Chitosan synthase activity of homologous protein of trehalose-6-phosphate synthase and application thereof

Abstract

The invention discloses chitinase activity and application of a trehalose-6-phosphate synthase homologous protein. The trehalose-6-phosphate synthase is selected from any one of the following proteins: A1) a protein as shown in any one of SEQ ID NO.1-8; a2) a protein which is obtained by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid as shown in any one of SEQ ID NO.1-8 and has chitin synthase activity; a3) a protein derived from Trichoderma, yeast, Aspergillus nidulans, Aspergillus fumigatus, Candida albicans, Magnaporthe oryzae, Alternaria alternata and Escherichia coli, having 98% or more of identity with the protein of A1) and having chitin synthase activity; and A4) a fusion protein obtained by connecting a label to the N end or C end of the sequence A1) or A2) or A3). The Tpss disclosed by the invention has chitin synthase activity, can be used for synthesizing chitin from UDP-GlcNAc, and has a very good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of gene functions and applications, and particularly to trehalose-6-phosphate synthase (Tps) and its application in chitin synthesis. Background Art

[0002] Chitin is a polysaccharide compound mainly present in the exoskeletons of crustaceans and insects, formed by the linkage of N-acetylglucosamine molecules, and it plays important structural and protective roles in organisms. Due to the biocompatibility and biodegradability of chitin, it is widely used in the medical field for wound healing and drug delivery. In addition, chitin also has antibacterial and antioxidant properties and can play a role in the food industry and packaging fields. In terms of environmental protection, the biodegradability of chitin makes it a viable alternative to traditional plastics, helping to reduce the environmental burden. Generally speaking, chitin, as a multifunctional biopolymer, shows broad application prospects in fields such as medicine, food, and environmental protection.

[0003] The metabolic pathway of trehalose is strictly regulated in the key physiological processes of insects, plants, and pathogenic fungi. This metabolic pathway is located upstream of the chitin metabolic pathway and is interconnected with chitin through trehalase. Specifically, trehalose is decomposed into glucose under the catalysis of trehalase, and then glucose-6-phosphate (G6P) is generated through the catalysis of hexokinase. G6P undergoes a pyrophosphorylation reaction to generate UDP-N-acetylglucosamine, which then enters the chitin biosynthesis pathway. This complex and coordinated metabolic pathway not only maintains the balance of trehalose and chitin in organisms but also participates in the key physiological processes related to organisms' adaptation to the environment and response to external stimuli. Trehalose-6-phosphate synthase is a key enzyme in the trehalose biosynthesis pathway, which catalyzes the transfer of the glucose ring part of the glycosyl donor substrate N-diphosphoglucose (NDPG) to the glucose ring of the glycosyl acceptor substrate G6P and promotes the formation of an α-1,1 glycosidic bond between the two. However, the role and application value of trehalose-6-phosphate in chitin synthesis have not been reported. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a trehalose-6-phosphate synthase and its application.

[0005] The technical solution adopted by the present invention to solve its technical problems is as follows:

[0006] In the first aspect, the present invention protects a class of trehalose-6-phosphate synthases (Tpss), which are any one of the following:

[0007] A1) A protein with an amino acid sequence shown in any of SEQ ID NO.1-8;

[0008] A2) A protein with an amino acid sequence shown in any of SEQ ID NO.1-8 in the sequence listing, which has been subjected to substitution, deletion or addition of one or several amino acid residues and has chitin synthase activity;

[0009] A3) A trehalose-6-phosphate synthase derived from Trichoderma, yeast, Aspergillus nidulans, Aspergillus fumigatus, Candida albicans, Magnaporthe oryzae, Alternaria alternata and Escherichia coli, which has more than 98% identity with the protein described in A1) and has chitin synthase activity;

[0010] A4) A fusion protein obtained by linking a tag to the N-terminus or C-terminus of the amino acid sequence in A1) or A2) or A3).

[0011] To facilitate the purification of the protein in A1), a tag as shown in the following table can be linked to the amino terminus or carboxyl terminus of the protein composed of the amino acid sequence shown in any of SEQ ID NO.1-8 in the sequence listing.

[0012] Table: Sequences of tags

[0013] Label Residue Sequence Poly-Arg 5-6 RRRRR Poly-His 2-10 HHHHHH FLAG 8 DYKDDDK Strep-tagⅡ 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0014] The protein in A2) above is a protein that has 75% or more identity with the amino acid sequence of the protein shown in any of SEQ ID No.1-8 and has the same function. The 75% or more identity means 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

[0015] The protein in A2) above can be artificially synthesized, or its coding gene can be synthesized first and then biologically expressed.

[0016] The coding gene of the protein in A2) above can be obtained by deleting the codons of one or several amino acid residues in the DNA sequence shown in any of SEQ ID No.9-16, and / or performing a missense mutation of one or several base pairs, and / or linking the coding sequence of the tag shown in the above table to its 5′ or 3′ end.

[0017] In a specific embodiment, the proteins can be denoted as TgTps1, AnTpsA, ScTps1, OtsA, MoTps1, AfTps1A, CaTps1 and AaTps1 respectively. Among them:

[0018] The amino acid sequence of TgTps1 is shown in SEQ ID NO.1, and its encoding gene is shown in SEQ ID NO.9;

[0019] The amino acid sequence of AnTpsA is shown in SEQ ID NO.2, and its encoding gene is shown in SEQ ID NO.10;

[0020] The amino acid sequence of ScTps1 is shown in SEQ ID NO.3, and its encoding gene is shown in SEQ ID NO.11;

[0021] The amino acid sequence of OtsA is shown in SEQ ID NO.4, and its encoding gene is shown in SEQ ID NO.12;

[0022] The amino acid sequence of MoTps1 is shown in SEQ ID NO.5, and its encoding gene is shown in SEQ ID NO.13;

[0023] The amino acid sequence of AfTps1A is shown in SEQ ID NO.6, and its encoding gene is shown in SEQ ID NO.14;

[0024] The amino acid sequence of CaTps1 is shown in SEQ ID NO.7, and its encoding gene is shown in SEQ ID NO.15;

[0025] The amino acid sequence of AaTps1 is shown in SEQ ID NO.8, and its encoding gene is shown in SEQ ID NO.16.

[0026] The present invention also provides biomaterials related to the aforementioned Tpss, and the biomaterials are any one of the following B1) to B8): B1) a nucleic acid molecule encoding any of the aforementioned proteins;

[0027] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0028] B3) a recombinant vector containing the nucleic acid molecule described in B1);

[0029] B4) a recombinant vector containing the expression cassette described in B2);

[0030] B5) a recombinant microorganism containing the nucleic acid molecule described in B1);

[0031] B6) a recombinant microorganism containing the expression cassette described in B2);

[0032] B7) a recombinant microorganism containing the recombinant vector described in B3);

[0033] B8) a recombinant microorganism containing the recombinant vector described in B4).

[0034] Among the above biological materials, the nucleic acid molecule may be any one of the following 1), 2), 3), or 4):

[0035] 1) A DNA molecule whose coding sequence is any one shown in SEQ ID No. 9-16 in the sequence listing;

[0036] 2) A DNA molecule shown in any one of SEQ ID No. 9-16 in the sequence listing;

[0037] 3) A DNA molecule having 75% or more identity with the nucleotide sequence defined in 1) or 2) and encoding TPSs;

[0038] 4) A DNA molecule that hybridizes with the nucleotide sequence defined in 1), 2), or 3) under stringent conditions and encodes TPSs.

[0039] Among them, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA;

[0040] Those of ordinary skill in the art can easily mutate the nucleotide sequence encoding the Tpss protein of the present invention by using known methods, such as directed evolution and point mutation methods. Those nucleotides that have been artificially modified and have 75% or higher identity with the nucleotide sequence of the Tpss protein isolated from the present invention, as long as they encode the Tpss protein and have the function of the Tpss protein, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0041] Among the above biological materials, the expression cassette (tpss gene expression cassette) containing the nucleic acid molecule encoding the Tpss protein in B2) refers to DNA that can express the Tpss protein in a host cell. This DNA may not only include a promoter that initiates the transcription of the tpss gene but also include a terminator that terminates the transcription of the tpss gene.

[0042] A recombinant vector containing the tpss gene expression cassette can be constructed using existing expression vectors.

[0043] Among the above biological materials, the vector may be a plasmid, cosmid, phage, or viral vector. The plasmid may specifically be the pET-28a(+) vector.

[0044] The recombinant vector in B3) may specifically be pET-28a-TgTps1, pET-28a-AnTpsA, pET-28a-ScTps1, pET-28a-OtsA, pET-28a-MoTps1, AfTps1A, pET-28a-CaTps1, and pET-28a-AaTps1. The above plasmids can express the fusion protein formed by TPSs shown in any one of SEQ.NO.1-8 in the sequence listing and His.

[0045] Among the above-mentioned biomaterials, the microorganism can be yeast, bacteria or fungi. Among them, the bacteria can be Escherichia coli, such as Escherichia coli Rosetta (DE3).

[0046] In a second aspect, the present invention also protects the use of the aforementioned Tpss as a chitin synthase.

[0047] In a third aspect, the present invention protects the use of the aforementioned Tpss or the aforementioned biomaterial in the preparation of chitin.

[0048] In a fourth aspect, the present invention protects the use of the aforementioned protein or any of the aforementioned biomaterials in catalyzing the production of chitin from uridine diphosphate N-acetylglucosamine (UDP-GlcNAc).

[0049] In a fifth aspect, the present invention protects the use of the aforementioned protein or any of the aforementioned biomaterials in the preparation of a product for catalyzing the production of chitin from UDP-GlcNAc.

[0050] In a sixth aspect, the present invention also protects a method for preparing a protein having chitin synthase activity, the method comprising the step of expressing the coding gene of Tpss in a biological cell to obtain a protein having chitin synthase activity.

[0051] In a specific embodiment, the cell is a microbial cell, a plant cell or a non-human animal cell

[0052] In a seventh aspect, the present invention also protects a method for preparing chitin, the method comprising: using the aforementioned Tpss to catalyze a reaction with UDP-GlcNAc as a substrate to obtain chitin.

[0053] In a specific embodiment, the catalytic reaction system can be: mixing Tpss, 10 mM UDP-GlcNAc, 10 mM HEPES, 10 mM KCl and 10 mM MgCl 2 and the reaction volume is 500 μl.

[0054] In a specific embodiment, the catalytic reaction can be carried out at 30 °C, and the reaction time can be 18 hours.

[0055] Experimental results show that the Tpss of the present invention has chitin synthase activity and can catalyze the synthesis of chitin from UDP-GlcNAc. The Tpss of the present invention and its coding gene have good application prospects. Description of the Drawings

[0056] Figure 1Purification diagram of trehalose-6-phosphate synthase protein. Among them, Figure a is AnTpsA, Figure b is ScTps1, Figure c is OtsA, Figure d is MoTps1, Figure e is AaTps1, Figure f is CaTps1, Figure g is AfTps1A, and Figure h is TgTps1.

[0057] Figure 2 Product identification diagram of chitin synthesized by AnTpsA. Among them, Figure a is the insoluble product, Figure b is the Fourier transform infrared spectrum, Figure c is the HRTEM image, and Figure d is the SAED image.

[0058] Figure 3 Trehalose-6-phosphate synthase chitin synthase activities in different species. Detailed implementation mode

[0059] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. The described embodiments are not all embodiments of the present invention, but only a part of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0060] Example: TPSs in fungi and bacteria both have chitin synthase activity

[0061] This embodiment provides 8 proteins with chitin synthase from Trichoderma, Aspergillus nidulans, Saccharomyces cerevisiae, Escherichia coli, Magnaporthe oryzae, Candida albicans, Aspergillus fumigatus, and Alternaria alternata, which are respectively denoted as TgTps1, AnTpsA, ScTps1, OtsA, MoTps1, AfTps1A, CaTps1, and AaTps1. The protein sequences of TgTps1, AnTpsA, ScTps1, OtsA, MoTps1, AfTps1A, CaTps1, and AaTps1 are shown in SEQ ID NO.1-8 in the sequence listing, and their encoding genes are shown in SEQ ID NO.9-16 in the sequence listing.

[0062] I. Construction of recombinant vectors

[0063] The sequences of TgTps1, AnTpsA, ScTps1, OtsA, MoTps1, AfTps1A, CaTps1 and AaTps1 were optimized for E. coli codons by Shanghai Bioengineering Co., Ltd., and the optimized sequences are shown as SEQ ID NO.17 - 24 in the sequence listing. pET-28a(+) was double-digested with restriction endonucleases BamHⅠ and XhoⅠ to obtain a linearized vector. The obtained linearized vector was respectively ligated with the optimized nucleic acid fragments, and the correctly sequenced recombinant vectors were respectively designated as pET-28a-TgTps1, pET-28a-AnTpsA, pET-28a-ScTps1, pET-28a-OtsA, pET-28a-MoTps1, pET-28a-AfTps1A, pET-28a-CaTps1 and pET-28a-AaTps1.

[0064] The characteristics of pET-28a-TgTps1, pET-28a-AnTpsA, pET-28a-ScTps1, pET-28a-OtsA, pET-28a-MoTps1, pET-28a-AfTps1A, pET-28a-CaTps1 and pET-28a-AaTps1 are described as follows: pET-28a-TgTps1, pET-28a-AnTpsA, pET-28a-ScTps1, pET-28a-OtsA, pET-28a-MoTps1, pET-28a-AfTps1A, pET-28a-CaTps1 and pET-28a-AaTps1 are recombinant vectors obtained by inserting the tpss genes shown in SEQ ID NO.17 - 24 into the vector pET-28a(+) using BamHⅠ and XhoⅠ respectively, and can respectively express fusion proteins formed by TPSs and His (designated as His-TgTps1, His-AnTpsA, His-ScTps1, His-OtsA, His-MoTps1, His-AfTps1A, His-CaTps1 and His-AaTps1).

[0065] II. Expression and purification of proteins

[0066] The pET-28a-TgTps1, pET-28a-AnTpsA, pET-28a-ScTps1, pET-28a-OtsA, pET-28a-MoTps1, pET-28a-AfTps1A, pET-28a-CaTps1, and pET-28a-AaTps1 obtained in Step 1 were separately introduced into Escherichia coli Rosetta(DE3) to obtain recombinant bacteria Rosetta(DE3)-pET-28a-TgTps1, Rosetta(DE3)-pET-28a-AnTpsA, Rosetta(DE3)-pET-28a-ScTps1, Rosetta(DE3)-pET-28a-OtsA, Rosetta(DE3)-pET-28a-MoTps1, Rosetta(DE3)-AfTps1A, Rosetta(DE3)-pET-28a-CaTps1, and Rosetta(DE3)-pET-28a-AaTps1. Protein expression and purification were performed on each recombinant bacterium, and the specific steps were as follows:

[0067] Using LB medium containing kanamycin, the recombinant bacteria were cultured until the medium became turbid. Subsequently, they were cultured on a large scale in LB medium containing 400 ml of kanamycin at a ratio of 1:100 until the bacteria reached an OD 600 value between 0.4 and 0.6, IPTG was added to a final concentration of 0.3 mM for induction, and the culture was continued at 23 °C for 8 hours. After the culture was completed, the bacterial cells were collected, centrifuged at 12,000 rpm for 3 minutes, and the supernatant was discarded. The obtained precipitate was resuspended with Lysis buffer (20 mM Na 2 HPO 4 , 5 mM imidazole, 300 mM NaCl, pH = 7.2), and protease inhibitors were added. Ultrasonic cell disruption was performed using an ultrasonic cell disruptor, with ultrasonic treatment for 5 seconds at a frequency of 20 Hz, paused for 5 seconds, for a total of 20 minutes until it reached a semi-transparent state. The cell debris was removed by centrifugation at 10,000 rpm for 30 minutes to obtain the supernatant, which was the crude protein solution. The crude protein solution was passed through a Ni 2+ affinity chromatography column, and finally eluted with a buffer containing 250 mM imidazole (20 mM Na 2 HPO 4 , 250 mM imidazole, 300 mM NaCl, pH = 7.2) to obtain purified His-TgTps1, His-AnTpsA, His-ScTps1, His-OtsA, His-MoTps1, His-AfTps1A, His-CaTps1, and His-AaTps1.

[0068] The results were as follows Figure 1 shown. Each purified protein was verified by SDS-PAGE electrophoresis and stained with Coomassie Brilliant Blue. After decolorization, a single target fusion protein was obtained

[0069] III. In vitro synthesis of chitin and structural observation

[0070] The reaction mixture consisted of 10 mM HEPES buffer (pH 7.8), 10 mM NaCl, 10 mM UDP-GlcNAc, and 10 μg AnTpsA, with a final volume of 500 μl. After 18 h of reaction, the reaction products in 100 reaction systems were collected by centrifugation at 15,000 rpm for 15 min. Chitin was separated from the reaction mixture by centrifugation, and then the separated chitin was continuously washed three times with 200 μl of 2% (w / v) sodium dodecyl sulfate (SDS) solution to remove attached substances and contaminants. Finally, it was washed with deionized water to further remove any residual SDS and other soluble components. The insoluble colloidal chitin was then lyophilized using a freeze dryer to obtain crystalline chitin. The chemical composition of the synthesized chitin was determined using attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR, Nicolet iS5, thermofisher, USA). The scanning wavelength was set at 1 cm -1 , and the scanning range was 4000 - 600 cm -1 , and α-chitin extracted from shrimp shells was used as a standard. The crystal structure of the synthesized chitin was further identified using a high-resolution transmission electron microscope (HRTEM, JEM-F200, Japan) equipped with an energy-dispersive X-ray spectrometer (EDX, X-max 80 Oxford, UK)

[0071] The results were as follows Figure 2 shown. After reacting at 30 °C for 20 h, a white product gradually appeared at the bottom of the centrifuge tube. Subsequently, the product was lyophilized to obtain the product. ATR-FTI characterization was performed to determine its chemical composition. The results showed that the main absorption peak was located at approximately 1650 cm -1 , corresponding to the C=O stretching (amide I) band, which was the same as that of α-chitin derived from shrimp shells. The crystal structure of the prepared sample was further analyzed by HRTEM. As shown in the figure, the 0.457 nm lattice spacing of the (110) crystal plane was visible within the selected area. The selected area electron diffraction (SAED) image showed distinct diffraction spots corresponding to the (110), (013), (132), and (063) planes, which was consistent with the results obtained from X-ray diffraction (XRD) analysis of the standard α-chitin. These results confirmed that AnTpsA converted UDP-GlcNAc into chitin in vitro

[0072] IV. Detection of Chitin Synthase Activity of TPSs in Different Organisms

[0073] Detect the chitin synthase activities of the fusion proteins His-TgTps1, His-AnTpsA, His-ScTps1, His-OtsA, His-MoTps1, His-AfTps1A, His-CaTps1 and His-AaTps1 obtained in Step 2 of the detection procedure.

[0074] An in vitro method was used to evaluate the chitin synthase activity of the purified Tps1, and the chitin synthase activity was reflected by the consumption rate of the chitin substrate UDP-GlcNAc. The reaction mixture consisted of 10 mM HEPES buffer (pH 7.8), 10 mM NaCl, 10 mM UDP-GlcNAc, and metal ions, and 10 μg of the corresponding protein was added, with a final volume of 500 μl. The samples were incubated at 30 °C for 20 minutes, and then the protein was removed using a protein ultrafiltration tube to terminate the reaction. Subsequently, the content of UDP-GlcNAc in the reaction solution was determined by LC-MS / MS, and thus the consumption of UDP-GlcNAc was calculated. The chitin synthase activity of TPSs was expressed as the consumption of UDP-GlcNAc per μg of protein per minute.

[0075] The results are as Figure 3 shown. After 20 minutes of reaction, UDP-GlcNAc in each reaction system was consumed, and the chitin synthase activities of His-TgTps1, His-AnTpsA, His-ScTps1, His-OtsA, His-MoTps1, His-AfTps1A, His-CaTps1 and His-AaTps1 were calculated, indicating that TPSs have chitin synthase activity in fungi and bacteria.

[0076] The protection scope of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, the changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the appended claims are used as the protection scope.

Claims

1. The protein is any one of the following: A1) A protein whose amino acid sequence is shown in any one of SEQ ID NO.1-8; A2) A protein which is obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid shown in any one of SEQ ID NO.1-8 in the sequence listing and has chitin synthase activity; A3) A trehalose-6-phosphate synthase which is derived from Trichoderma, yeast, Aspergillus nidulans, Aspergillus fumigatus, Candida albicans, Magnaporthe oryzae, Alternaria, Escherichia coli, has more than 98% identity with the protein described in A1) and has chitin synthase activity; A4) A fusion protein obtained by connecting a tag to the N-terminus or C-terminus of the amino acid sequence of A1) or A2) or A3).

2. The biological material related to the protein described in claim 1 is any one of the following B1) to B8): B1) A nucleic acid molecule encoding the protein described in any one of claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1); B4) A recombinant vector containing the expression cassette described in B2); B5) A recombinant microorganism containing the nucleic acid molecule described in B1); B6) A recombinant microorganism containing the expression cassette described in B2); B7) A recombinant microorganism containing the recombinant vector described in B3); B8) A recombinant microorganism containing the recombinant vector described in B4).

3. The biological material according to claim 2, characterized in that: the nucleic acid molecule is any one of the following 1) or 2) or 3) or 4): 1) A DNA molecule whose coding sequence is shown in any one of SEQ ID NO.9-16 in the sequence listing; 2) A DNA molecule shown in any one of SEQ ID NO.9-16 in the sequence listing; 3) A DNA molecule which has 75% or more identity with the nucleotide sequence defined in 1) or 2) and encodes the protein described in claim 1; 4) A DNA molecule which hybridizes with the nucleotide sequence defined in 1) or 2) or 3) under stringent conditions and encodes the protein described in claim 1.

4. The application of the protein described in claim 1 as a chitin synthase.

5. The application of the protein described in claim 1 or the biological material described in any one of claims 2-3 in the preparation of chitin.

6. The application of the protein described in claim 1 or the biological material described in any one of claims 2-3 in catalyzing the production of chitin from UDP-GlcNAc.

7. The application of the protein described in claim 1 or the biological material described in any one of claims 2-3 in the preparation of a product for catalyzing the production of chitin from UDP-GlcNAc.

8. A method for preparing a protein with chitin synthase activity, which comprises expressing the coding gene of the protein described in claim 1 in a biological cell to obtain a protein with chitin synthase activity; the cell is a microbial cell, a plant cell or a non-human animal cell.

9. A method for preparing chitin, characterized in that, the method comprises: using the protein described in claim 1 to catalyze a reaction with UDP-GlcNAc as a substrate to obtain chitin.

10. The method according to claim 9, characterized in that, The described catalytic reaction system is as follows: Mix the protein described in claim 1, 10 mM UDP-GlcNAc, 10 mM HEPES, 10 mM KCl, and 10 mM MgCl 2 together, with a reaction volume of 500 μl; preferably, the catalytic reaction temperature is 20 to 40 °C, preferably 30 °C, and the reaction time is 0.1 to 18 hours.

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