Mutant, nucleic acid molecule, expression vector and application thereof

Abstract

The invention relates to the field of bioengineering, in particular to a mutant, a nucleic acid molecule, an expression vector and application thereof. The invention provides a mutant which has one or more amino acid site mutations on the basis of wild type phenylalanine ammonialyase: the 272nd site, the 273rd site, the 411th site, the 412th site, the 413th site, the 415th site, the 500th site, the 507th site and the 511th site, and the amino acid sequence of the wild type phenylalanine ammonialyase is shown as SEQ ID NO: 1. According to the invention, a phenylalanine ammonialyase (PAL) coding gene is identified and represented in details from tobacco (Nicotiana tabacum), and the gene is not systematically researched and represented yet. A series of mutants are constructed through protein engineering research, and several heat-resistant mutants are outstanding in heat-resistant effect and have important significance.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and in particular to mutants, nucleic acid molecules, expression vectors, and their applications. Background Technology

[0002] The mechanism of action of phenylalanine ammonia-lyase (PAL) is to catalyze the deamination of L-phenylalanine to form trans-cinnamic acid. It is the initiating and rate-limiting enzyme in phenylpropanoid metabolism and plays a crucial role in various organisms. This reaction is reversible, meaning it can also catalyze ammonia addition. Therefore, PAL plays an important role in plant growth, development, disease resistance, and stress tolerance. In the medical field, PAL proteins are often used to treat phenylketonuria (PKU), a genetically modified disease (a therapeutic enzyme with biomedical applications), as well as in the synthesis of some pharmaceutical intermediates and high-value-added compounds.

[0003] Most in vitro-derived PALs have unavoidable limitations, such as low activity and poor environmental tolerance. Furthermore, plant-derived PALs may also exhibit low heterologous expression levels and the inability of the protein to function properly.

[0004] Protein engineering techniques can effectively improve the properties of enzyme proteins, thereby obtaining mutants with excellent performance to meet the industrial needs of enzyme applications. Summary of the Invention

[0005] In view of this, the present invention provides mutants, nucleic acid molecules, expression vectors, and their applications. The present invention has identified and characterized in detail a phenylalanine ammonia-lyase (PAL) encoding gene from tobacco (Nicotiana tabacum), a gene that has not yet been systematically studied and characterized. Through protein engineering research, we constructed a series of mutants, several of which are heat-resistant and exhibit outstanding heat resistance, which is of significant importance.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] The present invention provides a mutant having one or more amino acid site mutations at positions 272, 273, 411, 412, 413, 415, 500, 507 and 511, based on wild-type phenylalanine ammonia-lyase, wherein the amino acid sequence of the wild-type phenylalanine ammonia-lyase is shown in SEQ ID NO:1.

[0008] In some embodiments of the present invention, the sequence of SEQ ID NO:1 in the above mutant is:

[0009] In some embodiments of the present invention, the mutation at position 272 in the above-described mutant includes:

[0010] A272S; and / or

[0011] The mutation at position 273 includes: N273M; and / or

[0012] The mutation at position 411 includes: A411S; and / or

[0013] The mutation at position 412 includes: L412I; and / or

[0014] The mutation at position 413 includes: A413S; and / or

[0015] The mutation at position 415 includes: I415V; and / or

[0016] The 500th mutation includes: A500S; and / or

[0017] The mutation at position 507 includes: M507L; and / or

[0018] The mutation at position 511 includes Y511F.

[0019] In some embodiments of the present invention, the mutants described above include one or more of the following: A272S, N273M, A411S, L412I, A413S, I415V, A500S, M507L, Y511F, A272S-N273M, A411S-L412I, A411S-L412I-A413S, A411S-L412I-A413S-I415V, A500S-M507L, and A500S-M507L-Y511F.

[0020] In some embodiments of the present invention, the above-mentioned mutant has the following characteristics:

[0021] (1) An amino acid sequence as shown in any of SEQ ID NO:2 to SEQ ID NO:16; or

[0022] (2) An amino acid sequence obtained by substituting, deleting, or adding one or more amino groups to the amino acid sequence shown in (1), and which has the same or similar function as the amino acid sequence shown in (1); or

[0023] (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2).

[0024] In some embodiments of the present invention, in the above-mentioned mutant, the sequence of SEQ ID NO: 2 is: MAGVAQNGHQEMDFCVKVDPLNWEMAADSLKGSHLDEVKKMVAEFRKPVVKLGGETLTVAQVAAIAAKDNAKTVKVELSEGARAGVKASSDWVMDSMSKGTDSYGVTTGFGATSHRRTKNGGALQKELIRFLNAGVFGNGTESCHTLPQSGTRAAMLVRINTLLQGYSGIRFEILEAITKLLNHNVTPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAIGPNGETLNAEEAFRVAGVNSGFFELQPKEGLALVNGTAVGSGLASMVLFDsNILAVFSEVLSAIFAEVMNGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSSYVKAPQKLHETDPLQKPKQDRYALRTSPQWLGPQIEVIRSATKMIEREINSVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNARLALASIGKLMFAQFSELVNDYYNNGLPSNLTAGRNPSLDYGFKGSEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISARKTAEAVDILKLMSSTYLVALCQAIDLRHLEENLRNAVKNTVSQVAKRTLTMGANGELHPSRFCEKDLLRVVDREYVFRYADDACSANYPLMQKLRQVLVDHALENGENEKNANSSIFQKILAFEGELKAVLPKEVESARISLENGNPAIANRIKECRSYPLYRFVREELGAELLTGEKVRSPGEECDKVFTAMCNGQIIDSLLECLKEWNGAPLPIC. (A272S)

[0025] In some embodiments of the present invention, in the above-mentioned mutant, the sequence of SEQ ID NO:3 is: MAGVAQNGHQEMDFCVKVDPLNWEMAADSLKGSHLDEVKKMVAEFRKPVVKLGGETLTVAQVAAIAAKDNAKTVKVELSEGARAGVKASSDWVMDSMSKGTDSYGVTTGFGATSHRRTKNGGALQKELIRFLNAGVFGNGTESCHTLPQSGTRAAMLVRINTLLQGYSGIRFEILEAITKLLNHNVTPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAIGPNGETLNAEEAFRVAGVNSGFFELQPKEGLALVNGTAVGSGLASMVLFDAMILAVFSEVLSAIFAEVMNGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSSYVKAPQKLHETDPLQKPKQDRYALRTSPQWLGPQIEVIRSATKMIEREINSVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNARLALASIGKLMFAQFSELVNDYYNNGLPSNLTAGRNPSLDYGFKGSEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISARKTAEAVDILKLMSSTYLVALCQAIDLRHLEENLRNAVKNTVSQVAKRTLTMGANGELHPSRFCEKDLLRVVDREYVFRYADDACSANYPLMQKLRQVLVDHALENGENEKNANSSIFQKILAFEGELKAVLPKEVESARISLENGNPAIANRIKECRSYPLYRFVREELGAELLTGEKVRSPGEECDKVFTAMCNGQIIDSLLECLKEWNGAPLPIC. (N273M)

[0026] In some embodiments of the present invention, in the above-mentioned mutant, the sequence of SEQ ID NO: 4 is: MAGVAQNGHQEMDFCVKVDPLNWEMAADSLKGSHLDEVKKMVAEFRKPVVKLGGETLTVAQVAAIAAKDNAKTVKVELSEGARAGVKASSDWVMDSMSKGTDSYGVTTGFGATSHRRTKNGGALQKELIRFLNAGVFGNGTESCHTLPQSGTRAAMLVRINTLLQGYSGIRFEILEAITKLLNHNVTPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAIGPNGETLNAEEAFRVAGVNSGFFELQPKEGLALVNGTAVGSGLASMVLFDANILAVFSEVLSAIFAEVMNGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSSYVKAPQKLHETDPLQKPKQDRYALRTSPQWLGPQIEVIRSATKMIEREINSVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNARLSLASIGKLMFAQFSELVNDYYNNGLPSNLTAGRNPSLDYGFKGSEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISARKTAEAVDILKLMSSTYLVALCQAIDLRHLEENLRNAVKNTVSQVAKRTLTMGANGELHPSRFCEKDLLRVVDREYVFRYADDACSANYPLMQKLRQVLVDHALENGENEKNANSSIFQKILAFEGELKAVLPKEVESARISLENGNPAIANRIKECRSYPLYRFVREELGAELLTGEKVRSPGEECDKVFTAMCNGQIIDSLLECLKEWNGAPLPIC. (A411S)

[0027] In some embodiments of the present invention, in the above-mentioned mutant, the sequence of SEQ ID NO: 5 is: MAGVAQNGHQEMDFCVKVDPLNWEMAADSLKGSHLDEVKKMVAEFRKPVVKLGGETLTVAQVAAIAAKDNAKTVKVELSEGARAGVKASSDWVMDSMSKGTDSYGVTTGFGATSHRRTKNGGALQKELIRFLNAGVFGNGTESCHTLPQSGTRAAMLVRINTLLQGYSGIRFEILEAITKLLNHNVTPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAIGPNGETLNAEEAFRVAGVNSGFFELQPKEGLALVNGTAVGSGLASMVLFDANILAVFSEVLSAIFAEVMNGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSSYVKAPQKLHETDPLQKPKQDRYALRTSPQWLGPQIEVIRSATKMIEREINSVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNARLAIASIGKLMFAQFSELVNDYYNNGLPSNLTAGRNPSLDYGFKGSEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISARKTAEAVDILKLMSSTYLVALCQAIDLRHLEENLRNAVKNTVSQVAKRTLTMGANGELHPSRFCEKDLLRVVDREYVFRYADDACSANYPLMQKLRQVLVDHALENGENEKNANSSIFQKILAFEGELKAVLPKEVESARISLENGNPAIANRIKECRSYPLYRFVREELGAELLTGEKVRSPGEECDKVFTAMCNGQIIDSLLECLKEWNGAPLPIC. (L412I)

[0028] In some embodiments of the present invention, in the above-mentioned mutant, the sequence of SEQ ID NO: 6 is: MAGVAQNGHQEMDFCVKVDPLNWEMAADSLKGSHLDEVKKMVAEFRKPVVKLGGETLTVAQVAAIAAKDNAKTVKVELSEGARAGVKASSDWVMDSMSKGTDSYGVTTGFGATSHRRTKNGGALQKELIRFLNAGVFGNGTESCHTLPQSGTRAAMLVRINTLLQGYSGIRFEILEAITKLLNHNVTPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAIGPNGETLNAEEAFRVAGVNSGFFELQPKEGLALVNGTAVGSGLASMVLFDANILAVFSEVLSAIFAEVMNGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSSYVKAPQKLHETDPLQKPKQDRYALRTSPQWLGPQIEVIRSATKMIEREINSVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNARLALSSIGKLMFAQFSELVNDYYNNGLPSNLTAGRNPSLDYGFKGSEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISARKTAEAVDILKLMSSTYLVALCQAIDLRHLEENLRNAVKNTVSQVAKRTLTMGANGELHPSRFCEKDLLRVVDREYVFRYADDACSANYPLMQKLRQVLVDHALENGENEKNANSSIFQKILAFEGELKAVLPKEVESARISLENGNPAIANRIKECRSYPLYRFVREELGAELLTGEKVRSPGEECDKVFTAMCNGQIIDSLLECLKEWNGAPLPIC. (A413S)

[0029] In some embodiments of the present invention, in the above-mentioned mutant, the sequence of SEQ ID NO:7 is: MAGVAQNGHQEMDFCVKVDPLNWEMAADSLKGSHLDEVKKMVAEFRKPVVKLGGETLTVAQVAAIAAKDNAKTVKVELSEGARAGVKASSDWVMDSMSKGTDSYGVTTGFGATSHRRTKNGGALQKELIRFLNAGVFGNGTESCHTLPQSGTRAAMLVRINTLLQGYSGIRFEILEAITKLLNHNVTPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAIGPNGETLNAEEAFRVAGVNSGFFELQPKEGLALVNGTAVGSGLASMVLFDANILAVFSEVLSAIFAEVMNGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSSYVKAPQKLHETDPLQKPKQDRYALRTSPQWLGPQIEVIRSATKMIEREINSVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNARLALASVGKLMFAQFSELVNDYYNNGLPSNLTAGRNPSLDYGFKGSEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISARKTAEAVDILKLMSSTYLVALCQAIDLRHLEENLRNAVKNTVSQVAKRTLTMGANGELHPSRFCEKDLLRVVDREYVFRYADDACSANYPLMQKLRQVLVDHALENGENEKNANSSIFQKILAFEGELKAVLPKEVESARISLENGNPAIANRIKECRSYPLYRFVREELGAELLTGEKVRSPGEECDKVFTAMCNGQIIDSLLECLKEWNGAPLPIC. (I415V)

[0030] In some embodiments of the present invention, in the above-mentioned mutant, the sequence of SEQ ID NO:8 is: MAGVAQNGHQEMDFCVKVDPLNWEMAADSLKGSHLDEVKKMVAEFRKPVVKLGGETLTVAQVAAIAAKDNAKTVKVELSEGARAGVKASSDWVMDSMSKGTDSYGVTTGFGATSHRRTKNGGALQKELIRFLNAGVFGNGTESCHTLPQSGTRAAMLVRINTLLQGYSGIRFEILEAITKLLNHNVTPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAIGPNGETLNAEEAFRVAGVNSGFFELQPKEGLALVNGTAVGSGLASMVLFDANILAVFSEVLSAIFAEVMNGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSSYVKAPQKLHETDPLQKPKQDRYALRTSPQWLGPQIEVIRSATKMIEREINSVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNARLALASIGKLMFAQFSELVNDYYNNGLPSNLTAGRNPSLDYGFKGSEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISARKTAESVDILKLMSSTYLVALCQAIDLRHLEENLRNAVKNTVSQVAKRTLTMGANGELHPSRFCEKDLLRVVDREYVFRYADDACSANYPLMQKLRQVLVDHALENGENEKNANSSIFQKILAFEGELKAVLPKEVESARISLENGNPAIANRIKECRSYPLYRFVREELGAELLTGEKVRSPGEECDKVFTAMCNGQIIDSLLECLKEWNGAPLPIC. (A500S)

[0031] In some embodiments of the present invention, in the above-mentioned mutant, the sequence of SEQ ID NO:9 is: MAGVAQNGHQEMDFCVKVDPLNWEMAADSLKGSHLDEVKKMVAEFRKPVVKLGGETLTVAQVAAIAAKDNAKTVKVELSEGARAGVKASSDWVMDSMSKGTDSYGVTTGFGATSHRRTKNGGALQKELIRFLNAGVFGNGTESCHTLPQSGTRAAMLVRINTLLQGYSGIRFEILEAITKLLNHNVTPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAIGPNGETLNAEEAFRVAGVNSGFFELQPKEGLALVNGTAVGSGLASMVLFDANILAVFSEVLSAIFAEVMNGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSSYVKAPQKLHETDPLQKPKQDRYALRTSPQWLGPQIEVIRSATKMIEREINSVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNARLALASIGKLMFAQFSELVNDYYNNGLPSNLTAGRNPSLDYGFKGSEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISARKTAEAVDILKLLSSTYLVALCQAIDLRHLEENLRNAVKNTVSQVAKRTLTMGANGELHPSRFCEKDLLRVVDREYVFRYADDACSANYPLMQKLRQVLVDHALENGENEKNANSSIFQKILAFEGELKAVLPKEVESARISLENGNPAIANRIKECRSYPLYRFVREELGAELLTGEKVRSPGEECDKVFTAMCNGQIIDSLLECLKEWNGAPLPIC. (M507L)

[0032] In some embodiments of the present invention, in the above mutant, the sequence of SEQ ID NO: 10 is: MAGVAQNGHQEMDFCVKVDPLNWEMAADSLKGSHLDEVKKMVAEFRKPVVKLGGETLTVAQVAAIAAKDNAKTVKVELSEGARAGVKASSDWVMDSMSKGTDSYGVTTGFGATSHRRTKNGGALQKELIRFLNAGVFGNGTESCHTLPQSGTRAAMLVRINTLLQGYSGIRFEILEAITKLLNHNVTPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAIGPNGETLNAEEAFRVAGVNSGFFELQPKEGLALVNGTAVGSGLASMVLFDANILAVFSEVLSAIFAEVMNGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSSYVKAPQKLHETDPLQKPKQDRYALRTSPQWLGPQIEVIRSATKMIEREINSVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNARLALASIGKLMFAQFSELVNDYYNNGLPSNLTAGRNPSLDYGFKGSEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISARKTAEAVDILKLMSSTFLVALCQAIDLRHLEENLRNAVKNTVSQVAKRTLTMGANGELHPSRFCEKDLLRVVDREYVFRYADDACSANYPLMQKLRQVLVDHALENGENEKNANSSIFQKILAFEGELKAVLPKEVESARISLENGNPAIANRIKECRSYPLYRFVREELGAELLTGEKVRSPGEECDKVFTAMCNGQIIDSLLECLKEWNGAPLPIC. (Y511F)

[0033] In some embodiments of the present invention, in the above-mentioned mutant, the sequence of SEQ ID NO: 11 is: MAGVAQNGHQEMDFCVKVDPLNWEMAADSLKGSHLDEVKKMVAEFRKPVVKLGGETLTVAQVAAIAAKDNAKTVKVELSEGARAGVKASSDWVMDSMSKGTDSYGVTTGFGATSHRRTKNGGALQKELIRFLNAGVFGNGTESCHTLPQSGTRAAMLVRINTLLQGYSGIRFEILEAITKLLNHNVTPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAIGPNGETLNAEEAFRVAGVNSGFFELQPKEGLALVNGTAVGSGLASMVLFDSMILAVFSEVLSAIFAEVMNGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSSYVKAPQKLHETDPLQKPKQDRYALRTSPQWLGPQIEVIRSATKMIEREINSVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNARLALASIGKLMFAQFSELVNDYYNNGLPSNLTAGRNPSLDYGFKGSEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISARKTAEAVDILKLMSSTYLVALCQAIDLRHLEENLRNAVKNTVSQVAKRTLTMGANGELHPSRFCEKDLLRVVDREYVFRYADDACSANYPLMQKLRQVLVDHALENGENEKNANSSIFQKILAFEGELKAVLPKEVESARISLENGNPAIANRIKECRSYPLYRFVREELGAELLTGEKVRSPGEECDKVFTAMCNGQIIDSLLECLKEWNGAPLPIC. (A272S + N273M)

[0034] In some embodiments of the present invention, in the above-mentioned mutant, the sequence of SEQ ID NO: 12 is: MAGVAQNGHQEMDFCVKVDPLNWEMAADSLKGSHLDEVKKMVAEFRKPVVKLGGETLTVAQVAAIAAKDNAKTVKVELSEGARAGVKASSDWVMDSMSKGTDSYGVTTGFGATSHRRTKNGGALQKELIRFLNAGVFGNGTESCHTLPQSGTRAAMLVRINTLLQGYSGIRFEILEAITKLLNHNVTPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAIGPNGETLNAEEAFRVAGVNSGFFELQPKEGLALVNGTAVGSGLASMVLFDANILAVFSEVLSAIFAEVMNGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSSYVKAPQKLHETDPLQKPKQDRYALRTSPQWLGPQIEVIRSATKMIEREINSVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNARLsiASIGKLMFAQFSELVNDYYNNGLPSNLTAGRNPSLDYGFKGSEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISARKTAEAVDILKLMSSTYLVALCQAIDLRHLEENLRNAVKNTVSQVAKRTLTMGANGELHPSRFCEKDLLRVVDREYVFRYADDACSANYPLMQKLRQVLVDHALENGENEKNANSSIFQKILAFEGELKAVLPKEVESARISLENGNPAIANRIKECRSYPLYRFVREELGAELLTGEKVRSPGEECDKVFTAMCNGQIIDSLLECLKEWNGAPLPIC. (A411S+L412I)

[0035] In some embodiments of the present invention, in the above-mentioned mutant, the sequence of SEQ ID NO: 13 is: MAGVAQNGHQEMDFCVKVDPLNWEMAADSLKGSHLDEVKKMVAEFRKPVVKLGGETLTVAQVAAIAAKDNAKTVKVELSEGARAGVKASSDWVMDSMSKGTDSYGVTTGFGATSHRRTKNGGALQKELIRFLNAGVFGNGTESCHTLPQSGTRAAMLVRINTLLQGYSGIRFEILEAITKLLNHNVTPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAIGPNGETLNAEEAFRVAGVNSGFFELQPKEGLALVNGTAVGSGLASMVLFDANILAVFSEVLSAIFAEVMNGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSSYVKAPQKLHETDPLQKPKQDRYALRTSPQWLGPQIEVIRSATKMIEREINSVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNARLSISSIGKLMFAQFSELVNDYYNNGLPSNLTAGRNPSLDYGFKGSEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISARKTAEAVDILKLMSSTYLVALCQAIDLRHLEENLRNAVKNTVSQVAKRTLTMGANGELHPSRFCEKDLLRVVDREYVFRYADDACSANYPLMQKLRQVLVDHALENGENEKNANSSIFQKILAFEGELKAVLPKEVESARISLENGNPAIANRIKECRSYPLYRFVREELGAELLTGEKVRSPGEECDKVFTAMCNGQIIDSLLECLKEWNGAPLPIC. (A411S+L412I+A413S)

[0036] In some embodiments of the present invention, in the above-mentioned mutant, the sequence of SEQ ID NO: 14 is: MAGVAQNGHQEMDFCVKVDPLNWEMAADSLKGSHLDEVKKMVAEFRKPVVKLGGETLTVAQVAAIAAKDNAKTVKVELSEGARAGVKASSDWVMDSMSKGTDSYGVTTGFGATSHRRTKNGGALQKELIRFLNAGVFGNGTESCHTLPQSGTRAAMLVRINTLLQGYSGIRFEILEAITKLLNHNVTPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAIGPNGETLNAEEAFRVAGVNSGFFELQPKEGLALVNGTAVGSGLASMVLFDANILAVFSEVLSAIFAEVMNGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSSYVKAPQKLHETDPLQKPKQDRYALRTSPQWLGPQIEVIRSATKMIEREINSVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNARLSISSIGKLMFAQFSELVNDYYNNGLPSNLTAGRNPSLDYGFKGSEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISARKTAEAVDILKLMSSTYLVALCQAIDLRHLEENLRNAVKNTVSQVAKRTLTMGANGELHPSRFCEKDLLRVVDREYVFRYADDACSANYPLMQKLRQVLVDHALENGENEKNANSSIFQKILAFEGELKAVLPKEVESARISLENGNPAIANRIKECRSYPLYRFVREELGAELLTGEKVRSPGEECDKVFTAMCNGQIIDSLLECLKEWNGAPLPIC. (A411S+L412I+A413S+I415V)

[0037] In some embodiments of the present invention, in the above mutant, the sequence of SEQ ID NO: 15 is: MAGVAQNGHQEMDFCVKVDPLNWEMAADSLKGSHLDEVKKMVAEFRKPVVKLGGETLTVAQVAAIAAKDNAKTVKVELSEGARAGVKASSDWVMDSMSKGTDSYGVTTGFGATSHRRTKNGGALQKELIRFLNAGVFGNGTESCHTLPQSGTRAAMLVRINTLLQGYSGIRFEILEAITKLLNHNVTPCLPLRGTITASGDLVPLSYIAGLLTGRPNSKAIGPNGETLNAEEAFRVAGVNSGFFELQPKEGLALVNGTAVGSGLASMVLFDANILAVFSEVLSAIFAEVMNGKPEFTDHLTHKLKHHPGQIEAAAIMEHILDGSSYVKAPQKLHETDPLQKPKQDRYALRTSPQWLGPQIEVIRSATKMIEREINSVNDNPLIDVSRNKALHGGNFQGTPIGVSMDNARLALASIGKLMFAQFSELVNDYYNNGLPSNLTAGRNPSLDYGFKGSEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISARKTAEsVDILKLlSSTYLVALCQAIDLRHLEENLRNAVKNTVSQVAKRTLTMGANGELHPSRFCEKDLLRVVDREYVFRYADDACSANYPLMQKLRQVLVDHALENGENEKNANSSIFQKILAFEGELKAVLPKEVESARISLENGNPAIANRIKECRSYPLYRFVREELGAELLTGEKVRSPGEECDKVFTAMCNGQIIDSLLECLKEWNGAPLPIC. (A500S + M507L)

[0038] In some embodiments of the present invention, in the above-mentioned mutant, the sequence of SEQ ID NO:16 is: (A500S+M507L+Y511F).

[0039] The present invention also provides a nucleic acid molecule encoding the above-mentioned mutant.

[0040] In some embodiments of the present invention, the above-mentioned nucleic acid molecules have:

[0041] (4) A nucleotide sequence as shown in any of SEQ ID NO:17 to SEQ ID NO:24; or

[0042] (5) A nucleotide sequence obtained by modifying, substituting, deleting, or adding one or more bases to the nucleotide sequence described in (4); or

[0043] (6) A sequence having at least 80% homology to the nucleotide sequence described in (4) or (5); or

[0044] (7) The complementary sequence of the nucleotide sequence described in (4), (5) or (6).

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054] The present invention also provides an expression vector comprising the above-mentioned nucleic acid molecule.

[0055] The present invention also provides a host, transformation and / or transfection of the above expression vector.

[0056] In some embodiments of the present invention, the host includes a host cell.

[0057] The present invention also provides a method for preparing the above-mentioned mutant, wherein the above-mentioned host is inoculated, cultured, induced, and purified to obtain the mutant.

[0058] The present invention also provides products comprising: the above-described mutant, the above-described nucleic acid molecule, the above-described expression vector, the above-described host, and / or the mutant obtained by the above-described preparation method.

[0059] This invention provides a detailed identification and characterization of a phenylalanine ammonia-lyase (PAL) encoding gene from tobacco (Nicotiana tabacum), a gene that has not yet been systematically studied and characterized. Through protein engineering research, we constructed a series of mutants, several of which are heat-resistant and exhibit significant heat resistance. Attached Figure Description

[0060] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0061] Figure 1 The tolerance of different mutants and wild-type to temperatures ranging from 35 to 65°C was tested. The results showed that the activity of wild-type decreased significantly over time at different temperatures. The mutants did not show a significant decrease in activity after incubation for 120 minutes at 35°C and 45°C. At 55°C and 65°C, sequence No.12 showed the most outstanding heat resistance, followed by sequence No.2 and sequence No.15. Detailed Implementation

[0062] This invention discloses mutants, nucleic acid molecules, expression vectors, and their applications.

[0063] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0064] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0065] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0066] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0067] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0068] In the embodiments of this invention, all raw materials and reagents used can be purchased from the market.

[0069] The present invention will be further illustrated below with reference to the embodiments:

[0070] Example 1

[0071] A PAL coding sequence was selected from the tobacco (Nicotiana tabacum) genome for in vitro synthesis. Codon optimization was performed during synthesis, and the sequence was seamlessly ligated into the pET28a vector and transformed into *E. coli* BL21(DE3) competent cells. After thawing, 50 μL of the competent cells were added to an EP tube, followed by 1–5 μL of plasmid, and incubated on ice for 30 min. Then, the cells were heat-shocked at 42°C for 60 s, followed by an ice bath for 5 min (this is the heat shock method; other methods, such as electroporation, can also be used). 500 μL of antibiotic-free LB broth was added, and the cells were incubated at 37°C and 180 rpm for 30–60 min. Approximately 10–20 μL of the bacterial culture was plated and incubated overnight at 37°C. Single colonies were picked the next day for sequencing verification.

[0072] *E. coli* strain BL21(DE3) carrying the recombinant plasmid was cultured overnight in LB medium containing 50 μg / mL kanamycin at 37°C and 200 rpm. The overnight culture was then inoculated into fresh TB medium at 2% (v / v). When the OD600 value reached 0.8–1.0, protein expression was induced for approximately 6 hours at 25°C with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG). Cells were collected by centrifugation, resuspended in 50 mM Tris-HCl buffer, and then sonicated. Cell debris was removed by centrifugation at 12,000 rpm for 15 minutes at 4°C. The supernatant was subjected to Ni²⁺-NTA affinity chromatography, and the target protein was eluted using elution buffer containing 50 mM Tris (pH 7.5) and 500 mM imidazole. The purity of the eluted protein was confirmed by SDS-PAGE analysis. The purified protein was dialyzed with 20 mM Tris buffer (pH 8.0) to remove imidazole and excess salt, thus obtaining the purified protein.

[0073] Enzyme activity of all variants was determined in a 200 μL reaction system containing 1 μg of purified protein, 12.1 mM phenylalanine, and 20 mM Tris-HCl buffer (pH 8.0). The reaction was incubated at 35°C for 30 min, and absorbance changes were monitored at 290 nm. Enzyme activity units (U) were defined as the amount of enzyme that catalyzes the production of 1 μmol of product per minute.

[0074] The PAL protein we characterized has an optimal temperature of 55°C, exceeding the performance range of existing heat-resistant PAL mutants. It retains over 85% of its activity within the 45–65°C range. Activity is still detectable at 75°C, but with significant loss.

[0075] Through computer-aided design and screening, nine sites were selected for modification, resulting in a maximum 7.5-fold increase in mutant activity at 55℃. Simultaneously, we designed a combinatorial mutant, which showed a maximum 10.7-fold increase at 55℃ and a 6.5-fold increase at 75℃.

[0076] Table 1

[0077]

[0078] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Mutant, characterized in that, one or more of the following amino acid positions: 272, 273, 411, 412, 413, 415, 500, 507, and 511, based on a wild-type phenylalanine ammonia-lyase having an amino acid sequence as shown in SEQ ID NO:

1.

2. The mutant of claim 1, wherein the mutation at position 272 comprises: A272S; and / or the mutation at position 273 comprises: N273M; and / or the mutation at position 411 comprises: A411S; and / or the mutation at position 412 comprises: L412I; and / or the mutation at position 413 comprises: A413S; and / or the mutation at position 415 comprises: I415V; and / or the mutation at position 500 comprises: A500S; and / or the mutation at position 507 comprises: M507L; and / or the mutation at position 511 comprises: Y511F.

3. Mutant according to claim 1 or 2, characterized in that comprises: one or more of A272S, N273M, A411S, L412I, A413S, I415V, A500S, M507L, Y511F, A272S-N273M, A411S-L412I, A411S-L412I-A413S, A411S-L412I-A413S-I415V, A500S-M507L, and A500S-M507L-Y511F.

4. Mutant according to any one of claims 1 to 3, characterized in that has: (1) an amino acid sequence as shown in any one of SEQ ID NO: 2 to SEQ ID NO: 16; or (2) an amino acid sequence obtained by substitution, deletion, or addition of one or more amino acids to the amino acid sequence as shown in (1), and which has the same or similar function as the amino acid sequence as shown in (1); or (3) an amino acid sequence having at least 80% identity to the amino acid sequence as shown in (1) or (2).

5. A nucleic acid molecule encoding the mutant as claimed in any one of claims 1 to 4.

6. The nucleic acid molecule of claim 5, wherein, has: (4) a nucleotide sequence as shown in any one of SEQ ID NO: 17 to SEQ ID NO: 24; or (5) a nucleotide sequence obtained by modification, substitution, deletion, or addition of one or more bases to the nucleotide sequence as shown in (4); or (6) a sequence having at least 80% homology to the nucleotide sequence as shown in (4) or (5); or (7) a complementary sequence of the nucleotide sequence as shown in (4), (5), or (6).

7. An expression vector, characterized in that, comprises: the nucleic acid molecule as claimed in claim 5 or 6.

8. A host, characterized in that, transforming and / or transfecting the expression vector as claimed in claim 7.

9. The method of producing the mutant according to any one of claims 1 to 4, wherein obtaining the mutant after inoculating, culturing, inducing, and purifying the host as claimed in claim 8.

10. A product characterized by, comprises: the mutant as claimed in any one of claims 1 to 4, the nucleic acid molecule as claimed in claim 5 or 6, the expression vector as claimed in claim 7, the host as claimed in claim 8, and / or the mutant obtained by the preparation method as claimed in claim 9.

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