CN113969292A

CN113969292A - Engineering probiotics for the treatment of phenylketonuria and its construction method and application

Info

Publication number: CN113969292A
Application number: CN202111347505.1A
Authority: CN
Inventors: 付加芳; 曹广祥; 马欣; 王杰; 林赓
Original assignee: Shandong First Medical University and Shandong Academy of Medical Sciences
Current assignee: Shandong First Medical University and Shandong Academy of Medical Sciences
Priority date: 2021-11-15
Filing date: 2021-11-15
Publication date: 2022-01-25
Anticipated expiration: 2041-11-15
Also published as: CN113969292B

Abstract

The invention relates to the field of medicine and biotechnology, in particular to engineering probiotics for treating phenylketonuria and a construction method and application thereof; the recombinant expression vector comprises a recombinant expression vector I and a recombinant expression vector II; the first recombinant expression vector is an expression vector containing a phenylalanine ammonia lyase gene PAL nucleotide sequence, a protein secretion processing structural domain coding gene prtP nucleotide sequence and a lactobacillus acidophilus constitutive promoter SL; the second recombinant expression vector is an expression vector containing a phenylalanine ammonia lyase gene PAL nucleotide sequence, a phenylalanine transfer pump gene pheP and a lactobacillus acidophilus constitutive promoter SL; the engineering probiotics which can secrete phenylalanine ammonia lyase to the outside of the bacteria body in the gastrointestinal tract is constructed for the first time, so that the phenylalanine in the digested food in the gastrointestinal tract is effectively degraded, the phenylalanine intake of a patient is reduced, and the method is used for treating PKU.

Description

Engineering probiotics for treating phenylketonuria and construction method and application thereof

Technical Field

The invention relates to the field of medicines and biotechnology, in particular to engineering probiotics for treating phenylketonuria and a construction method and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

PKU is a congenital phenylalanine metabolic disorder disease, and the only clinical therapy aiming at phenylketonuria patients at present is to adopt a low-phenylalanine PA diet strategy, feed low-PA milk powder in infancy, follow a low-protein diet in infancy and adult stages and control the intake of phenylalanine; however, strict low-protein diet therapy strategies have great side effects on adolescent development and adults, influence the physical and psychological health of patients, and have poor quality of life and low compliance.

At present, there are some PKU treatment strategies at the experimental stage, for example, infecting mice with an adenovirus vector carrying phenylalanine hydroxylase PAH gene to restore mouse liver PAH activity, but the scheme has the general defect of gene therapy; the recombinant PAH is expressed by using escherichia coli, the blood PA concentration is reduced after oral administration, but the recombinant PAH protein is decomposed by protease in the digestive tract after oral administration. In addition, in the scheme of treating PKU by using escherichia coli engineering probiotics in the prior art, a phenylalanine ammonia lyase gene and a phenylalanine transport pump gene are integrated on an escherichia coli genome to construct escherichia coli engineering probiotics, a microbial inoculum is prepared and enters the intestinal tract of a patient through oral administration, phenylalanine is absorbed into the microbial inoculum from the intestinal tract by the engineering bacteria, and then PA is degraded under the action of phenylalanine ammonia lyase, so that the PA intake of the patient is reduced. However, the existing escherichia coli engineering bacteria for treating phenylketonuria are mainly planted in large intestines, and in addition, the existing escherichia coli engineering bacteria can degrade phenylalanine only by absorbing phenylalanine into bacteria, so that the degradation efficiency of the engineering bacteria on phenylalanine is limited.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide engineering probiotics for treating phenylketonuria and a construction method and application thereof.

In order to achieve the above object, the technical solution of the present invention is as follows:

in a first aspect of the present invention, there is provided a recombinant expression vector comprising a first recombinant expression vector and a second recombinant expression vector;

the first recombinant expression vector is an expression vector containing a phenylalanine ammonia lyase gene (PAL) nucleotide sequence, a protein secretion processing structural domain coding gene prtP nucleotide sequence and a lactobacillus acidophilus constitutive promoter SL;

the second recombinant expression vector is an expression vector containing a phenylalanine lyase gene PAL nucleotide sequence, a phenylalanine transport pump gene (pheP) and a lactobacillus acidophilus constitutive promoter SL.

Preferably, the phenylalanine ammonia lyase gene PAL is derived from Photorhabdus asymbycia ATCC43949, and the nucleotide sequence is shown as SEQ ID NO: 1 is shown.

Preferably, the protein secretion processing domain encoding gene prtP is derived from Lactcaseibacterium paracasei ATCC 334, and the nucleotide sequence is shown in SEQ ID NO: 2, respectively.

Preferably, the phenylalanine transport pump gene pheP is derived from Escherichia coli K-12, and the nucleotide sequence is shown in SEQ ID NO: 3, respectively.

Preferably, the Lactobacillus acidophilus constitutive promoter SL is derived from Lactobacillus acidophilus NCFM, and the nucleotide sequence is shown in SEQ ID NO: 4, respectively.

In a second aspect of the invention, there is provided an engineered probiotic for the treatment of phenylketonuria, said engineered probiotic being selected from lactobacillus acidophilus comprising a recombinant expression vector according to the first aspect.

In one or more embodiments of the present invention, a method for constructing the above engineered probiotics comprises the following steps:

(1) placing the protein secretion processing structural domain gene prtP and PAL gene, or phenylalanine transfer pump gene pheP and PAL gene in the downstream of lactobacillus acidophilus constitutive promoter SL;

(2) connecting the gene fragment to a pORI28 vector by adopting the conventional molecular biology technology;

(3) the lactobacillus acidophilus NCFM is used as a chassis bacterium, and the genes are integrated on a genome by a homologous recombination method to obtain a protein secretion processing structural domain gene prtP and PAL integrated strain or a gene PAL and gene pheP integrated strain.

In a third aspect of the present invention, there is provided a use of the recombinant expression vector of the first aspect or the engineered probiotic of the second aspect in the preparation of a medicament for the treatment of phenylketonuria.

In a fourth aspect of the present invention, there is provided a pharmaceutical composition comprising the engineered probiotic bacteria of the second aspect and a pharmaceutically acceptable excipient.

The specific embodiment of the invention has the following beneficial effects:

the invention constructs engineering probiotics which can secrete phenylalanine lyase to the outside of the bacteria in the gastrointestinal tract for the first time, effectively degrades phenylalanine in digested food in the gastrointestinal tract, utilizes the characteristic that lactobacillus acidophilus is mainly planted in the places of protein digestion and amino acid absorption, namely the stomach and the small intestine, effectively degrades phenylalanine in the food, reduces the phenylalanine intake of a patient, and can be used for treating PKU;

the phenylalanine lyase gene PAL from Photorhabdus asymbycica ATCC43949 has higher activity of the enzyme expressed in Lactobacillus acidophilus, and can effectively degrade phenylalanine, thereby achieving the purpose of treating phenylketonuria;

the phenylalanine ammonia lyase gene PAL from Photorhabdus asymbiotica ATCC43949 and the phenylalanine transfer pump gene pheP from Escherichia coli K-12 can enhance the absorption and degradation effects of engineering probiotics on phenylalanine in food and reduce the intake of phenylalanine of patients after being integrated into lactobacillus acidophilus;

the phenylalanine ammonia lyase gene PAL from Photorhabdus asymbiotica ATCC43949 and the protein secretion processing domain gene prtP from Lactcaseibacillus paracasei ATCC 334 are combined and integrated into lactobacillus acidophilus to obtain engineering probiotics which can secrete phenylalanine lyase to the outside of bacteria in the gastrointestinal tract, so that phenylalanine in digested food in the gastrointestinal tract is effectively degraded, the phenylalanine intake of a patient is reduced, and the pharmaceutical composition is used for treating PKU;

the engineering probiotics provided by the invention is beneficial to treating phenylketonuria patients, improves the life quality of the patients, and has important clinical treatment value and social benefit.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is the phenylalanine concentration in blood of PKU mice in example 1;

FIG. 2 is the phenylalanine concentration in blood of PKU mice in example 2.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

In one embodiment of the present invention, a recombinant expression vector is provided, comprising a first recombinant expression vector and a second recombinant expression vector;

the second recombinant expression vector is an expression vector containing a nucleotide sequence of phenylalanine ammonia lyase gene (PAL), a phenylalanine transport pump gene (pheP) and a constitutive promoter SL of lactobacillus acidophilus.

SEQ ID NO：1：

atgaacgctaaagatatccagccaactattattattaatgaaaatggttttatctctctggaagatatctatcagattgcgacaaatcaaaaaaaagtagaaatatcaacgggtgtcatagatattttgaatcgtggtcgtgaaaaattagaggaaaaattaaacttaggagaggtgatatatgggattaatacaggatttgggggaaatgccaatttagttgtgccgtttgagaaaatctcagaacaccagaaaaatctgttaacttttctttctgctggcactggggactatatgtccaaaacttgtattagagcttcacaatttactgtgttactttctatttgcaaaggttggtcagcaacaaggccaattgtcgctcaaacgatagtcgatcatcttaaccatgatattgtgcctctggttccccgatatggctccgtgggagcaagcggtgatttaattcctctatcttatattgcacgtgcattatgtggtattggcaaggttaattatatgggagaggaaattaacgctgctgaagcaattaaacgtgcaggattaactccgttatcattacaagcaaaagaaggtctcgctctcattaatggcactcgggtaatgtcaggtatcagttcagttaccgtgattaaactggaaaaactattaaaagcgacaatctcttctatagcacttgctgtcgaagcattactcgcatctcatgaacattatgatgttcgaattcagcaagtaaaaaatcaccctggtcagaaagcagtagcgagtacattgcgtaatttattggaaggttcgacacagaccagtttattagaaggtattaaagaacaagcgaataaagcttgtcgtcaccaagaagttacccgactaaatgataccttacaagaagtttactcaattcgttgtgcgccgcaaatattaggcatagttccggaatctttagctacagctcgaaaaatattggaacgtgaagttatttctgctaatgataatccattgattgatccggaaaatggtgatgttctacacggtgggaattttatgggacaatatgttgcccggactatggatgcattgaagttggatattgctttaattgccaatcatcttcatgcaatcgtagcactgatgatggatagccgtttttctcgtggattaccaaattcattaagtccgacgcctggtatgtatcaaggtctgaaaggtgtccaactttcacaaacagctttagttgctgcaattcgccatgactgttctgcatctggtatacatactttagctacagagcagtataatcaagatattgtcagcttgggtttacatgccgcacaagacgttctggagatggaacaaaaattacgtaacattgttgcaatgacaattctggtagtttgtcaggccatttatctccgcggcaatattagtaagcttgctcctgaaaccgctaaattttaccatgcagtacgtgaaattagtcctcctctggaagccgatcgtgcattggatcaagatataatccggattgccgatgcaattattaatgataatcttcctttaccagaaatcattcttgaagaataa

SEQ ID NO：2：

atgcaaaggaaaaagaaagggctatcgatcttgttagccggtacagtcgctttaggggcgctggctgtcttgccagtcggcgaaatccaagcaaaggcggctatctcgcagcaaactaaaggatcatcactcgcaaatacggttaaggccgcgactgctaagcaagcggccactgacacaaccgcagcgacaacgaatcaagcgattgccacacagttggcggctaaaggtattgattacaataagctgaataaagttcagcagcaagatacttatgttgacgtcattgttcaaatgagcgcagcgcctgcctctgaaaacggcactttaagaactgattactccagcacggcggagattcagcaggagaccaataaagtgatcgcggctcaggcaagcgttaaggcagctgttgaacaagtcacccaacaaactgccggtgaaagttatggctatgtcgttaacggcttttcaactaaagttagggttgttgatatccctaaactgaaacaaattgccggagttaaaacagtcacattggcgaaagtttactatccgactgatgctaaggctaactcgatggcgaatgtgcaggccgtatgg

SEQ ID NO：3：

atgaaaaacgcgtcaaccgtatcggaagatactgcgtcgaatcaagagccgacgcttcatcgcggattacataaccgtcatattcaactgattgcgttgggtggcgcaattggtactggtctgtttcttggcattggcccggcgattcagatggcgggtccggctgtattgctgggctacggcgtcgccgggatcatcgctttcctgattatgcgccagcttggcgaaatggtggttgaggagccggtatccggttcatttgcccactttgcctataaatactggggaccgtttgcgggcttcctctctggctggaactactgggtaatgttcgtgctggtgggaatggcagagctgaccgctgcgggcatctatatgcagtactggttcccggatgttccaacgtggatttgggctgccgccttctttattatcatcaacgccgttaacctggtgaacgtgcgcttatatggcgaaaccgagttctggtttgcgttgattaaagtgctggcaatcatcggtatgatcggctttggcctgtggctgctgttttctggtcacggcggcgagaaagccagtatcgacaacctctggcgctacggtggtttcttcgccaccggctggaatgggctgattttgtcgctggcggtaattatgttctccttcggcggtctggagctgattgggattactgccgctgaagcgcgcgatccggaaaaaagcattccaaaagcggtaaatcaggtggtgtatcgcatcctgctgttttacatcggttcactggtggttttactggcgctctatccgtgggtggaagtgaaatccaacagtagcccgtttgtgatgattttccataatctcgacagcaacgtggtagcttctgcgctgaacttcgtcattctggtagcatcgctgtcagtgtataacagcggggtttactctaacagccgcatgctgtttggcctttctgtgcagggtaatgcgccgaagtttttgactcgcgtcagccgtcgcggtgtgccgattaactcgctgatgctttccggagcgatcacttcgctggtggtgttaatcaactatctgctgccgcaaaaagcgtttggtctgctgatggcgctggtggtagcaacgctgctgttgaactggattatgatctgtctggcgcatctgcgttttcgtgcagcgatgcgacgtcaggggcgtgaaacacagtttaaggcgctgctctatccgttcggcaactatctctgcattgccttcctcggcatgattttgctgctgatgtgcacgatggatgatatgcgcttgtcagcgatcctgctgccggtgtggattgtattcctgtttatggcatttaaaacgctgcgtcggaaataa

SEQ ID NO：4：

Aggcgtgttgcctgtacgcatgctgattcttcagcaagactactacctcatgagagttatagactcatggatcttgctttgaagggttttgtacattataggctcctatcacatgctgaacctatggcctattacatttttttatatttcaaggaggaaaagaccac

In the invention, the phenylalanine ammonia lyase gene PAL is derived from Photorhabdus asymbycia ATCC43949, the inventor finds that the activities of enzymes obtained by expressing genes coding phenylalanine lyases from different sources in lactobacillus acidophilus are different, and the inventor finds that the activities of the enzymes obtained by expressing genes coding the Phellin ammonia lyase from different sources in lactobacillus acidophilus are higher, so that phenylalanine can be effectively degraded, and the aim of treating phenylketonuria is fulfilled.

Preferably, the recombinant expression vector is obtained by connecting a phenylalanine ammonia lyase gene (PAL) nucleotide sequence, a protein secretion processing domain coding gene prtP nucleotide sequence, a lactobacillus acidophilus constitutive promoter SL and a pORI28 vector;

the recombinant expression vector II is obtained by connecting a phenylalanine ammonia lyase gene (PAL) nucleotide sequence, a phenylalanine transport pump gene (pheP), a lactobacillus acidophilus constitutive promoter SL and a pORI28 vector;

in one embodiment of the invention, the engineering probiotics for treating phenylketonuria are provided, and are selected from lactobacillus acidophilus containing the recombinant expression vector;

preferably, said lactobacillus acidophilus is selected from lactobacillus acidophilus NCFM;

according to the invention, the phenylalanine ammonia lyase gene PAL from Photorhabdus asymbycica ATCC43949 and the phenylalanine transport pump gene pheP from Escherichia coli K-12 are combined, or the phenylalanine ammonia lyase gene PAL is combined with the protein secretion processing domain gene prtP from Lactcaceae paracasei ATCC 334, and the combined gene is integrated into Lactobacillus acidophilus to obtain the engineering probiotics capable of secreting phenylalanine ammonia lyase out of thalli in the gastrointestinal tract, so that phenylalanine in digested food in the gastrointestinal tract is effectively degraded, the phenylalanine intake of a patient is reduced, and the phenylalanine ammonia lyase is used for treating PKU.

Preferably, the promoter SL, the gene prtP, the gene pheP and the gene PAL are synthesized by a whole gene synthesis method.

Preferably, the lactobacillus is lactobacillus acidophilus; the engineering probiotics provided by the invention can secrete phenylalanine lyase into stomach and small intestine, and more effectively degrade PA in food.

In an embodiment of the present invention, an application of the recombinant expression vector or the engineering probiotic in preparation of a drug for treating phenylketonuria is provided.

Preferably, the medicament is in a solid dosage form and the mode of administration is oral.

The engineering probiotics can introduce phenylalanine lyase out of the bacteria in the gastrointestinal tract to degrade PA in the digested food.

In an embodiment of the present invention, a pharmaceutical composition is provided, which includes the above engineering probiotics and pharmaceutically acceptable excipients.

The invention will be further explained and illustrated with reference to specific examples.

Example 1

Engineering probiotic LA205 construction

A lactobacillus acidophilus NCFM starting strain is integrated with a protein secretion processing structural domain gene prtP and a phenylalanine lyase PAL gene on a genome, and the method comprises the following steps:

in this example, the whole gene synthesis and sequencing were carried out by Shanghai Bioengineering Co., Ltd. Molecular biological experiments including competent cell preparation, transformation, etc. were carried out according to the molecular cloning protocols (third edition).

Designing a gene segment SPAL containing a promoter SL (DNA sequence NO3), a protein secretion processing structural domain gene prtP (DNA sequence NO1) and a phenylalanine ammonia lyase gene PAL (DNA sequence NO2), obtaining a DNA segment by using a whole-gene synthesis method, constructing the DNA segment into a cloning vector pUC57, and obtaining a pUC57-SPAL vector.

And (3) carrying out PCR amplification by using a lactobacillus acidophilus NCFM genome as a template and L-F and L-R as primers to obtain an upstream homology arm L-arm. And (3) performing PCR amplification by using the NCFM genome as a template and R-F and R-R as primers to obtain the downstream homology arm R-arm. The SPAL fragment was obtained by PCR amplification using pUC57-SPAL vector as template and SPAL-F and SPAL-R as primers. The L-arm, R-arm and SPAL fragments were cloned into the vector pORI28 by a recombinase method to obtain the vector pORI 28-SPAL.

The pORI28-SPAL vector and the pTRK669 helper plasmid are transferred into the Lactobacillus acidophilus NCFM strain through electrotransformation to obtain a transformant, and the recombinant strain integrating the secretory phenylalanine lyase gene into a genome is screened by Val-F and Val-R through PCR. And eliminating plasmids through continuous passage at 42 ℃ and on an antibiotic-free culture medium to finally obtain the engineering bacteria LA 205.

TABLE 1 primer List

Primer name	Primer sequence (5 '→ 3')
		L-F	TCTAGACTTTGTTTCTAACTCTCCAG
L-R	CTGGAGAGTTAGAAACAAAGTCTAGA
		R-F	TCAGGCAAGATTATTAAACC
R-R	AGATCTGAAATCAATTTCTTAATTGA
		SPAL-F	CAAAGGTGTAACCAGTTAAAGGCGTGTTGCCTGTACGCA
SPAL-R	GGTTTAATAATCTTGCCTGATTATTCTTCAAGAATGATTT
		Val-F	AGAATTAACTCAAGGTCG
Val-R	ATCACGGTAACTGAACTG

Degradation activity of engineering probiotics LA205 on phenylalanine under in vitro condition

Inoculating NCFM probiotic and LA205 engineering probiotic into MRS broth culture medium, culturing at 37 deg.C and 250rpm to OD₆₀₀The cells were collected by centrifugation and washed 2 times with PBS buffer 1.0. Quantitatively transferring to low concentration MRS culture medium (containing 1/10MRS culture medium nutrient components) containing 50mg/L phenylalanine, culturing at 37 deg.C and 250rpm,samples were taken at 0, 2, 4, 6 and 8 hours, respectively, and the supernatant samples were collected by centrifugation and assayed for phenylalanine concentration by HPLC. Each treatment was performed in 3 replicates and the results are shown in table 2. The result shows that the decrease speed of the phenylalanine concentration in the treatment of the LA205 engineering probiotics is obviously higher than that in the treatment of NCFM probiotics, which indicates that the LA205 engineering probiotics has better phenylalanine degrading capability.

TABLE 2 mean phenylalanine concentration (mg/L) under probiotic treatment conditions

Time	NCFM	LA1314
			Initiation of	53.04	53.63
At 2 nd hour	50.61	47.51
			At 4 th hour	52.26	38.43
At 6 th hour	45.71	28.06
			At 8 th hour	40.36	10.57

Simultaneously inoculating NCFM probiotic and LA205 engineering probiotic into MRS broth culture medium, culturing at 37 deg.C and 250rpm to OD₆₀₀The supernatant was collected by centrifugation and filtered through a sterile filter to obtain a sterile filtrate. Then, 50mg/L phenylalanine was added to the sterile filtrate, and the mixture was shaken at 37 ℃ and 250rpm, and sampled at 0 hour, 1 hour, 2 hours, 3 hours and 4 hours, and the samples were subjected to HPLC to detect the phenylalanine concentration. Each treatment was performed in 3 replicates and the results are shown in table 3. The result shows that the aseptic filtrate of the LA205 engineering probiotics can effectively degrade phenylalanine, and the LA205 engineering probiotics can secrete phenylalanine lyase to the outside of the bacteria.

TABLE 3 phenylalanine concentration Change (mg/L) in cell-free filtrate

Time	NCFM	LA1314
			Initiation of	50.24	51.43
At 1 hour	50.61	41.25
			At 2 nd hour	51.06	32.53
At 3 rd hour	50.41	28.02
			At 4 th hour	49.57	8.53

Effect of engineered Probiotics LA205 on phenylalanine concentration in blood of Parapyrnuria mice

The phenylketonuria mouse model is purchased from biotechnology company and has the genotype of Pah^enu2. The model mouse has typical symptoms of phenylketonuria, and Pah is fed under normal condition^enu2The phenylalanine content in the blood of the mouse at the 6 th week of birth is 1200 mu mol/L, the phenylalanine content in the blood at the 8 th week reaches 1500 mu mol/L, the weight and the body type are obviously smaller, and the hair color gradually shows brown yellow.

The experimental sample is 8 weeks old Pah^enu2Mice were fed with phenylalanine-free feed and fed with a daily intragastric administration of 100. mu.l of phenylalanine-containing purified water as a supplement. After the animal experiment, the mice were divided into 3 groups, and the mice were gavaged for 2 times every day, and 200 μ l of physiological saline, NCFM probiotic or LA205 engineering probiotic were gavaged, respectively, with the dose of probiotic being about 1 x 10 per time¹⁰cfu/mouse. Periodically sampling blood every day, and checking the phenylalanine concentration in the blood. The results of the mean concentrations of phenylalanine in blood are shown in FIG. 1. The results show Pah after oral administration of LA205 engineered probiotic bacteria^enu2The phenylalanine concentration in the blood of the mouse is obviously reduced compared with the oral physiological saline and NCFM probiotics, which shows that the LA205 engineering probiotics can degrade phenylalanine in food and reduce the intake of phenylalanine in the mouse.

In conclusion, the lactobacillus acidophilus engineering probiotic LA205 constructed by the invention can secrete phenylalanine lyase to the outside of the bacteria, degrade phenylalanine in gastrointestinal tract digestion food, obviously reduce the phenylalanine concentration in blood of a mouse with a phenylketosis gene, and is expected to be applied to treatment of phenylketonuria.

Example 2

Construction of engineered probiotic LA1314

A lactobacillus acidophilus NCFM starting strain is used for integrating a phenylalanine lyase gene PAL and a phenylalanine transfer pump gene pheP on a genome, and the method comprises the following steps:

in the embodiment, the whole gene synthesis and sequencing are completed by Shanghai biological engineering Co., Ltd; molecular biological experiments including competent cell preparation, transformation, etc. were carried out according to the molecular cloning protocols (third edition).

Designing a gene segment PALP containing a promoter SL (DNA sequence NO3), a phenylalanine lyase gene PAL (DNA sequence NO1) and a phenylalanine transfer pump gene pheP (DNA sequence NO2), obtaining a DNA segment by using a whole-gene synthesis method, constructing the DNA segment into a cloning vector pUC57, and obtaining a pUC57-PALP vector.

And (3) carrying out PCR amplification by using a lactobacillus acidophilus NCFM genome as a template and L-F and L-R as primers to obtain an upstream homology arm L-arm. And (3) performing PCR amplification by using the NCFM genome as a template and R-F and R-R as primers to obtain the downstream homology arm R-arm. And (3) carrying out PCR amplification by using a pUC57-PALP vector as a template and PALP-F and PALP-R as primers to obtain a PALP fragment. The L-arm, R-arm and PALP fragments were cloned into the pORI28 vector by recombinase method to obtain the pORI28-PALP vector.

The pORI28-PALP vector and pTRK669 helper plasmid are transferred into Lactobacillus acidophilus NCFM strain by electrotransformation to obtain transformant, and Val-F and Val-R are used for PCR screening of recombinant strain in which phenylalanine lyase gene PAL and phenylalanine transfer pump gene pheP are integrated into genome. Plasmid was then eliminated by serial passage at 42 ℃ and on antibiotic-free medium, finally obtaining the engineered bacterium LA 1314.

TABLE 4 primer List

Primer name	Primer sequence (5 '→ 3')
		L-F	TCTAGACTTTGGTTTCTAACTCTCCAG
L-R	CTGGAGAGTTAGAAACAAAGTCTAGA
		R-F	TCAGGCAAGATTATTAAACC
R-R	AGATCTGAAATCAATTTCTTAATTGA
		PALP-F	CAAAGGTGTAACCAGTTAAAGGCGTGTTGCCTGTACGCA
PALP-R	GGTTTAATAATCTTGCCTGAAAATTTAAAACACAAAAAAAG
		Val-F	AGAATTAACTCAAGGTCG
Val-R	ATCACGGTAACTGAACTG

Degradation activity of engineering probiotics LA1314 on phenylalanine under in vitro condition

Inoculating NCFM probiotic and LA1314 engineering probiotic respectively in MRS broth culture medium, culturing at 37 deg.C and 250rpm to OD₆₀₀The cells were collected by centrifugation and washed 2 times with PBS buffer 1.0. Finally, the suspension was diluted to OD with 0.5% glucose and 50mg/L phenylalanine in PBS buffer₆₀₀＝1.0，37℃、Culturing at 250rpm, sampling at 0 hr, 1 hr, 2 hr, 3 hr and 4 hr, centrifuging to collect supernatant, and detecting phenylalanine concentration by HPLC. Each treatment was performed in 3 replicates and the results are shown in table 5. The result shows that the decrease speed of the phenylalanine concentration in the treatment of the LA1314 engineering probiotics is obviously higher than that in the treatment of NCFM probiotics, which indicates that the LA1314 engineering probiotics has better phenylalanine degrading capability.

TABLE 5 mean phenylalanine concentration (mg/L) under probiotic treatment conditions

Time	NCFM	LA1314
			Initiation of	51.74	52.13
At 1 hour	45.61	30.24
			At 2 nd hour	42.16	12.43
At 3 rd hour	40.75	8.96
			At 4 th hour	38.46	0.57

Effect of engineered probiotic LA1314 on phenylalanine concentration in P-pyruvuria mice blood

The experimental sample is 8 weeks old Pah^enu2Mice were fed with phenylalanine-free feed and fed with a daily intragastric administration of 100. mu.l of phenylalanine-containing purified water as a supplement. After the animal experiment was started, mice were divided into 3 groups, and the gavage was performed 2 times per day, and 200 μ l of physiological saline, NCFM probiotic or LA1314 engineered probiotic was administered at a dose of about 1 x 10 per time¹⁰cfu/mouse. Periodically sampling blood every day, and checking the phenylalanine concentration in the blood. The results of the mean concentrations of phenylalanine in blood are shown in FIG. 1. The results show Pah after oral administration of LA1314 engineered probiotics^enu2The phenylalanine concentration in the blood of the mice is obviously reduced compared with oral physiological saline and NCFM probiotics, which shows that the LA1314 engineering probiotics can degrade phenylalanine in food and reduce the intake of phenylalanine in the mice.

In conclusion, the lactobacillus acidophilus engineering probiotic LA1314 constructed by the invention can degrade phenylalanine, remarkably reduce the concentration of phenylalanine in blood of a mouse with a phenylketonuria gene, and is expected to be applied to treatment of phenylketonuria.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Shandong first medical university (Shandong province medical science institute)

<120> engineering probiotics for treating phenylketonuria and construction method and application thereof

<130> 202127698

<160> 14

<170> PatentIn version 3.3

<210> 1

<211> 1599

<212> DNA

<213> PAL

<400> 1

atgaacgcta aagatatcca gccaactatt attattaatg aaaatggttt tatctctctg 60

gaagatatct atcagattgc gacaaatcaa aaaaaagtag aaatatcaac gggtgtcata 120

gatattttga atcgtggtcg tgaaaaatta gaggaaaaat taaacttagg agaggtgata 180

tatgggatta atacaggatt tgggggaaat gccaatttag ttgtgccgtt tgagaaaatc 240

tcagaacacc agaaaaatct gttaactttt ctttctgctg gcactgggga ctatatgtcc 300

aaaacttgta ttagagcttc acaatttact gtgttacttt ctatttgcaa aggttggtca 360

gcaacaaggc caattgtcgc tcaaacgata gtcgatcatc ttaaccatga tattgtgcct 420

ctggttcccc gatatggctc cgtgggagca agcggtgatt taattcctct atcttatatt 480

gcacgtgcat tatgtggtat tggcaaggtt aattatatgg gagaggaaat taacgctgct 540

gaagcaatta aacgtgcagg attaactccg ttatcattac aagcaaaaga aggtctcgct 600

ctcattaatg gcactcgggt aatgtcaggt atcagttcag ttaccgtgat taaactggaa 660

aaactattaa aagcgacaat ctcttctata gcacttgctg tcgaagcatt actcgcatct 720

catgaacatt atgatgttcg aattcagcaa gtaaaaaatc accctggtca gaaagcagta 780

gcgagtacat tgcgtaattt attggaaggt tcgacacaga ccagtttatt agaaggtatt 840

aaagaacaag cgaataaagc ttgtcgtcac caagaagtta cccgactaaa tgatacctta 900

caagaagttt actcaattcg ttgtgcgccg caaatattag gcatagttcc ggaatcttta 960

gctacagctc gaaaaatatt ggaacgtgaa gttatttctg ctaatgataa tccattgatt 1020

gatccggaaa atggtgatgt tctacacggt gggaatttta tgggacaata tgttgcccgg 1080

actatggatg cattgaagtt ggatattgct ttaattgcca atcatcttca tgcaatcgta 1140

gcactgatga tggatagccg tttttctcgt ggattaccaa attcattaag tccgacgcct 1200

ggtatgtatc aaggtctgaa aggtgtccaa ctttcacaaa cagctttagt tgctgcaatt 1260

cgccatgact gttctgcatc tggtatacat actttagcta cagagcagta taatcaagat 1320

attgtcagct tgggtttaca tgccgcacaa gacgttctgg agatggaaca aaaattacgt 1380

aacattgttg caatgacaat tctggtagtt tgtcaggcca tttatctccg cggcaatatt 1440

agtaagcttg ctcctgaaac cgctaaattt taccatgcag tacgtgaaat tagtcctcct 1500

ctggaagccg atcgtgcatt ggatcaagat ataatccgga ttgccgatgc aattattaat 1560

gataatcttc ctttaccaga aatcattctt gaagaataa 1599

<210> 2

<211> 603

<212> DNA

<213> prtP

<400> 2

atgcaaagga aaaagaaagg gctatcgatc ttgttagccg gtacagtcgc tttaggggcg 60

ctggctgtct tgccagtcgg cgaaatccaa gcaaaggcgg ctatctcgca gcaaactaaa 120

ggatcatcac tcgcaaatac ggttaaggcc gcgactgcta agcaagcggc cactgacaca 180

accgcagcga caacgaatca agcgattgcc acacagttgg cggctaaagg tattgattac 240

aataagctga ataaagttca gcagcaagat acttatgttg acgtcattgt tcaaatgagc 300

gcagcgcctg cctctgaaaa cggcacttta agaactgatt actccagcac ggcggagatt 360

cagcaggaga ccaataaagt gatcgcggct caggcaagcg ttaaggcagc tgttgaacaa 420

gtcacccaac aaactgccgg tgaaagttat ggctatgtcg ttaacggctt ttcaactaaa 480

gttagggttg ttgatatccc taaactgaaa caaattgccg gagttaaaac agtcacattg 540

gcgaaagttt actatccgac tgatgctaag gctaactcga tggcgaatgt gcaggccgta 600

tgg 603

<210> 3

<211> 1377

<212> DNA

<213> pheP

<400> 3

atgaaaaacg cgtcaaccgt atcggaagat actgcgtcga atcaagagcc gacgcttcat 60

cgcggattac ataaccgtca tattcaactg attgcgttgg gtggcgcaat tggtactggt 120

ctgtttcttg gcattggccc ggcgattcag atggcgggtc cggctgtatt gctgggctac 180

ggcgtcgccg ggatcatcgc tttcctgatt atgcgccagc ttggcgaaat ggtggttgag 240

gagccggtat ccggttcatt tgcccacttt gcctataaat actggggacc gtttgcgggc 300

ttcctctctg gctggaacta ctgggtaatg ttcgtgctgg tgggaatggc agagctgacc 360

gctgcgggca tctatatgca gtactggttc ccggatgttc caacgtggat ttgggctgcc 420

gccttcttta ttatcatcaa cgccgttaac ctggtgaacg tgcgcttata tggcgaaacc 480

gagttctggt ttgcgttgat taaagtgctg gcaatcatcg gtatgatcgg ctttggcctg 540

tggctgctgt tttctggtca cggcggcgag aaagccagta tcgacaacct ctggcgctac 600

ggtggtttct tcgccaccgg ctggaatggg ctgattttgt cgctggcggt aattatgttc 660

tccttcggcg gtctggagct gattgggatt actgccgctg aagcgcgcga tccggaaaaa 720

agcattccaa aagcggtaaa tcaggtggtg tatcgcatcc tgctgtttta catcggttca 780

ctggtggttt tactggcgct ctatccgtgg gtggaagtga aatccaacag tagcccgttt 840

gtgatgattt tccataatct cgacagcaac gtggtagctt ctgcgctgaa cttcgtcatt 900

ctggtagcat cgctgtcagt gtataacagc ggggtttact ctaacagccg catgctgttt 960

ggcctttctg tgcagggtaa tgcgccgaag tttttgactc gcgtcagccg tcgcggtgtg 1020

ccgattaact cgctgatgct ttccggagcg atcacttcgc tggtggtgtt aatcaactat 1080

ctgctgccgc aaaaagcgtt tggtctgctg atggcgctgg tggtagcaac gctgctgttg 1140

aactggatta tgatctgtct ggcgcatctg cgttttcgtg cagcgatgcg acgtcagggg 1200

cgtgaaacac agtttaaggc gctgctctat ccgttcggca actatctctg cattgccttc 1260

ctcggcatga ttttgctgct gatgtgcacg atggatgata tgcgcttgtc agcgatcctg 1320

ctgccggtgt ggattgtatt cctgtttatg gcatttaaaa cgctgcgtcg gaaataa 1377

<210> 4

<211> 167

<212> DNA

<213> promoter SL

<400> 4

aggcgtgttg cctgtacgca tgctgattct tcagcaagac tactacctca tgagagttat 60

agactcatgg atcttgcttt gaagggtttt gtacattata ggctcctatc acatgctgaa 120

cctatggcct attacatttt tttatatttc aaggaggaaa agaccac 167

<210> 5

<211> 26

<212> DNA

<213> L-F

<400> 5

tctagacttt gtttctaact ctccag 26

<210> 6

<211> 26

<212> DNA

<213> L-R

<400> 6

ctggagagtt agaaacaaag tctaga 26

<210> 7

<211> 20

<212> DNA

<213> R-F

<400> 7

tcaggcaaga ttattaaacc 20

<210> 8

<211> 26

<212> DNA

<213> R-R

<400> 8

agatctgaaa tcaatttctt aattga 26

<210> 9

<211> 39

<212> DNA

<213> SPAL-F

<400> 9

caaaggtgta accagttaaa ggcgtgttgc ctgtacgca 39

<210> 10

<211> 40

<212> DNA

<213> SPAL-R

<400> 10

ggtttaataa tcttgcctga ttattcttca agaatgattt 40

<210> 11

<211> 18

<212> DNA

<213> Val-F

<400> 11

agaattaact caaggtcg 18

<210> 12

<211> 18

<212> DNA

<213> Val-R

<400> 12

atcacggtaa ctgaactg 18

<210> 13

<211> 39

<212> DNA

<213> PALP-F

<400> 13

caaaggtgta accagttaaa ggcgtgttgc ctgtacgca 39

<210> 14

<211> 41

<212> DNA

<213> PALP-R

<400> 14

ggtttaataa tcttgcctga aaatttaaaa cacaaaaaaa g 41

Claims

1. a recombinant expression vector, is characterized in that, described recombinant expression vector comprises recombinant expression vector one and recombinant expression vector two;

One of the recombinant expression vectors is a nucleotide sequence comprising a phenylalanine ammonia-lyase (PAL) gene, a prtP nucleotide sequence encoding a protein secretion processing domain, and a Lactobacillus acidophilus constitutive promoter. Expression vector of subSL;

The second recombinant expression vector is an expression vector comprising the nucleotide sequence of the phenylalanine ammonia lyase gene PAL, the phenylalanine transporter (pheP) gene and the constitutive promoter SL of Lactobacillus acidophilus.

2 . The recombinant expression vector of claim 1 , wherein the phenylalanine ammonia lyase gene PAL is derived from Photorhabdus asymbiotica ATCC43949, and the nucleotide sequence is shown in SEQ ID NO: 1. 3 .

3 . The recombinant expression vector according to claim 1 , wherein the gene prtP encoding the protein secretion processing domain is derived from Lacticaseibacillus paracasei ATCC 334, and the nucleotide sequence is shown in SEQ ID NO: 2. 4 .

4 . The recombinant expression vector of claim 1 , wherein the phenylalanine transport pump gene pheP is derived from Escherichia coli K-12, and the nucleotide sequence is shown in SEQ ID NO: 3. 5 .

5 . The recombinant expression vector of claim 1 , wherein the Lactobacillus acidophilus constitutive promoter SL is derived from Lactobacillus acidophilus NCFM, and the nucleotide sequence is shown in SEQ ID NO: 4. 6 .

6. The recombinant expression vector according to claim 1, wherein the recombinant expression vector is composed of phenylalanine ammonia lyase gene PAL nucleotide sequence, protein secretion processing domain coding gene prtP nucleotide Sequence, Lactobacillus acidophilus constitutive promoter SL and pORI28 vector are connected to obtain;

The second recombinant expression vector is obtained by connecting the nucleotide sequence of the phenylalanine ammonia lyase gene, the phenylalanine transport pump gene pheP, the Lactobacillus acidophilus constitutive promoter SL and the pORI28 vector.

7. An engineering probiotic for the treatment of phenylketonuria, wherein the engineering probiotic is selected from the Lactobacillus acidophilus containing the recombinant expression vector described in the first aspect;

Preferably, the Lactobacillus acidophilus is selected from Lactobacillus acidophilus NCFM.

8. a method of constructing the described engineering probiotics of claim 7, is characterized in that, comprises the following steps:

(1) Place the protein secretion processing domain gene prtP and PAL gene, or the phenylalanine transport pump gene pheP and PAL gene downstream of the constitutive promoter SL of Lactobacillus acidophilus;

(2) using conventional molecular biology techniques to connect the above-mentioned gene fragment to the pORI28 vector;

(3) using Lactobacillus acidophilus NCFM as the chassis bacteria, by homologous recombination method, the above-mentioned genes are integrated into the genome to obtain protein secretion processing domain gene prtP and PAL gene integration strain or gene PAL and gene pheP integration strain;

Preferably, a total gene synthesis method is used to synthesize promoter SL, gene prtP, gene pheP and gene PAL;

Preferably, the Lactobacillus is Lactobacillus acidophilus.

9. the application of the recombinant expression vector described in claim 1 or the engineering probiotics described in claim 7 in the preparation of phenylketonuria therapeutic medicine;

Preferably, the medicament is in a solid dosage form and is administered orally.

10. A pharmaceutical composition, characterized in that it comprises the engineered probiotic bacteria of claim 7 and pharmaceutically acceptable excipients.