acid. This solves the problems of low stability and limited decarboxylation activity of existing HmfF enzymes, providing a novel enzymatic catalytic tool for the biodegradation of terephthalic acid.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a furanyl dicarboxylic acid decarboxylase HmfF-N10, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] Secondly, the present invention provides a gene encoding the aforementioned furanyl dicarboxylic acid decarboxylase HmfF-N10.

[0008] Based on the above technical solution, the nucleotide sequence of the gene is further shown in SEQ ID NO.2.

[0009] Thirdly, the present invention provides a recombinant expression vector into which the above-mentioned gene is inserted.

[0010] Based on the above technical solution, the vector further includes an isopropyltransferase gene.

[0011] Based on the above technical solution, the vector further includes the pACYCDuet-1 expression vector.

[0012] Fourthly, the present invention provides a recombinant engineered bacterium carrying the above-mentioned recombinant expression vector and capable of expressing furanyldicarboxylase HmfF-N10.

[0013] Based on the above technical solution, the recombinant engineered bacteria is Escherichia coli.

[0014] Fifthly, the present invention provides the application of the above-mentioned furanyl dicarboxylase HmfF-N10, recombinant expression vector or recombinant engineered bacteria in catalyzing the decarboxylation reaction of terephthalic acid.

[0015] Based on the above technical solution, the decarboxylation reaction is further carried out in a conventional buffer solution with a pH of 6.5 to 8.5 and a reaction temperature of 30 to 80°C, preferably 40 to 60°C.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The furanyl dicarboxylate decarboxylase HmfF-N10 obtained by the present invention using ancestral sequence reconstruction technology has good thermal stability and a melting temperature as high as 86.83℃, which greatly improves the practicality of the enzyme.

[0018] 2. The furanyl dicarboxylase HmfF-N10 obtained by the present invention using ancestral sequence reconstruction technology not only naturally catalyzes the decarboxylation reaction of furanyl dicarboxylase, but also exhibits catalytic activity in the decarboxylation process of terephthalic acid, thereby enhancing the application potential of this enzyme in the decomposition of terephthalic acid. Attached Figure Description

[0019] Figure 1 Phylogenetic tree of furanyl dicarboxylate decarboxylase HmfF.

[0020] Figure 2 The cavity volumes of HmfF ancestral enzymes N9, N10, and N13 are correlated with the predicted melting temperature Tm.

[0021] Figure 3 This is an electrophoresis image of HmfF-N10 expressed in Escherichia coli during the purification process.

[0022] Figure 4 This is a comparison of the thermal stability curves of HmfF-N10 and PtHmfF.

[0023] Figure 5 The graph shows the catalytic rate of HmfF-N10 as a function of temperature.

[0024] Figure 6 The specific activity diagram of HmfF-N10 and PtHmfF catalyzing terephthalic acid is shown. Detailed Implementation

[0025] To better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below. It should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a clearer and more thorough understanding of the present invention and to fully convey the present invention to those skilled in the art.

[0026] Example 1: Ancestor Sequence Reconstruction

[0027] Sequences of various known prenylated flavinmononucleotide (prFMN)-dependent furanyl dicarboxylase (HmfF) homologous proteins were retrieved and screened from the UniProt protein database. To ensure the representativeness and accuracy of the analysis, sequences covering multiple related species with sequence similarity within a certain range were selected to avoid high redundancy while ensuring evolutionary diversity. MAFFT sequence analysis was used to align the selected 93 sequences from different species, with sequence similarity ranging from 26% to 90%. A phylogenetic tree of furanyl dicarboxylase homologous sequences was constructed using the maximum likelihood method. The phylogenetic tree is shown below. Figure 1 As shown, this phylogenetic tree reflects the evolutionary relationships and ancestral nodes of enzyme sequences among different species. The tree is divided into five clusters based on developmental relationships, and it was found that sequence similarity gradually increases as the tree moves clockwise. The first two clusters show low levels of sequence similarity both between and within clusters, with most similarities around 35% and a very few exceeding 60%. The third cluster shows sequence similarities around 50% between and within clusters, while approximately three-quarters of the sequences in the latter two clusters show similarities above 50%. To identify ancestral enzymes with evolutionary potential, we aim for a relatively moderate level of overall similarity among the modern enzymes in their branches, which facilitates further rational analysis of the active pocket. Based on the above sequence analysis, the screening scope for ancestral enzymes is narrowed down to the third cluster. Furthermore, based on the sequence relationships of modern enzymes and their evolutionary relationships within the phylogenetic tree, three ancestral enzymes—N9 (amino acid sequence as shown in SEQ ID NO:3), N10 (amino acid sequence as shown in SEQ ID NO:1), and N13 (amino acid sequence as shown in SEQ ID NO:4)—were initially selected. The cavity volume and melting temperature (Tm) of three ancestral enzymes, N9, N10, and N13, were predicted using online bioinformatics websites (Cavity Plus and DeepSTABp). The results are as follows: Figure 2 As shown. While increasing the volume of the enzyme's substrate-binding pocket cavity can enhance its capacity to accommodate a broad range of substrates (especially larger non-natural substrates), excessive space can lead to loose binding between the enzyme and substrate, resulting in decreased catalytic activity. Compared to N13, N9 and N10 have predicted Tm values ​​above 80℃, indicating potential for improved thermal stability. Considering all these factors, the ancestral enzyme was reconstructed using N10, which has a mid-sized cavity and potential thermal stability, and was named HmfF-N10, with the amino acid sequence shown in SEQ ID NO:1.

[0028] To achieve gene synthesis and heterologous expression of the ancestral enzyme, the amino acid sequence was reverse-translated to obtain the corresponding nucleotide sequence. Considering the codon usage preferences of the target expression host (such as E. coli), the nucleotide sequence was optimized to improve gene expression efficiency. The final encoded gene sequence is shown in SEQ ID NO:2.

[0029] Example 2: Construction of the HmfF-N10 gene

[0030] The HmfF-N10 gene (nucleotide sequence shown in SEQ ID NO.2) and the gene of the isopropyltransferase UbiX from *Pseudomonas aeruginosa* were cloned into the BamⅠ / HindⅢ and NdeⅠ / XhoⅠ restriction sites of the expression vector pACYCDuet-1, respectively, to construct recombinant plasmids. HmfF belongs to the UbiD family, and enzymes in this family typically exhibit catalytic activity co-expressed with UbiX. UbiX is responsible for the cofactor prFMN required for the synthesis of UbiD. prFMN is transferred to UbiD family enzymes such as HmfF and Fdc1, activating them and enabling them to exert catalytic decarboxylation activity (Archives of Biochemistry and Biophysics, 2017, 632, 209-221). The amino acid sequence of the *Pseudomonas aeruginosa* isopropyltransferase UbiX (amino acid sequence shown in SEQ ID NO.5) can be obtained from protein databases such as UniProt. The pACYCDuet-1 expression vector is a conventional vector used in gene cloning and can be obtained through public purchase or other means. The cloning method is a conventional molecular cloning method, and the recombinant plasmid was constructed and then sequenced and verified by a nucleic acid sequencing company.

[0031] Example 3: HmfF-N10 Expression and Whole-Cell Catalyst Acquisition

[0032] The recombinant plasmid constructed in Example 2 was transformed into *E. coli* competent cells BL21(DE3) using a heat shock transformation method. The plasmid was then plated on LB agar plates containing 50 μg / mL chloramphenicol resistance and incubated upside down at 37°C for 12 h. Single colonies from the plate were transferred to 5 mL of sterile LB liquid medium containing 50 μg / mL chloramphenicol and incubated overnight at 37°C. 1 mL of this medium was then inoculated into 100 mL of sterile LB liquid medium containing 50 μg / mL chloramphenicol and incubated at 37°C for another 8 h. Finally, 10 mL of this medium was inoculated into 1 L of sterile LB liquid medium containing 50 μg / mL chloramphenicol and incubated for approximately 3 h. OD... 600 When the concentration reaches 0.6–0.8, IPTG is added to a final concentration of 0.4 mM for induction. The cells are cultured overnight at 16°C and centrifuged to obtain HmfF-N10 cells, which are the whole-cell catalysts.

[0033] Example 4: Purification method of HmfF-N10

[0034] The bacterial cells obtained in Example 3 were resuspended in 50 mL of buffer A (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM MnCl2, 5% glycerol), and then 50 μL of β-mercaptoethanol was added for sonication for 15 min at a power of 300 W. After sonication, the mixture was centrifuged at 4 °C and 12000 rpm for 30 min to obtain a supernatant containing the target protein. The supernatant was first subjected to Ni... 2 + Affinity chromatography purification was performed, with the wash and eluent containing 32 mM and 350 mM imidazole, respectively. The collected eluent was concentrated to 5 mL by ultrafiltration and then purified using an AKTA pure protein purification system with a Superdex 200 gel filtration column. The protein collected from the gel chromatography column was concentrated to a specific volume and stored at -80°C. Samples were retained during this process, and the purity of the purified protein was verified using SDS-PAGE. The electrophoresis results are shown below. Figure 3 As shown, a protein band appeared near 50 kDa, which matched the molecular weight calculated from the sequence, indicating that the purified HmfF-N10 protein was successfully obtained.

[0035] Example 5: Determination of the enzymatic stability of HmfF-N10

[0036] The purified HmfF-N10 was diluted to 1 mg / mL with buffer A (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM NCl2, 5% glycerol), and centrifuged at 4 °C and 12000 rpm for 10 min to remove air bubbles and aggregates. The treated protein was aspirated by capillary tube, and its thermostability changes within 25–95 °C were measured using a high-throughput multi-parameter protein stability analyzer. Three independent biological replicates were set up. The Tm value obtained by Boltzmann function fitting was 86.83 ± 0.30 °C, which is 15 °C higher than the previously reported Tm value of HmfF (PtHmfF) derived from Pelotomaculum thermopropionicum (ACS Catalysis, 2019, 9, 4, 2854-2865). Figure 4 ).

[0037] The thermal stability of HmfF-N10 was determined by measuring its catalytic rate against its natural decarboxylation substrate, 2,5-furandicarboxylic acid, at temperatures ranging from 30 to 100 °C. This decarboxylation reaction was carried out in buffer A (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM MnCl2, 5% glycerol) for 2 h. The reaction system consisted of 1 mL of 1 mM 2,5-furandicarboxylic acid and 730 μL of whole-cell catalyst. The whole-cell catalyst was obtained by dissolving 100 mL of LB liquid culture medium in 5 mL of buffer A and then sonicating the resulting cells. The decarboxylation yield versus temperature curve is shown below. Figure 5 As shown, HmfF-N10 reached its maximum catalytic rate at 60-70℃ and still retained 60% of its relative catalytic activity at 80℃. These thermal stability experiments demonstrate that HmfF-N10 exhibits excellent thermal stability.

[0038] Example 6: High-performance liquid chromatography analysis of terephthalic acid substrates and products

[0039] Analysis was performed using an Agilent 1260 Infinity II high-performance liquid chromatography system. The column was a Z0RBAX SB-C18 column (4.6 × 150 mm, 5 μm). The detection wavelength was 240 nm, the injection volume was 10 μL, the flow rate was 0.8 mL / min, and the column temperature was 30 °C. A series of standard solutions (0.01–1.5 mM) of terephthalic acid and benzoic acid were prepared. Terephthalic acid and benzoic acid were successfully separated by isocratic elution for 16 min in a mobile phase of a mixture of 0.1% trifluoroacetic acid and acetonitrile (v / v = 0.8:0.2).

[0040] Example 7: Determination of the decarboxylation activity of HmfF-N10 catalyzing terephthalic acid

[0041] The activity of 0.934 mg of purified HmfF-N10 enzyme in catalyzing the decarboxylation of 5 mM terephthalic acid to benzoic acid was determined under pH 7.5 and temperature 50℃. The reaction buffer was 10 mM HEPES (pH 7.5), the reaction system was 1 mL, and the reaction time was 15 h. The control group was made by replacing HmfF-N10 with PtHmfF, and all other conditions remained unchanged.

[0042] After the reaction was completed, it was terminated with four volumes of a methanol-acetonitrile mixture (v / v = 1:1). The product concentration was confirmed by HPLC using the detection method described in Example 6. Calculations showed that PtHmfF exhibited almost no catalytic decarboxylation activity for terephthalic acid, and the HmfF-N10 enzyme specific activity was 0.246 U / mg. Figure 6 ), where U is defined as μmol / h.

Claims

1. A furan dicarboxylic acid decarboxylase HmfF-N10, characterized by, The amino acid sequence is shown as SEQ ID NO.

1.

2. A gene encoding the furan dicarboxylic acid decarboxylase HmfF-N10 according to claim 1.

3. The gene of claim 2, wherein, The nucleotide sequence of the gene is shown as SEQ ID NO.

2.

4. A recombinant expression vector inserted with the gene according to claim 2 or 3.

5. The recombinant expression vector of claim 4, wherein, The vector comprises a pACYCDuet-1 expression vector.

6. A recombinant engineering bacterium carrying the recombinant expression vector according to claim 4 or 5 and capable of expressing the furan dicarboxylic acid decarboxylase HmfF-N10.

7. The recombineering bacteria of claim 6, wherein, The recombinant engineering bacterium is Escherichia coli.

8. Use of the furan dicarboxylic acid decarboxylase HmfF-N10 according to claim 1, the recombinant expression vector according to claim 4 or 5, or the recombinant engineering bacterium according to claim 6 or 7 in catalyzing a terephthalic acid decarboxylation reaction.

9. Use according to claim 8, characterized in that, The decarboxylation reaction is carried out in a conventional buffer, the pH of the buffer is 6.5-8.5, and the reaction temperature is 30-80℃.

10. Use according to claim 9, characterized in that, The reaction temperature is 40-60℃.

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