Recombinant polypeptide and recombinant cell both expressing aromatic amino acid degrading enzyme, and use of recombinant polypeptide and recombinant cell

Abstract

A recombinant polypeptide and a recombinant cell both expressing an aromatic amino acid degrading enzyme, and a use of the recombinant polypeptide and the recombinant cell, relating to the field of biotechnology. The sequence of the aromatic amino acid degrading enzyme XAL derived from Phanerochaete chrysosporium is taken as a starting sequence, a series of XAL mutations that can improve the enzyme activity and stability are found by means of screening and verification of mutation sites, the mutants are introduced into the genetically engineered bacteria, and then degradation activity verification is performed; and it is found that compared with a control strain of a transformed non-mutant enzyme, the constructed recombinant strain has the amino acid degradation activity significantly improved. The present invention further relates to a use of a recombinant XAL polypeptide or a recombinant cell, and a composition of the recombinant XAL polypeptide or the recombinant cell for the treatment and industrial purposes.

Description

A recombinant polypeptide and recombinant cell expressing aromatic amino acid degrading enzyme and their application Technical Field

[0001] The present invention relates to a recombinant polypeptide and a recombinant cell expressing an aromatic amino acid degrading enzyme and applications thereof, belonging to the field of biotechnology. Background Art

[0002] Phenylalanine degrading enzyme (PAL), along with histidine degrading enzyme (HAL) and tyrosine degrading enzyme (TAL), are members of the aromatic amino acid degrading enzyme family (EC 4.3.1.23-1.25 and 4.3.1.3). Aromatic amino acid degrading enzyme (XAL) possesses the activities of phenylalanine degrading enzyme (PAL), histidine degrading enzyme (HAL), and tyrosine degrading enzyme (TAL), degrading aromatic amino acids.

[0003] Phenylketonuria (PKU) is the most common inborn error in amino acid metabolism and an autosomal recessive genetic disorder. It is caused by mutations in the phenylalanine hydroxylase gene, which leads to reduced enzyme activity and accumulation of phenylalanine and its metabolites in the body. Symptoms typically begin 3-6 months after birth, with clinical manifestations including intellectual and motor developmental delays, decreased skin and hair pigmentation, and foul-smelling urine. If left untreated, high levels of phenylalanine accumulation can lead to serious medical problems, such as epileptic seizures and intellectual disability. Currently, most common drugs for treating phenylketonuria are chemical drugs, which have disadvantages such as high toxic side effects and easy development of drug resistance. Among biological drugs, the current effective drug for treating PKU is the injection of PEG-modified recombinant PAL protein (PEG-PAL, polyethylene glycol-conjugated phenylalanine ammonia lyase), which can reduce the concentration of phenylalanine in human plasma to a relatively safe concentration level. However, PEG-PAL can cause a more severe immune response. Different from the invasive preparations and treatments of PEG-PAL, there are also studies on oral PAL preparations both domestically and internationally. The advantage of oral delivery is that there is no immune rejection reaction, but the disadvantage is also more obvious. Oral PAL will be broken down by trypsin, pepsin, chymotrypsin, etc. in the gastrointestinal tract.

[0004] Tyrosinemia is caused by enzyme defects in the tyrosine metabolism pathway, leading to elevated plasma tyrosine concentrations. Enzyme defects at different steps can lead to a variety of clinically diverse disorders, including damage to multiple organs, including the brain, liver, kidneys, and bones, with poor prognosis and high mortality and disability rates. Tyrosine is derived from both dietary intake and endogenous synthesis. The substrate for endogenous synthesis is derived from the dietary essential amino acid tyrosine. Currently, there is only one marketed drug for tyrosinemia: nitisinone (NTBC). NTBC is a competitive inhibitor of 4-hydroxyphenylpyruvate dioxygenase, inhibiting the normal catabolism of tyrosine in HT-1 patients and preventing the accumulation of toxic metabolites, thereby preventing liver and kidney damage and improving survival. During nitisinone treatment, patients must restrict their dietary tyrosine and tyrosine intake. Common adverse reactions reported in studies include thrombocytopenia, leukopenia, and visual disturbances, including conjunctivitis, corneal opacity, keratitis, and photophobia. However, because NTBC is a 4-hydroxyphenylpyruvate dioxygenase inhibitor, it inhibits the metabolism of tyrosine to 4-hydroxyphenylpyruvate, leading to elevated blood tyrosine levels. If blood tyrosine levels exceed 500 μmol / L, protein intake should be controlled and a tyrosine-free diet or tyrosine-formulated nutritional powder should be administered. Otherwise, elevated blood tyrosine levels can lead to corneal damage. Treatment options for tyrosinemia include liver transplantation and gene therapy. Liver transplantation has been used for over 20 years and can significantly improve liver, kidney, and neurological symptoms. In recent years, the need for liver transplantation has gradually decreased with the use of NTBC. Furthermore, children who undergo liver transplantation still have an approximately 5%-10% mortality rate and require lifelong immunosuppressive therapy. This approach is limited by surgical conditions and the limited availability of donors, making clinical application difficult. Therefore, liver transplantation is considered an option only for children with severe liver failure who have failed to respond to nitisinone or who cannot receive nitisinone for other reasons, and who have evidence of liver malignancy. Gene therapy is currently still in the animal research stage.

[0005] The present invention is based on the lack of therapeutic drugs for phenylketonuria and tyrosinemia. However, as genetic defects, phenylketonuria and tyrosinemia currently have no specific cure. Existing treatments all involve reducing phenylalanine or tyrosine intake through dietary restriction or special diets, thereby alleviating symptoms. However, special or restricted diets can significantly impact the nutritional health of patients because the food is bland. Therefore, finding a way to allow patients to consume lower amounts of phenylalanine or tyrosine under normal dietary conditions is particularly important. Therefore, if we want to treat the above diseases from the perspective of food-induced metabolism, the first problem to be solved is how to improve the catalytic activity and stability of the XAL protein while reducing the amount of drug used. Therefore, the targeted modification of natural PAL is particularly important.

[0006] Summary of the Invention

[0007] To solve the above problems, the present invention mutates the aromatic amino acid degrading enzyme from Phanerochaete chrysosporium to obtain its mutant, thereby improving the catalytic activity, enzyme stability, thermal stability, acid tolerance, etc. of the enzyme, while also improving its performance in drug formulation, which has important economic value and social significance.

[0008] The present invention provides a recombinant XAL polypeptide and its mutants, biologically active fragments and analogs, as well as a pharmaceutical combination of recombinant cells expressing the recombinant XAL polypeptide and its mutants, biologically active fragments and analogs.

[0009] The present invention provides polynucleotides encoding recombinant aromatic amino acid degrading enzyme polypeptides, recombinant polypeptides, and / or compositions thereof, recombinant cells expressing recombinant aromatic amino acid degrading enzyme polypeptides, and / or compositions thereof. In some embodiments, the recombinant XAL polypeptide is optimized to provide enhanced catalytic activity, reduced susceptibility to proteolysis, and increased tolerance to storage at elevated temperatures. The present invention also provides therapeutic uses of recombinant cells expressing recombinant XAL and / or compositions thereof.

[0010] One existing method for lowering phenylalanine in the blood involves the injection of recombinant PAL and PAL variants modified by PEGylation (PEG-PAL). PAL variants that can be used in PEG-PAL compositions include wild-type Nostoc punctiforme (NpPAL), Anabaena variabilis (AvPAL), and variants of Rhodosporidium toruloides (RtPAL). There are no reports regarding methods for lowering tyrosine in the blood.

[0011] A key advantage of oral enzyme formulations is reduced exposure of the enzyme to the immune system, thereby minimizing the immune response observed after injection. However, a major limitation for oral formulations is the loss of enzyme activity in the stomach and intestinal lumen. To be effective and functional, these enzymes must withstand acidic pH and protease degradation. In some previous studies, some enzymes had relatively low specific activity at pH 7.0, partly due to protease degradation.

[0012] Despite progress in the development of various oral enzyme formulations, there remains a need for oral enzymes with improved properties. These improved properties include, but are not limited to, a longer half-life, increased catalytic activity, improved stability to digestive tract conditions and storage conditions, and reduced aggregation. Furthermore, the use of large amounts of enzymes not only compromises formulation but also increases production and patient costs. Therefore, improving enzyme activity and tolerability is crucial. Furthermore, the mainstream view is that using probiotic cells as a chassis to construct recombinant cells is more therapeutically beneficial than heterologous expression of recombinant proteins.

[0013] Aromatic amino acid degrading enzyme (XAL) can also be used as a treatment for tyrosine metabolism disorders such as type I tyrosinemia, type II tyrosinemia, type III tyrosinemia, and alkaptonuria, which are all autosomal metabolic genetic disorders in which a type of enzyme involved in tyrosine degradation is partially or ineffective due to a mutation in the corresponding gene. The deletion of this gene causes elevated levels of tyrosine and other tyrosine metabolites in the blood. Because tyrosine is derived from phenylalanine, limiting phenylalanine and tyrosine intake is beneficial to patients with tyrosine metabolism disorders.

[0014] ①Recombinant XAL polypeptide:

[0015] The recombinant XAL polypeptide of the present invention is derived from Phanerochaete chrysosporium.

[0016] In some embodiments, the recombinant XAL polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO: 4. In some embodiments, the amino acid differences are at 1, 2, 3, 4, 5, 6, 7, 8 or more amino acid positions. In some embodiments, a recombinant XAL polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO. 4 and has at least one amino acid residue difference compared to SEQ ID NO. 4 at one or more amino acid positions selected from the group consisting of: 48, 210, 374, 384, 394, 522, 526, 585, 586, 601. In some embodiments, the amino acid substitution is selected from at least one of F48V, I210F, D374A, F384S, S394G, A522V, I526S, I585L, L586P, D601G, or a combination thereof, wherein amino acid positions are numbered relative to SEQ ID NO. 4.

[0017] In some embodiments, the recombinant XAL polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO: 6. In some embodiments, the amino acid differences are at 1, 2, 3, 4, 5, 6, 7, 8 or more amino acid positions. In some embodiments, the recombinant XAL polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO. 6, and has at least one amino acid residue difference compared to SEQ ID NO. 6 at one or more amino acid positions selected from the group consisting of: 62, 93, 250, 359, 370, 409, 468, 624, 690, 708. In some embodiments, the amino acid substitution is selected from at least one of K62N, E93D, S250N, E370V, N409T, L468I, D624Y, G690S, I708F, or a combination thereof, wherein amino acid positions are numbered relative to SEQ ID NO. 6.

[0018] In some embodiments, the recombinant XAL polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO: 8. In some embodiments, the amino acid differences are at 1, 2, 3, 4, 5, 6, 7, 8 or more amino acid positions. In some embodiments, a recombinant XAL polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO. 8 and has at least one amino acid residue difference compared to SEQ ID NO. 8 at one or more amino acid positions selected from the group consisting of: 147, 382, ​​383, 433, 582, 678. In some embodiments, the amino acid substitution is selected from at least one of G147D, A382T, Q383K, A433T, L582P, E678D, or a combination thereof, wherein amino acid positions are numbered relative to SEQ ID NO. 8.

[0019] In some embodiments, the recombinant XAL polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO: 10. In some embodiments, the amino acid differences are at 1, 2, 3, 4, 5, 6, 7, 8 or more amino acid positions. In some embodiments, a recombinant XAL polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO. 10 and has at least one amino acid residue difference compared to SEQ ID NO. 10 at one or more amino acid positions selected from the group consisting of: 127, 167, 170, 199, 204, 400, 488. In some embodiments, the amino acid substitution is selected from at least one of D127A, A167T, S170C, I199L, E204G, T400P, A488S, or a combination thereof, wherein amino acid positions are numbered relative to SEQ ID NO. 10.

[0020] In some embodiments, the recombinant XAL polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO: 12. In some embodiments, the amino acid differences are at 1, 2, 3, 4, 5, 6, 7, 8 or more amino acid positions. In some embodiments, a recombinant XAL polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO. 12, and has at least one amino acid residue difference compared to SEQ ID NO. 12 at one or more amino acid positions selected from the group consisting of: 67, 202, 413, 571, 573, 590. In some embodiments, the amino acid substitution is selected from at least one of D67H, M202L, D413E, H571Y, P573S, I590F, or a combination thereof, wherein amino acid positions are numbered relative to SEQ ID NO. 12.

[0021] In some embodiments, the recombinant XAL polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO: 14. In some embodiments, the amino acid differences are at 1, 2, 3, 4, 5, 6, 7, 8 or more amino acid positions. In some embodiments, a recombinant XAL polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO. 14, and has at least one amino acid residue difference compared to SEQ ID NO. 14 at one or more amino acid positions selected from the group consisting of: 5, 141, 170, 183, 417, 482, 500, 524. In some embodiments, the amino acid substitution is selected from at least one of I5M, L141M, S170Y, R183P, G417S, V482A, V500F, Y524H, or a combination thereof, wherein amino acid positions are numbered relative to SEQ ID NO. 14.

[0022] In some embodiments, the recombinant XAL polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO: 16. In some embodiments, the amino acid differences are at 1, 2, 3, 4, 5, 6, 7, 8 or more amino acid positions. In some embodiments, a recombinant XAL polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO. 16, and has at least one amino acid residue difference compared to SEQ ID NO. 16 at one or more amino acid positions selected from the group consisting of: 476, 180, 213, 322, 341, 356, 722, 727. In some embodiments, the amino acid substitution is selected from at least one of N476T, I180S, L213R, G322S, V341L, V356E, G722A, V727F, or a combination thereof, wherein amino acid positions are numbered relative to SEQ ID NO. 16.

[0023] In some embodiments, the recombinant XAL polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO: 18. In some embodiments, the amino acid differences are at 1, 2, 3, 4, 5, 6, 7, 8 or more amino acid positions. In some embodiments, a recombinant XAL polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO. 18, and has at least one amino acid residue difference compared to SEQ ID NO. 18 at one or more amino acid positions selected from the group consisting of: 91, 122, 207, 225, 476, 537, 677, 684, 731. In some embodiments, the amino acid substitution is selected from at least one of N91S, F122I, R207S, D225H, N476K, A537V, Y677D, L684M, L731V, or a combination thereof, wherein amino acid positions are numbered relative to SEQ ID NO. 18.

[0024] In some embodiments, the recombinant XAL polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO: 20. In some embodiments, the amino acid differences are at 1, 2, 3, 4, 5, 6, 7, 8 or more amino acid positions. In some embodiments, a recombinant XAL polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO. 20 and has at least one amino acid residue difference compared to SEQ ID NO. 20 at one or more amino acid positions selected from the group consisting of: 81, 241, 297, 480, 481, 707, 729. In some embodiments, the amino acid substitution is selected from at least one of R81H, A241S, N297K, K480E, G481D, I707V, M729I, or a combination thereof, wherein amino acid positions are numbered relative to SEQ ID NO. 20.

[0025] In some embodiments, the recombinant XAL polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO: 22. In some embodiments, the amino acid differences are at 1, 2, 3, 4, 5, 6, 7, 8 or more amino acid positions. In some embodiments, a recombinant XAL polypeptide exhibiting at least one improved property has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO. 22, and has at least one amino acid residue difference compared to SEQ ID NO. 22 at one or more amino acid positions selected from the group consisting of: 61, 184, 203, 331, 388, 585, 629. In some embodiments, the amino acid substitution is selected from at least one of G61R, M184I, G203C, T331P, Q388H, I585T, S629C, or a combination thereof, wherein amino acid positions are numbered relative to SEQ ID NO. 22.

[0026] ② Mutants with reduced sensitivity to proteolysis:

[0027] In some embodiments, the recombinant XAL polypeptide of the invention has the corresponding enzymatic activity, exhibits reduced susceptibility to proteolysis, and comprises: a) an amino acid sequence having at least 85% sequence identity to the reference sequence SEQ ID NO. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22; and b) an amino acid residue difference at one or more amino acid positions compared to SEQ ID NO. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22.

[0028] In some embodiments, a recombinant XAL polypeptide exhibiting reduced susceptibility to proteolysis has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and has at least one amino acid position (similar to SEQ ID NO. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22) that is identical to SEQ ID NO. No. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 have at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity and differ in the amino acid residues at 1, 2, 3, 4, 5, 6, 7, 8 or more amino acid positions).

[0029] In some embodiments, the proteolytic susceptibility of the recombinant XAL polypeptide is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, or at least 90% compared to the proteolytic susceptibility of wild-type XAL (e.g., PcXAL having SEQ ID NO. 2) or a reference XAL polypeptide under essentially identical conditions. Proteolytic activity can be measured using any suitable method known in the art, including, but not limited to, the methods described in the Examples.

[0030] In some embodiments, the recombinant XAL polypeptide has reduced sensitivity to a combination of one or more proteases, including but not limited to pepsin, trypsin, chymotrypsin, carboxypeptidase A / B, peptidase, etc., when both a reference XAL and a recombinant XAL with reduced sensitivity are compared and exposed to substantially the same amount of proteases under substantially the same conditions.

[0031] ③ Mutants that improve protein thermal stability:

[0032] In some embodiments, the recombinant XAL polypeptide of the present invention has corresponding enzymatic activity, exhibits improved protein thermostability, and comprises: a) an amino acid sequence having at least 85% sequence identity to the reference sequence SEQ ID NO. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22; and b) an amino acid residue difference at one or more amino acid positions compared to SEQ ID NO. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22.

[0033] In some embodiments, the recombinant XAL polypeptide exhibiting improved protein thermostability has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and has at least one amino acid position (similar to SEQ ID NO. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22) that is identical to SEQ ID NO. No. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 have at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity and differ in the amino acid residues at 1, 2, 3, 4, 5, 6, 7, 8 or more amino acid positions).

[0034] In some embodiments, the improved protein thermostability of the recombinant XAL polypeptide is compared to the protein thermostability of wild-type XAL (e.g., PcXAL having SEQ ID NO. 2) or a reference XAL polypeptide under substantially identical conditions at 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C. Improved protein thermostability can be measured using any suitable method known in the art, including but not limited to the methods described in the Examples. In some embodiments, any suitable assay can be used with the present invention, including but not limited to those provided herein.

[0035] ④ Mutants with improved acid tolerance:

[0036] In some embodiments, the recombinant XAL polypeptide of the invention has corresponding enzymatic activity, exhibits improved acid tolerance, and comprises: a) an amino acid sequence having at least 85% sequence identity to the reference sequence SEQ ID NO. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22; and b) an amino acid residue difference at one or more amino acid positions compared to SEQ ID NO. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22.

[0037] In some embodiments, the recombinant XAL polypeptide exhibiting improved acid tolerance has at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity to SEQ ID NO. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and has at least one amino acid position (similar to SEQ ID NO. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22) that is identical to SEQ ID NO. No. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 have at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater amino acid sequence identity and differ in the amino acid residues at 1, 2, 3, 4, 5, 6, 7, 8 or more amino acid positions).

[0038] In some embodiments, the improved acid tolerance of the recombinant XAL polypeptide is compared to the acid tolerance of wild-type XAL (e.g., PcXAL having SEQ ID NO. 2) or a reference XAL polypeptide under substantially identical conditions at pH 1.5, pH 2, pH 2.5, pH 3, pH 3.5, pH 4, pH 5, pH 6, pH 7, or pH 8. The improved acid tolerance can be measured using any suitable method known in the art, including, but not limited to, the methods described in the Examples. In some embodiments, any suitable assay can be used in the present invention, including, but not limited to, those provided herein.

[0039] ⑤ Polynucleotide encoding recombinant polypeptide, expression vector, and host cell:

[0040] In some embodiments, the polynucleotide is linked to one or more heterologous regulatory sequences to create a recombinant polynucleotide capable of expressing a recombinant polypeptide. In some embodiments, at least one heterologous expression construct encoding a recombinant XAL polypeptide is introduced into a corresponding host cell to express the corresponding XAL polypeptide.

[0041] In some embodiments, the polynucleotide sequence encoding the XAL polypeptide is codon-optimized according to the host cell. Preferred codons used in bacteria are typically used for expression in bacteria. Thus, the codon-optimized polynucleotide encoding the recombinant XAL polypeptide contains preferred codons at approximately 45%, 55%, 65%, 75%, 85%, or greater than 95% of the codon positions in the entire coding region.

[0042] In some embodiments, the polynucleotide encoding the recombinant XAL polypeptide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs. 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21. In some embodiments, the polynucleotide encoding the recombinant XAL polypeptide has at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotide residue identity to SEQ ID NOs. 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21.

[0043] In some embodiments, any recombinant XAL polynucleotide encoding the present invention is manipulated in various ways to promote expression of a XAL polypeptide. In some embodiments, a polynucleotide encoding a XAL polypeptide comprises an expression vector in which one or more control sequences are present to regulate expression of the XAL polynucleotide and / or polypeptide. Depending on the expression vector utilized, manipulation of the isolated polynucleotide prior to insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art.

[0044] In some embodiments, control sequences include a promoter, a polyadenylation sequence, a leader peptide sequence, a signal peptide sequence, and a transcription terminator, among others.

[0045] In some embodiments, suitable promoters and leader peptide sequences are selected based on the host cell. For bacterial host cells, suitable promoters for directing the present disclosure include, but are not limited to, promoters obtained from the following: the Escherichia coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus licheniformis α-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens α-amylase gene (amyQ), the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and prokaryotic β-lactamase genes, and the tac promoter. Exemplary promoters for filamentous fungal host cells include, but are not limited to, promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline proteinase, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium oxysporum trypsin-like protease promoters, and mutant, truncated, and hybrid promoters thereof.

[0046] In some embodiments, a terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the XAL polypeptide. Any suitable terminator that is functional in the host cell of choice may be used in the present invention.

[0047] In some embodiments, the leader sequence is operably linked to a nucleic acid sequence encoding a XAL polypeptide.

[0048] Any suitable leader sequence that is functional in the host cell of choice may be used in the present invention.

[0049] In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide.

[0050] In some embodiments, the control sequence is also a propeptide coding region encoding an amino acid sequence located at the amino terminus of the polypeptide. The resulting polypeptide is referred to as a "propolypeptide." The propeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region can be obtained from any suitable source, including but not limited to the following genes: Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae α-factor.

[0051] In some embodiments, regulatory sequences are also utilized that facilitate the regulation of polypeptide expression relative to the growth of the host cell. Regulatory systems are systems that cause gene expression to be turned on or off in response to chemical or physical stimuli. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to, the lac, tac, and trp operator systems.

[0052] In some embodiments, the present invention relates to recombinant expression vectors comprising a promoter, a terminator, an origin of replication, a leader peptide, a polynucleotide encoding a recombinant XAL polypeptide, and, depending on the type of host into which the polynucleotide is to be introduced, one or more expression control regions. In some embodiments, the various nucleic acids and control sequences are ligated together to form a recombinant expression vector, which includes one or more restriction enzyme sites for insertion into or substitution of the nucleic acid sequence encoding the XAL polypeptide.

[0053] In some embodiments, the recombinant expression vector can be any suitable vector, such as a plasmid or a virus (the vector can be a linear plasmid or a closed circular plasmid), and the choice of the recombinant vector generally depends on the compatibility of the vector with the host cell to be transferred into the vector.

[0054] In some embodiments, the expression vector is an autonomously replicating vector, the replication of which is independent of chromosomal replication, such as a plasmid, a minichromosome, an artificial chromosome.In some embodiments, a single or more vectors or plasmids are utilized.

[0055] In some embodiments, recombinant XAL polypeptides disclosed herein can be obtained by subjecting a polynucleotide encoding a naturally occurring or recombinant XAL polypeptide to any suitable mutagenesis and / or directed evolution method known in the art and / or as described herein. In some embodiments, the directed evolution process includes mutagenic PCR, cassette mutagenesis, staggered extension procedures, in vitro recombination, and other methods to generate libraries of variants that can be expressed, screened, and assayed. Any suitable mutagenesis and directed evolution methods can be used in the present invention and are well known in the art.

[0056] Mutagenesis and directed evolution methods can be readily applied to polynucleotides encoding XAL to generate libraries of variants that can be expressed, screened, and assayed. Any suitable mutagenesis and directed evolution methods can be used in the present invention and are well known in the art.

[0057] In some embodiments, mutants obtained after mutagenesis are screened by subjecting them to specified assay conditions and measuring the amount of enzyme activity remaining after heat treatment, acid treatment, or other suitable assay conditions. Clones containing polynucleotides encoding XAL polypeptides are then sequenced and used for recombinant polypeptide expression. Enzymes in the mutant library can be analyzed using any suitable method known in the art, such as HPLC.

[0058] In some embodiments, the protein purification method used is any one or more of the well-known techniques, including sonication, filtration, salting out, ultracentrifugation, and chromatography, among others, to recover the recombinant XAL polypeptide expressed in the host cells from the cells and / or culture medium.

[0059] In some embodiments, the recombinant XAL polypeptide is produced in a host cell by a method comprising culturing a host cell comprising a polynucleotide sequence encoding a recombinant XAL polypeptide described herein in a culture medium and under conditions conducive to production of the recombinant XAL polypeptide, and recovering the recombinant XAL polypeptide from the cell and / or culture medium.

[0060] In some embodiments, the present invention provides a method for producing a recombinant XAL polypeptide, the method comprising culturing a recombinant cell comprising a polynucleotide sequence encoding a recombinant XAL polypeptide under suitable culture conditions to produce the recombinant XAL polypeptide, and optionally recovering the recombinant XAL polypeptide from the culture and / or cultured bacterial cells, wherein the recombinant XAL polypeptide has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a reference sequence of SEQ ID NO. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and one or more amino acid residue differences compared to SEQ ID NO. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 as provided herein. In some embodiments, the host cell produces more than one recombinant XAL polypeptide.

[0061] In some embodiments, after the recombinant XAL polypeptides are recovered from the recombinant host cells and / or culture medium, they are further purified by any suitable method known in the art. In some other embodiments, the purified XAL polypeptides are combined with other ingredients to provide compositions and formulations comprising the recombinant XAL polypeptides, such as pharmaceutical compositions, for various applications and uses, as appropriate.

[0062] In some embodiments, compositions comprising the recombinant XAL polypeptides of the present invention include one or more commonly used carrier compounds, including, but not limited to, sugars, starches, celluloses, gums, and / or proteins. Additional components in oral formulations may include colorants and / or sweeteners and lubricants, as well as enteric coatings. In some embodiments, disintegrants or solubilizers are included. In some embodiments, particularly in liquid formulations, the recombinant XAL polypeptides are formulated with various additional components, including, but not limited to, preservatives, thickeners, humectants, alcohols, suspending agents, fatty acids, and / or emulsifiers.

[0063] In some embodiments, XAL recombinant protein can be used as a therapeutic protein for the treatment of phenylketonuria, tyrosinemia type I, tyrosinemia type II, tyrosinemia type III, and alkaptonuria. If patients with phenylalanine and tyrosine metabolism disorders are not treated early, high levels of phenylalanine, tyrosine, and some of their breakdown products can lead to significant medical problems, including liver and kidney failure, liver cancer, intellectual disability, and even death.

[0064] In some embodiments, the present invention provides recombinant XAL polypeptides suitable for reducing phenylalanine and tyrosine concentrations in the blood, cerebrospinal fluid, and the like of patients with phenylketonuria, tyrosinemia, and alkaptonuria. The dosage of recombinant XAL polypeptide administered to patients with elevated blood phenylalanine and tyrosine levels depends on the patient's genotype, condition, and other factors known to those skilled in the art. In some embodiments, the compositions are intended for single or repeated administration to patients with phenylketonuria, tyrosinemia, and alkaptonuria.

[0065] In some embodiments, the concentration of the recombinant XAL polypeptide in the composition administered to the patient is sufficient to effectively treat, ameliorate, and / or prevent symptoms of a disease (e.g., type I, type II, or type III tyrosinemia or alkaptonuria, disease, and / or symptoms). In some embodiments, the recombinant XAL polypeptide is administered to a patient with phenylketonuria, tyrosinemia, or alkaptonuria in combination with nitisinone or other pharmaceutical and / or dietary compositions.

[0066] Specifically:

[0067] A recombinant polypeptide having aromatic amino acid degrading enzyme (XAL) activity, the recombinant polypeptide comprising: a) an amino acid sequence having at least 85% sequence homology with a reference sequence of SEQ ID NO. 2 or a functional fragment thereof; b) a polypeptide sequence comprising at least one amino acid residue difference at one or more amino acid positions compared to SEQ ID NO. 2 or a functional fragment thereof; and c) the recombinant polypeptide exhibits an improved property selected from the following compared to the reference sequence of SEQ ID NO. 2: i) enhanced catalytic activity, ii) reduced sensitivity to proteolysis, iii) increased tolerance to acidic pH, or any combination of i), ii), and iii).

[0068] Further, wherein the one or more amino acid positions are selected from 5, 48, 61, 62, 67, 81, 91, 93, 122, 127, 141, 147, 167, 170, 180, 183, 184, 199, 202, 203, 204, 207, 210, 213, 225, 241, 250, 297, 322, 331, 341, 356, 359, 370, 374, 382, ​​383, 384, 385 8, 394, 400, 409, 413, 417, 433, 468, 476, 480, 481, 482, 488, 500, 522, 524, 526, 537, 571, 573, 582, 585, 586, 590, 601, 624, 629, 677, 678, 684, 690, 707, 708, 722, 727, 729, 731 and / or any combination thereof, wherein the amino acid positions are numbered with reference to SEQ ID NO. 2.

[0069] Further, when optimally aligned with the polypeptide of SEQ ID NO. 2 compared to the amino acid residue difference selected from the following substitutions: I5M, F48V, G61R, K62N, D67H, R81H, N91S, E93D, F122I, D127A, L141M, G147D, A167T, S170C, S170Y, I180S, R183P, M184I, I199L, M202L, G203C, E204G, R207S, I210F, L213R, D225H, A241S, S250N, N297K, G322S, T331P, V341L, V356E, E359V, E370V, D374A, A382T, Q 383K, F384S, Q388H, S394G, T400P, N409T, D413E, G417S, A433T, L468I, N4 76T, N476K, K480E, G481D, V482A, A488S, V500F, A522V, Y524H, I526S, A53 7V, H571Y, P573S, L582P, I585L, I585T, L586P, I590F, D601G, D624Y, S629 C, Y677D, E678D, L684M, G690S, I707V, I708F, G722A, V727F, M729I, L731V.

[0070] Further, the recombinant polypeptide has at least 85% sequence identity to SEQ ID NO. 2; and at positions I5 or F48, G61, K62, D67, R81, N91, E93, F122, D127, L141, G147, A167, S170, I180, R183, M184, I199, M202, G203, E204, R207, I210, L213, D225, A241, S250, N297, G322, T331, V341, V356, E359, E370, D374, A382, F The amino acid residue differences are at 384, Q388, S394, T400, N409, D413, G417, A433, L468, N476, K480, G481, V482, A488, V500, A522, Y524, I526, A537, H571, P573, L582, I585, L586, I590, D601, D624, S629, Y677, E678, L684, G690, I707, I708, G722, V727, M729, and L731.

[0071] Furthermore, the amino acid residue differences are I5M or F48V, G61R, K62N, D67H, R81H, N91S, E93D, F122I, D127A, L141M, G147D, A167T, S170C, S170Y, I180S, R183P, M184I, I199L, M202L, G203C, E204G, R207S, I210F, L 213R, D225H, A241S, S250N, N297K, G322S, T331P, V341L, V356E, E359V, E370V, D374A, A382T, Q383K, F384S, Q388H, S394G, T400P, N 409T, D413E, G417S, A433T, L468I, N476T, N476K, K480E, G481D, V482A, A488S, V500F, A522V, Y524H, I526S, A537V, H571Y, P573S, L582P, I585L, I585T, L586P, I590F, D601G, D624Y, S629C, Y677D, E678D, L684M, G690S, I707V, I708F, G722A, V727F, M729I, L731V.

[0072] Furthermore, the improved property is selected from the group consisting of enhancing the ability to degrade phenylalanine and / or tyrosine.

[0073] Furthermore, the reference sequence is a wild-type PcXAL derived from Phanerochaete chrysosporium.

[0074] Furthermore, the recombinant polypeptide comprises an amino acid sequence having at least about 90% sequence identity with SEQ ID NO. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or a functional fragment thereof.

[0075] Furthermore, a protein tag is fused to the N-terminus of the amino acid sequence of the recombinant polypeptide.

[0076] Furthermore, the protein tag is selected from 6★His tag, HA tag, MBP tag, SUMO tag, Myc tag, GST tag, Flag tag or GroE tag, etc. Preferably, the nucleotide sequence thereof is sequence optimized.

[0077] A polynucleotide sequence encoding at least one recombinant polypeptide as described above.

[0078] Further, the sequence is operably linked to a control sequence.

[0079] Furthermore, the polynucleotide is codon-optimized.

[0080] An expression vector comprising at least one polynucleotide sequence as described above, preferably, and at least one control sequence.

[0081] Furthermore, the control sequence is a promoter, a terminator, a leader peptide, etc.

[0082] Furthermore, the promoter is a heterologous promoter.

[0083] A recombinant cell, comprising at least one of the above-mentioned polynucleotide sequences and / or the above-mentioned vector.

[0084] Furthermore, host cells of the recombinant cells include, but are not limited to, fungal cells, algal cells, insect cells, and plant cells. Suitable fungal host cells include, but are not limited to, Ascomycota, Basidiomycota, Deuteromycota, Zygomycota, and Imperfectomycetes.

[0085] Further, the recombinant cell is a yeast cell, including but not limited to cells of species of Candida, Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, and Yarrowia. In some embodiments of the present invention, the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Kluyveromyces, Schizosaccharomyces pombe, Pichia pastoris, Pichia membranaceus, Pichia stipitis, Pichia methanolica, Pichia angustifolia, Kluyveromyces lactis, Candida albicans, or Yarrowia lipolytica.

[0086] Further, the recombinant cell is a prokaryotic cell. Suitable prokaryotic cells include, but are not limited to, Gram-positive, Gram-negative, and Gram-variable bacteria. Any suitable bacterial organism can be used in the present invention, including but not limited to Agrobacterium, Alicyclobacillus, Anabaena, Histocystis, Acinetobacter, Thermus acidophilus, Arthrobacter, Azotobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera, Campylobacter, Clostridium, Corynebacterium, Chromobacterium, Coccoccus, Escherichia, Enterococcus, Enterobacter, Fusobacterium, Faecalibacterium, Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter, Gram Rabella, Lactobacillus, Lactococcus, Thuringia, Micrococcus, Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus, Saccharomonas, Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobacter, Thermosynechococcus, Thermococcus, Xanthomonas, Yersinia, Zymomonas.

[0087] Further, the recombinant cell is: Agrobacterium, Acinetobacter, Azotobacter, Bacillus, Bifidobacterium, Buchnera, Geobacillus, Campylobacter, Clostridium, Corynebacterium, Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus, Lactococcus, Pantoea, Pseudomonas, Staphylococcus, Salmonella, Streptococcus, Streptomyces or Zymomonas.

[0088] Furthermore, the recombinant cells are non-pathogenic to humans or industrial production strains and are suitable for use in the present invention. In some embodiments of the present invention, the bacterial host cell is an Agrobacterium species, such as Agrobacterium radiobacterium, Agrobacterium rhizogenes, or Agrobacterium rubus.

[0089] Further, the recombinant cell is a Bacillus species, such as Bacillus thuringiensis, Bacillus anthracis, Bacillus megaterium, Bacillus subtilis, Bacillus lentus, Bacillus circulans, Bacillus brevis, Bacillus laurens, Bacillus coagulans, Bacillus brevis, Bacillus firmus, Bacillus licheniformis, Bacillus clausii, Bacillus stearothermophilus, Bacillus alkalophilus, and Bacillus amyloliquefaciens.

[0090] Furthermore, the recombinant cell is a Clostridium species, such as Clostridium tetani, Clostridium côte d'Ivoire, Clostridium perfringens, or Clostridium beijerinckii.

[0091] Further, the recombinant cell is a Streptomyces species, such as Streptomyces dichromogenes, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces aureogenes, Streptomyces aureus, Streptomyces fungicidins, Streptomyces griseus, and Streptomyces lividans.

[0092] Furthermore, the recombinant cell is an Escherichia species, such as Escherichia coli.

[0093] Preferably, the host cell can be any probiotic bacteria.

[0094] A method for producing the above-mentioned mutant comprises the following steps: using a plasmid containing a wild-type encoding gene as a template, mutating the wild-type gene by site-directed mutagenesis to obtain a plasmid containing a mutant encoding gene, transferring the plasmid into a host cell, and culturing the recombinant cell to obtain the mutant.

[0095] Of course, those skilled in the art will appreciate that the present invention also provides a method for improving the activity or stability of an aromatic amino acid degrading enzyme, using SEQ ID NO. 2 or its corresponding nucleotide sequence as a starting sequence, and performing the following steps on 5, 48, 61, 62, 67, 81, 91, 93, 122, 127, 141, 147, 167, 170, 180, 183, 184, 199, 202, 203, 204, 207, 210, 213, 225, 241, 250, 297, 322, 331, 341, 356, 359, 370, 374, 382, ​​383, 384. 4, 388, 394, 400, 409, 413, 417, 433, 468, 476, 480, 481, 482, 488, 500, 522, 524, 526, 537, 571, 573, 582, 585, 586, 590, 601, 624, 629, 677, 678, 684, 690, 707, 708, 722, 727, 729, 731, or a combination thereof.

[0096] Furthermore, the activity is amino acid degradation activity, and preferably, the amino acid is phenylalanine and / or tyrosine.

[0097] Furthermore, the stability is one of the following or a combination thereof: enzyme stability (especially to enzymes contained in the stomach and intestine), thermal stability, and acid resistance stability.

[0098] The above-mentioned recombinant polypeptide and / or its composition, recombinant polypeptide encoding gene and / or its composition, recombinant vector containing recombinant polypeptide and / or its composition, recombinant cell and / or its composition are used to treat phenylketonuria and / or tyrosinemia.

[0099] Furthermore, it is suitable for treating elevated blood levels of phenylalanine and / or tyrosine.

[0100] Furthermore, the medicine prepared from the above substances is suitable for oral administration.

[0101] Furthermore, the drug may be in the form of tablets, capsules, oral liquid preparations, granules and pellets.

[0102] Furthermore, the tablets and capsules should also contain an enteric coating.

[0103] Furthermore, the above drugs can be used alone or co-administered with another compound having a therapeutic effect.

[0104] A pharmaceutical composition comprising at least one of the above-mentioned ingredients having therapeutic effects.

[0105] Further, after providing the composition to a subject, the concentration of phenylalanine and / or tyrosine in the blood of the subject is reduced.

[0106] Furthermore, after administration of the pharmaceutical composition, the symptoms of phenylketonuria and / or hypertyrosinemia are improved.

[0107] Furthermore, the subject is able to consume a diet that is less restricted in phenylalanine and tyrosine content than would be required of a subject who is not provided with at least one pharmaceutical composition comprising at least one recombinant polypeptide component, and / or recombinant cellular component.

[0108] Beneficial effects of the present invention:

[0109] The present invention provides a recombinant aromatic amino acid degrading enzyme polypeptide. Compared with the unmodified polypeptide, the recombinant polypeptide has multiple effects such as increased enzyme activity, improved thermal stability, improved enzyme stability, and improved acid tolerance. It not only significantly improves the amino acid degradation effect, but also increases the stability in storage and application. Based on this characteristic, the recombinant polypeptide or its composition is more effective in the treatment of phenylketonuria or tyrosinemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Figure 1 shows the results of blood phenylalanine level detection in model mice after oral administration of different cells. DETAILED DESCRIPTION

[0111] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0112] The scheme that the present invention relates to is as follows:

[0113] The present invention provides recombinant polypeptides comprising at least one substitution or set of substitutions at one or more amino acid positions selected from the group consisting of: 5, 48, 61, 62, 67, 81, 91, 93, 122, 127, 141, 147, 167, 170, 180, 183, 184, 199, 202, 203, 204, 207, 210, 213, 225, 241, 250, 297, 322, 331, 341, 356, 359, 37 0, 374, 382, ​​383, 384, 388, 394, 400, 409, 413, 417, 433, 468, 476, 480, 481, 482, 488, 500, 522, 524, 526, 537, 571, 573, 582, 585, 586, 590, 601, 624, 629, 677, 678, 684, 690, 707, 708, 722, 727, 729, 731, wherein the amino acid positions refer to SEQ ID NO.

[0114] In some embodiments, the recombinant polypeptide is a mutant of an aromatic amino acid degrading enzyme derived from Phanerochaete chrysosporium. In some further embodiments, the recombinant polypeptide of the present invention exhibits greater aromatic amino acid degrading activity than the wild-type PcXAL polypeptide. In some further embodiments, the recombinant polypeptide of the present invention is more thermostable than the wild-type PcXAL polypeptide. In some further embodiments, the recombinant polypeptide is more resistant to proteolysis than the wild-type PcXAL polypeptide. In still other embodiments, the recombinant polypeptide is more resistant to proteolysis by at least one digestive tract enzyme than the wild-type PcXAL polypeptide. In still other embodiments, the recombinant polypeptide is more acid-stable than the wild-type PcXAL polypeptide.

[0115] The present invention also provides a recombinant polynucleotide sequence encoding at least one recombinant polypeptide provided herein. In some embodiments, the recombinant polynucleotide encodes at least one recombinant polypeptide provided herein. In some further embodiments, the recombinant polynucleotide encodes at least one recombinant phenylalanine ammonia lyase polypeptide provided herein. In some further embodiments, the recombinant polynucleotide sequence is codon-optimized.

[0116] The present invention provides at least one protein tag. In some embodiments, the recombinant aromatic amino acid lyase carries any protein tag. In some embodiments, the recombinant aromatic amino acid lyase expresses a protein tag fused to its N-terminus. In some embodiments, the protein tag is SUMO, GST, or GroE. In other embodiments, the addition of a protein tag can increase protein expression and activity.

[0117] The present invention also provides an expression vector comprising at least one recombinant polynucleotide sequence provided herein. In some embodiments, the expression vector carries the recombinant polynucleotide sequence provided herein. In some other embodiments, the expression vector further comprises at least one control sequence. In some other embodiments, the control sequence comprises a promoter. In some embodiments, the promoter is a heterologous promoter.

[0118] The present invention also provides host cells transformed with at least one polynucleotide sequence provided herein.In some embodiments, the host cell is transformed with a polynucleotide sequence provided herein.

[0119] In some embodiments, the dosage form can be a pill, tablet, capsule, gelatin, liquid or emulsion. In some embodiments, the pill, tablet, capsule or gelatin further comprises an enteric coating. In some embodiments, the composition is suitable for oral and / or injection administration. In yet other embodiments, the composition can be administered alone and / or co-administered with other therapeutically effective compounds.

[0120] The present invention also provides the use of any of the compositions provided herein, the compositions comprising at least one recombinant polypeptide and / or recombinant cell. In some embodiments, any composition provided herein, alone or in any combination. It is not intended that the present invention be limited to any particular use.

[0121] The methods used in the following examples are:

[0122] (1) Construction of recombinant aromatic amino acid degrading enzyme mutants

[0123] Recombinant XAL polypeptides having the properties disclosed herein can be obtained by subjecting a polynucleotide encoding a naturally occurring or recombinant XAL polypeptide to any suitable mutagenesis and / or directed evolution method known in the art and / or as described herein. Exemplary directed evolution techniques are mutagenesis and / or DNA shuffling. Other directed evolution procedures that may also be used include mutagenic PCR, cassette mutagenesis, staggered extension procedures, in vitro recombination, and other approaches. Mutagenesis and directed evolution methods can be readily applied to polynucleotides encoding XAL to generate libraries of variants that can be expressed, screened, and assayed. Any suitable mutagenesis and directed evolution methods can be used in the present invention and are well known in the art.

[0124] (2) Amino acid detection method

[0125] Tyrosine detection method: Tyrosine was used as the reaction substrate. By detecting the reduction of tyrosine in the reaction system, a liquid chromatography detection method was established and optimized. The detection was performed using a Shimadzu LC-2050C high-performance liquid chromatography. The detection conditions and parameters are as follows:

[0126] Chromatographic column: SHIMADZU C18 column (250 mm × 4.6 mm, 5 μm);

[0127] Mobile phase: 1% acetic acid and acetonitrile;

[0128] Column temperature: 40°C;

[0129] Flow rate: 0.8 mL / min

[0130] Gradient elution:

[0131] 0-8 min, 1.5% acetic acid-acetonitrile (95:5) gradually changed to 1.5% acetic acid-acetonitrile (0:100);

[0132] 8-13 minutes, maintain 1.5% acetic acid-acetonitrile (0:100)

[0133] 13-14 minutes, 1.5% acetic acid-acetonitrile (0:100) gradually changed to 1.5% acetic acid-acetonitrile (95:5);

[0134] 14-23 minutes, maintain 1.5% acetic acid-acetonitrile (95:5)

[0135] Detection: UV, wavelength 280nm

[0136] Injection volume: 10 μL

[0137] Phenylalanine detection method: Phenylalanine was used as the reaction substrate. By detecting the reduction of phenylalanine in the reaction system, a liquid chromatography detection method was established and optimized. This method can achieve the simultaneous detection of phenylalanine and cinnamic acid. The detection was performed using a Shimadzu LC-2050C high-performance liquid chromatography. The detection conditions and parameters are as follows:

[0138] Chromatographic column: Eclipse XDB-C18 column (250 mm × 4.6 mm, 5 μm);

[0139] Mobile phase: 1% acetic acid and acetonitrile;

[0140] Column temperature: 40°C;

[0141] Flow rate: 0.8 mL / min;

[0142] Gradient elution:

[0143] 0-8 min, 1% acetic acid-acetonitrile (95:5) gradually changed to 1.5% acetic acid-acetonitrile (0:100);

[0144] 8-13 minutes, maintain 1% acetic acid-acetonitrile (0:100);

[0145] 13-14 minutes, 1% acetic acid-acetonitrile (0:100) gradually changed to 1.5% acetic acid-acetonitrile (95:5);

[0146] 14-23 minutes, maintain 1% acetic acid-acetonitrile (95:5).

[0147] Detection: UV, wavelength 260nm;

[0148] Injection volume: 10 μL.

[0149] (3) The materials involved in the following embodiments are as follows:

[0150] SOC medium: 2% tryptone, 0.5% yeast extract, 0.05% NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM D-glucose, adjusted to pH 7.5.

[0151] Artificial intestinal fluid and artificial gastric fluid were purchased from Nanjing Yuanzhixin Biotechnology Co., Ltd.

[0152] Example 1 Acquisition of PcXAL gene and construction of mutation library

[0153] The codon-optimized Phanerochaete chrysosporium aromatic amino acid degrading enzyme (PcXAL) gene sequence (SEQ ID NO. 1) for Enterobacterial expression was synthesized and cloned into the E. coli expression vector pGEX4T to provide pGEX4T-PcXAL. The plasmid was then transformed into E. coli strain EcN1917. A library of gene variants was generated from this plasmid construct using directed evolution techniques commonly known to those skilled in the art.

[0154] Example 2 High-throughput culture of recombinant polypeptides

[0155] Transformed E. coli cells were plated onto SOC screening plates for culture. After overnight incubation at 37°C, colonies were picked and cultured in 96-well deep-well plates containing 500 μl of SOC liquid screening medium. The cells were cultured in a shaker at 200 rpm and 30°C for 2-3 hours. The cells were then induced with 40 μL of 10 mM IPTG in sterile water and cultured overnight in a shaker at 200 rpm and 30°C for 12-15 hours. The next day, the cells were harvested at 4000 rpm for 20 minutes, the supernatant discarded, and the cells frozen at -80°C prior to analysis.

[0156] Example 3 Whole cell disruption and purification of recombinant polypeptides

[0157] A volume of lysis buffer is added to the cell pellet, mixed by inversion, and incubated at room temperature for 1-2 hours. The supernatant is then centrifuged at 4000 rpm for 20 minutes. The supernatant is analyzed by SDS-PAGE to confirm the correct band size, and protein purification is performed using FPLC. In some embodiments, larger amounts of cells are disrupted using methods such as ultrasonication, which are not listed here.

[0158] Example 4 Activity detection of whole cells expressing recombinant polypeptides

[0159] The activity of the engineered polypeptide was determined by measuring the formation of trans-cinnamic acid (phenylalanine product) and p-coumaric acid (tyrosine product). 10 CFU) were mixed with PBS (pH 7.0) containing 50 mM phenylalanine / tyrosine by vortexing (reaction volume: 2 mL). After incubation for a period of time, the cells were assayed by HPLC. The results are shown in Table 1. In some embodiments, the activity of various recombinant cells expressing recombinant polypeptides was assayed using different concentrations of phenylalanine / tyrosine as substrates, which are not listed here (negative mutations generated in high-throughput screening are not reflected in this article, the same applies below).

[0160] In Table 1, the activities of the recombinant cells expressing the original PcXAL polypeptide are compared with those of the whole cells, and are defined as follows: "★" = phenylalanine and tyrosine degradation activities increased by more than 0.95 times but less than 1 times; "★★" = phenylalanine and tyrosine degradation activities increased by more than 1 times but less than 1.25 times; "★★★" = phenylalanine and tyrosine degradation activities increased by more than 1.25 times.

[0161] Example 5 Recombinant polypeptide activity detection

[0162] The activity of the recombinant polypeptides was determined by measuring the formation of trans-cinnamic acid (phenylalanine product) and p-coumaric acid (tyrosine product). 0.5 mg of the recombinant polypeptide was mixed with PBS (pH 7.0) containing 50 mM phenylalanine / tyrosine by vortexing (reaction volume: 2 mL). After incubation, the mixture was assayed by HPLC. The results are shown in Table 2. In some embodiments, the degradation activities of various recombinant polypeptides were assayed using different concentrations of phenylalanine / tyrosine as substrates, which are not listed here.

[0163] In Table 2, the activities of the original PcXAL polypeptide are compared and defined as follows: "★" = phenylalanine and tyrosine degradation activities increased by more than 0.95 times but less than 1 times; "★★" = phenylalanine and tyrosine degradation activities increased by more than 1 times but less than 1.25 times; "★★★" = phenylalanine and tyrosine degradation activities increased by more than 1.25 times.

[0164] Example 6 Detection of protease sensitivity of recombinant polypeptides

[0165] Artificial intestinal fluid contains chymotrypsin, trypsin, sodium phosphate, sodium taurocholate, and calcium chloride to simulate the intestinal environment. After mixing the recombinant polypeptide with artificial intestinal fluid (50 mM phenylalanine / tyrosine, reaction volume 2 mL), incubation was performed for a period of time, and the activity was tested using HPLC. The results are shown in Table 3. In some embodiments, other proteases were used to test the protease sensitivity of various recombinant polypeptides, which are not listed here one by one.

[0166] In Table 3, the activities of the PcXAL polypeptides after treatment with artificial intestinal fluid for 0.5 hours and 1 hour, respectively, were compared with those of the original PcXAL polypeptide and are defined as follows: "★" = phenylalanine and tyrosine degradation activities increased by more than 0.95 times but less than 1 times; "★★" = phenylalanine and tyrosine degradation activities increased by more than 1 times but less than 1.25 times; "★★★" = phenylalanine and tyrosine degradation activities increased by more than 1.25 times.

[0167] Example 7 Detection of Thermal Stability of Recombinant Polypeptides

[0168] The high-throughput growth recombinant polypeptide lysate was incubated at 55°C for 0.5-1 hour, centrifuged, and the supernatant was transferred to a 96-well microtiter plate, frozen, and lyophilized into a dry powder. The lyophilized enzyme powder was incubated in a shaker at 50°C for up to 7 days. Thereafter, the lyophilized enzyme was shaken and mixed in 2 mL of PBS (pH 7.0) containing 50 mM phenylalanine / tyrosine. After incubation for a period of time, its activity was tested using HPLC. The results are shown in Table 4. In some embodiments, the thermal stability of various recombinant polypeptides and the recombinant polypeptides at different temperatures were tested, which are not listed here one by one.

[0169] In Table 4, the activities of the enzymes treated at 55°C for 0.5 hours and 1 hour, respectively, and then prepared into lyophilized enzyme powder and incubated at 50°C for 7 days were compared with those of the original PcXAL polypeptide, and are defined as follows: "★" = phenylalanine and tyrosine degradation activities increased by more than 0.95 times but less than 1 times; "★★" = phenylalanine and tyrosine degradation activities increased by more than 1 times but less than 1.25 times; "★★★" = phenylalanine and tyrosine degradation activities increased by more than 1.25 times.

[0170] Example 8 Detection of Acid Tolerance of Recombinant Polypeptides

[0171] The pH of artificial gastric fluid is 2.5. A PBS solution containing the recombinant polypeptide was mixed with artificial intestinal fluid at a ratio of 1:1 to simulate a feeding environment (50 mM phenylalanine / tyrosine in the presence of 2 mL). After incubation for a period of time, the activity was tested using HPLC. The results are shown in Table 5. In some embodiments, various recombinant polypeptides and their tolerance to different pH levels were tested, which are not listed here.

[0172] In Table 5, the activities of the recombinant peptides were compared with those of the original PcXAL peptide after a PBS solution was mixed with artificial intestinal fluid at a ratio of 1:1 and incubated for 0.5 and 1 hour, respectively, and were defined as follows: "★" = phenylalanine and tyrosine degradation activities increased by more than 0.95 times but less than 1 times; "★★" = phenylalanine and tyrosine degradation activities increased by more than 1 times but less than 1.25 times; "★★★" = phenylalanine and tyrosine degradation activities increased by more than 1.25 times.

[0173] Example 9 Detection of phenylalanine levels in blood of model mice

[0174] The host cells, recombinant cells expressing the original PcXAL polypeptide, and recombinant cells expressing the recombinant polypeptide were administered orally to mice with phenylalanine metabolism dysfunction. Phenylalanine levels in the blood of the model mice were measured after a period of time. The degradation activity of recombinant E. coli expressing the optimal mutant 14 is shown in Figure 1. In some embodiments, phenylalanine levels in the blood of mice were measured after oral administration of various recombinant cells expressing the recombinant polypeptide, which are not listed here.

[0175] Figure 1 shows A as a single dose and B as continuous dosing. In a phenylketonuria mouse model (PKU mouse), both single and continuous administration of recombinant cells expressing the original PcXAL polypeptide and recombinant cells expressing the recombinant polypeptide inhibited phenylalanine absorption after a single oral dose in PKU mice, reducing blood phenylalanine levels. Compared to the blank control group, the inhibition rate exceeded 30% two hours after oral administration of phenylalanine, and the effect persisted for at least four hours. The recombinant cells expressing the recombinant polypeptide demonstrated superior efficacy compared to those expressing the original PcXAL polypeptide. These results demonstrate that oral administration of recombinant cells expressing the recombinant polypeptide effectively inhibits phenylalanine absorption into the bloodstream through the digestive tract in the PKU mouse model, thereby reducing the elevated blood phenylalanine levels following oral administration and achieving the goal of treating phenylketonuria.

[0176] We also administered recombinant cells and tyrosine to a PKU mouse model with tyrosine metabolism dysfunction. After a period of time, we measured the tyrosine levels in the blood of the model mice and found that the degree of tyrosine degradation in the blood by recombinant E. coli expressing mutant 14 was much higher than that by recombinant cells expressing the original PcXAL polypeptide, with a statistically significant difference.

[0177] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A recombinant polypeptide expressing an aromatic amino acid degrading enzyme, characterized in that, The aromatic amino acid degrading enzyme is obtained by mutating the amino acid sequence shown in SEQ ID NO.1, and the mutated sites are any one or a combination of the following: 5, 48, 61, 62, 67, 81, 91, 93, 122, 127, 141, 147, 167, 170, 180, 183, 184, 199, 202, 203, 204, 207, 210, 213, 225, 241, 250, 297, 322, 331, 341, 356, 359, 370, 374, 382, 383, 384, 388, 394, 400, 409, 413, 417, 433, 468, 476, 480, 481, 482, 488, 500, 522, 524, 526, 537, 571, 573, 582, 585, 586, 590, 601, 624, 629, 677, 678, 684, 690, 707, 708, 722, 727, 729, 731.

2. The recombinant polypeptide according to claim 1, wherein The mutation is any one or a combination of the following: I5M, F48V, G61R, K62N, D67H, R81H, N91S, E93D, F122I, D127A, L141M, G147D, A167T, S170C, S170Y, I180S, R183P, M184I, I199L, M202L, G203C, E204G, R207S, I210F, L213R, D225H, A241S, S250N, N297K, G322S, T331P, V341L, V356E, E359V, E370V, D374A, A382T, Q383K, F384S, Q388H, S394G, T400P, N409T, D413E, G417S, A433T, L468I, N476T, N476K, K480E, G481D, V482A, A488S, V500F, A522V, Y524H, I526S, A537V, H571Y, P573S, L582P, I585L, I585T, L586P, I590F, D601G, D624Y, S629C, Y677D, E678D, L684M, G690S, I707V, I708F, G722A, V727F, M729I, L731V.

3. The recombinant polypeptide according to claim 1, characterized in that, The mutation is any one or a combination of the following: (1) Containing mutations of F48V, I210F, D374A, F384S, S394G, A522V, I526S, I585L, L586P, D601G, or a sequence with a homology of not less than 90% to the mutated sequence; (2) Comprising K62N, E93D, S250N, E359V, E370V, N409T, L468I, D624Y, G690S, I708F mutations, or a sequence with a homology of not less than 90% to the mutated sequence; (3) Comprising G147D, A382T, Q383K, A433T, L582P, E678D mutations, or a sequence with a homology of not less than 90% to the mutated sequence; (4) Comprising D127A, A167T, S170C, I199L, E204G, T400P, A488S mutations, or a sequence with a homology of not less than 90% to the mutated sequence; (5) Comprising D67H, M202L, D413E, H571Y, P573S, I590F mutations, or a sequence with a homology of not less than 90% to the mutated sequence; (6) Comprising I5M, L141M, S170Y, R183P, G417S, V482A, V500F, Y524H mutations, or a sequence with a homology of not less than 90% to the mutated sequence; (7) Comprising N476T, I180S, L213R, G322S, V341L, V356E, G722A, V727F mutations, or a sequence with a homology of not less than 90% to the mutated sequence; (8) Comprising N91S, F122I, R207S, D225H, N476K, A537V, Y677D, L684M, L731V mutations, or a sequence with a homology of not less than 90% to the mutated sequence; (9) Comprising R81H, A241S, N297K, K480E, G481D, I707V, M729I mutations, or a sequence with a homology of not less than 90% to the mutated sequence; (10) Comprising G61R, M184I, G203C, T331P, Q388H, I585T, S629C mutations, or a sequence with a homology of not less than 90% to the mutated sequence.

4. The recombinant polypeptide according to claim 1, wherein The mutant enzyme exhibits at least one of the following characteristics relative to the wild-type enzyme: (1) Improved amino acid degradation activity; (2) Improved enzyme stability; (3) Improved thermal stability; (4) Improved acid tolerance.

5. The recombinant polypeptide according to claim 1, characterized in that, An amino acid sequence of an aromatic amino acid degrading enzyme is fused with an expression protein tag at the N-terminus.

6. A polynucleotide encoding the recombinant polypeptide according to any one of claims 1-5.

7. An expression vector carrying the polynucleotide according to claim 6.

8. The expression vector according to claim 7, characterized in that, The expression vector contains at least one control sequence.

9. The expression vector according to claim 8, wherein The control sequence includes at least one of a promoter, a terminator, a signal peptide or a leader peptide.

10. A recombinant cell comprising the recombinant polypeptide according to any one of claims 1-5, the polynucleotide according to claim 6, or the expression vector according to any one of claims 7-9.

11. Use of the recombinant polypeptide according to any one of claims 1-5, the polynucleotide according to claim 6, the expression vector according to any one of claims 7-9, or the recombinant cell according to claim 10 in the preparation of a product for tyrosine and / or phenylalanine degradation. Use of the recombinant polypeptide according to any one of claims 1-5, the polynucleotide according to claim 6, the expression vector according to any one of claims 7-9, or the recombinant cell according to claim 10 in the preparation of a prophylactic or therapeutic drug for phenylketonuria and / or tyrosinemia.

13. A pharmaceutical composition, characterized in that, Containing the recombinant polypeptide according to any one of claims 1-5, the polynucleotide according to claim 6, the expression vector according to any one of claims 7-9, or the recombinant cell according to claim 10.

14. The pharmaceutical composition according to claim 13, wherein, The pharmaceutical composition contains a pharmaceutically acceptable carrier.

15. The pharmaceutical composition according to claim 13, wherein, Orally administer the pharmaceutical composition.

16. A method for improving the activity or stability of aromatic amino acid degrading enzymes, characterized in that, Comprising the following steps: mutating the amino acid sequence set forth in SEQ ID NO.2, said mutation being a mutation at any one site or a combination thereof among positions 5, 48, 61, 62, 67, 81, 91, 93, 122, 127, 141, 147, 167, 170, 180, 183, 184, 199, 202, 203, 204, 207, 210, 213, 225, 241, 250, 297, 322, 331, 341, 356, 359, 370, 374, 382, 383, 384, 388, 394, 400, 409, 413, 417, 433, 468, 476, 480, 481, 482, 488, 500, 522, 524, 526, 537, 571, 573, 582, 585, 586, 590, 601, 624, 629, 677, 678, 684, 690, 707, 708, 722, 727, 729, 731.

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