CN112662607A - Engineering probiotics with surface display of phenylalanine ammonia lyase - Google Patents

Engineering probiotics with surface display of phenylalanine ammonia lyase Download PDF

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CN112662607A
CN112662607A CN202110017460.5A CN202110017460A CN112662607A CN 112662607 A CN112662607 A CN 112662607A CN 202110017460 A CN202110017460 A CN 202110017460A CN 112662607 A CN112662607 A CN 112662607A
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蒋宇
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Abstract

The invention discloses an engineering probiotic with surface display phenylalanine ammonia lyase, which is Escherichia coli Nissle1917 derivative bacteria, wherein a gene argR is knocked out on a genome and/or mutation of a gene argA (Y19C) is generated, and an L-phenylalanine ammonia lyase gene stlA, an L-phenylalanine transport protein gene pheP, an L-amino acid deaminase gene pma and an efflux pump gene acrA are integrated. The engineering probiotic can be used for treating phenylketonuria.

Description

Engineering probiotics with surface display of phenylalanine ammonia lyase
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to engineering probiotics with surface display of phenylalanine ammonia lyase, a construction method of the engineering probiotics and application of the engineering probiotics in preparation of a medicine for treating phenylketonuria.
Background
Phenylketonuria (PKU) is a congenital disorder of phenylalanine metabolism, an autosomal recessive disease. In china, the incidence of PKU in newborns is probably 1/11000, and has been listed as a disease item that newborns must check.
Phenylketonuria is caused by deficiency of hepatic phenylalanine hydroxylase (PAH) or mutation of tetrahydrobiopterin synthase and dihydrobiopterin reductase. Normally, phenylalanine catalyzes the production of tyrosine by PAH, and then synthesizes thyroid, adrenal gland, melanin, and the like through a tyrosine metabolic pathway. The mutation of PAH causes phenylalanine to generate metabolic disorder in liver, and phenylalanine and alpha-ketoglutaric acid are accumulated in blood and tissues and excreted into urine, and in addition, metabolites of the phenylalanine and the alpha-ketoglutaric acid are accumulated in central nerve to generate toxicity, so that the patient infant is induced to generate excitation restlessness, hyperactivity, mental disorder and the like.
Current treatment for PKU is primarily diet. Phenylalanine is one of essential amino acids of human body, is mainly obtained from food, and the PKU patients cannot eat phenylalanine-free food, so that in order to ensure normal growth and development of the body, the PKU patients need to eat low-phenylalanine diet, but the food therapy has the problems of difficult long-term adherence, heavy economic burden and the like. With the development of molecular biology technology, gene therapy has entered into experimental stage, for example, recombinant adenovirus carrying cDNA expressing PAH gene is placed in mice to restore liver PAH activity, but at present there is mainly a problem of low transport efficiency.
Disclosure of Invention
Probiotics is a large class of drugs, and generally achieves the effects of treating diseases and rehabilitation and health care by oral administration of live bacteria preparations. The probiotic medicinal preparation has the advantages of convenient administration, good taste, easy acceptance by patients, high compliance and continuous proliferation in intestinal tracts so as to stably play a therapeutic role.
Coli Nissle1917 (abbreviated EcN or Nissle 1917) is a non-pathogenic e.coli and also a probiotic. The applicant reports in patent CN202011457369.7 that the Nissle1917 engineering probiotics for treating phenylketonuria has the degradation capability to phenylalanine, and enhances the effect of treating phenylketonuria. The inventor continuously utilizes the genetic engineering technology to modify Nissle1917 engineering probiotics, so that the Nissle1917 engineering probiotics have the surface display of phenylalanine ammonia lyase and further improve the capacity of degrading phenylalanine. Specifically, the invention comprises the following technical scheme:
an engineered probiotic, which is an escherichia coli Nissle 1917-derived bacterium, provided with surface-displayed phenylalanine ammonia-lyase (PAL).
The engineering probiotics take escherichia coli Nissle1917 as a substrate bacterium, and the escherichia coli Nissle1917 engineering probiotics with the surface displaying L-phenylalanine ammonia lyase PAL is constructed through plasmid expression or genome integration; or Nissle1917 engineering probiotics obtained by performing genetic engineering modification of surface display phenylalanine ammonia lyase PAL on the basis of Escherichia coli Nissle1917 engineering bacteria which already have phenylalanine ammonia lyase PAL in cells.
For example, the escherichia coli Nissle1917 engineering probiotics displaying the L-phenylalanine ammonia lyase gene stlA on the surface are constructed by using the escherichia coli Nissle1917 as a chassis bacterium through plasmid expression or genome integration. The plasmid may be, for example, pINP-stlA plasmid, which, after transformation into Nissle1917(EcN), yields the recombinantly engineered probiotic EcN/pINP-stlA.
The Escherichia coli Nissle1917 engineering bacteria with the intracellular phenylalanine ammonia lyase PAL can be constructed by the following steps: an exogenous L-phenylalanine ammonia lyase gene stlA, an exogenous L-phenylalanine transport protein gene pheP and an exogenous L-amino acid deaminase gene pma are integrated on a genome of Escherichia coli Nissle 1917.
Preferably, an endogenous (i.e. Nissle 1917-derived) efflux pump gene acrA is also integrated; and/or
The gene argR is knocked out and/or mutation of the gene argA (Y19C) is generated; and/or
And optimizing RBS sequences of stlA, pheP and acrA genes.
Preferably, the nucleotide sequence of the L-phenylalanine ammonia lyase (Genbank number KGM29850.1) gene stlA is SEQ ID NO 1;
the nucleotide sequence of the L-phenylalanine transport protein (Genbank number QPA14453.1) gene pheP is SEQ ID NO. 2;
the nucleotide sequence of the L-amino acid deaminase (Genbank number AAA86752.1) gene pma is SEQ ID NO. 3.
The nucleotide sequence of the efflux pump protein (Genbank number WP _001295833.1) gene acrA can be SEQ ID NO: 4.
Further, the above RBS sequence optimization may be such that the RBS (ribosome binding site) sequence of stlA gene in the genome is optimized from GCTAGCAGGATACTTCCAATCCATGGCAACAAAACAAAAAGTAGAGGAGGTAAAT to CTCGCGAGAATTAAGAAGAAAGGAGGTTTTTTTT (SEQ ID NO: 5); the RBS sequence of the pheP gene was optimized from GCTAGCAGGATACTTCCAATCCATGGCAACAAAACAAAAAGTAGAGGAGGTAAAT to GGAGTTATCTCTCCCGGGTCACAATATTAAGGAGGTTTTATTT (SEQ ID NO: 6); the RBS sequence of the acrA gene was optimized from GCTAGCAGGATACTTCCAATCCATGGCAACAAAACAAAAAGTAGAGGAGGTAAAT to GAGGCTAACAGGCACATTCAATAAGGAGGTTTTTT (SEQ ID NO: 7).
The dihydrodipicolinate synthase gene dapA can also be knocked out from the genome of the Escherichia coli Nissle1917 derivative.
Preferably, the genotype of the engineered probiotic is: EcN (malP:: Pj23119H-stlA, yicS:: Pj23119H-stlA, malE:: Pj23119H-stlA, rthC: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119H-pheP, agaI:: Pj23119H-pheP, araBD:: Para-pma, dapA:: inaK-stlA, argA,. DELTA.argR, lacZ:: Pj 23119H-acrA).
A second aspect of the present invention provides a method of constructing the above engineered probiotic, which may comprise the steps of:
A. taking engineering probiotics EcN/pINP-stlA containing surface display stlA plasmid pINP-stlA as chassis bacteria, knocking in L-phenylalanine ammonia lyase gene stlA at more than one site, preferably at more than two sites, preferably at more than three sites, preferably at more than four sites, preferably at five sites in malP site, yicS site, malE site, rhtC site and exo site of genome respectively to obtain stlA gene integration strain;
B. knocking in the L-phenylalanine endogenous protein gene pheP at more than one site, preferably two sites, of the lacZ site and the agaI site of the genome of the stlA gene integrated strain obtained in the step A to obtain a stlA + pheP integrated strain;
C. knocking in an L-amino acid deaminase gene pma at an araBD locus of a genome of the stlA + pheP integrated strain obtained in the step B to obtain the stlA + pheP + pma integrated strain;
D. knocking out a dihydrodipicolinate synthase gene dapA in the genome of the stlA + pheP + pma integrated strain obtained in the step C to obtain a stlA + pheP + pma delta dapA strain;
E. for the stlA + pheP + pma Δ dapA strain obtained in step D, mutation was performed (Y19C) on the argA site of its genome to give a stlA + pheP + pma Δ dapA argA strain;
F. knocking out argR in the genome of the stlA + pheP + pma delta dapA argA strain obtained in the step E to obtain the stlA + pheP + pma delta dapA argA delta argR strain.
Preferably, the above method may further comprise the steps of:
G. for the stlA + pheP + pma Δ dapA argA Δ argR strain obtained in step F, the efflux pump gene acrA was knocked in at the lacZ site of its genome to give a stlA + pheP + pma + acrA Δ dapA argA Δ argR strain.
The above method may further comprise the steps of:
H. and G, optimizing the RBS sequence of the stlA gene in the genome of the stlA + pheP + pma + acrA delta dapA argA delta argR strain obtained in the step G from the optimized sequence of SEQ ID NO. 5, optimizing the RBS sequence of the pheP gene to the optimized sequence of SEQ ID NO. 6 and optimizing the RBS sequence of the acrA gene to the optimized sequence of SEQ ID NO. 7 to obtain the RBS optimized strain.
I. And (D) knocking in the dapA locus of the genome of the RBS optimized strain obtained in the step (H) to display the stlA gene on the surface to obtain the engineering probiotics with the surface displayed phenylalanine ammonia lyase.
The knocking-in and knocking-out of each gene can be implemented by a gene editing technology, and the gene editing adopts a CRISPR-Cas9 system, a CRISPR-Cpf1 system, a CRISPR-Cas related transposition system INTEGRATE system or a CAST system.
The third aspect of the invention provides the application of the engineering probiotics in the preparation of drugs for treating phenylketonuria.
The above-mentioned drugs are preferably in oral dosage forms, and can be administered orally. Accordingly, the pharmaceutical dosage forms are oral dosage forms suitable for oral administration while maintaining probiotic activity, including solid granules, tablets, and liquid live bacterial formulations.
Optionally, the medicament also comprises at least one auxiliary therapeutic agent in addition to the active pharmaceutical ingredient engineering probiotics, thereby forming a compound medicament. Preferably, the adjunctive therapeutic agent is another pharmaceutical ingredient used to treat phenylketonuria without compromising probiotic activity.
In vitro experiments show that the engineering probiotics constructed by the invention can obviously degrade phenylalanine to generate trans-cinnamic acid, and is hopeful to be developed into an effective medicament for treating phenylketonuria.
Drawings
Fig. 1 is a bar graph comparing the original strain e.coli Nissle1917 and the ability of each engineered bacterium to degrade phenylalanine to produce trans-cinnamic acid.
Detailed Description
The inventor enables the recombinant Nissle1917(EcN) engineering bacteria to express the phenylalanine ammonia-lyase gene by genome modification on the basis of the original Escherichia coli Nissle1917 without the ability of degrading phenylalanine, thereby having the ability of degrading phenylalanine; the recombinant Nissle1917(EcN) engineering bacterium can express phenylalanine ammonia lyase genes in cells and can display L-phenylalanine ammonia lyase PAL on the surface. The recombinant Nissle1917(EcN) engineering bacteria with the surface displaying L-phenylalanine ammonia lyase PAL can have stronger phenylalanine degrading capability, thereby being used as engineering probiotics. Further, the arginine synthesis pathway can be enhanced by inactivating the gene argR and making an anti-feedback inhibition mutation on argA (Y19C); then, the strain can express L-phenylalanine endogenous protein genes and/or L-amino acid deaminase genes through genetic engineering, and the engineering probiotics TYS009 and TYS010 with obviously improved phenylalanine degradation capability are obtained. Has been described in patent CN202011457369.7, the entire content of which is incorporated herein.
On the basis of the engineering probiotics TYS010, the invention further strengthens phenylalanine efflux pump genes such as acrA, and optimizes RBS sequences of stlA, pheP and acrA genes to obtain the engineering probiotics TYS013 with the surface display phenylalanine ammonia lyase PAL so as to further improve the ability of degrading phenylalanine.
For the sake of brevity, "engineered probiotic" is sometimes referred to herein simply as "(genetically) engineered bacteria" or "probiotics," which are intended to have the same meaning and may be used interchangeably.
The argR gene is an arginine operon regulating gene which is ubiquitous in bacteria and has different functions in different bacteria, and the inventor researches and discovers that the inhibition of the argR gene on the arginine synthesis can be relieved to a certain extent by knocking out the negative regulating gene in the genome of Nissle1917 of escherichia coli.
Mutation of the gene argA, Y19C encoding N-acetylglutamate synthase (NAGS) has the effect of relieving arginine feedback inhibition.
In a specific embodiment, a constitutive promoter Pj23119 is introduced to the genome of probiotic E.coli Nissle1917 to regulate and control a phenylalanine ammonia lyase gene (stlA) derived from mycobacterium luminescens (Photorhabdus luminescens) and a phenylalanine specific transporter gene (pheP) derived from Escherichia coli MG1655, so that intracellular phenylalanine transportation can be effectively improved, and the intracellular phenylalanine is converted into trans-cinnamic acid; simultaneously, an L-amino acid deaminase gene (pma) derived from Proteus mirabilis HI4320 is introduced, so that phenylalanine can be degraded into phenylpyruvic acid; the argR is knocked out and argA (Y19C) is mutated to strengthen an arginine synthesis path, and ammonia released when phenylalanine is converted into trans-cinnamic acid and phenylpyruvic acid can be effectively utilized, so that the conversion reaction of phenylalanine into trans-cinnamic acid and phenylalanine is promoted, and the aim of effectively degrading phenylalanine is fulfilled. By enhancing the trans-cinnamic acid efflux pump gene arcA, the intracellular trans-cinnamic acid content is effectively reduced, and the conversion reaction from phenylalanine to trans-cinnamic acid is further promoted. And the expression of the genes is improved by optimizing the ribosome binding site RBS of stlA, pheP and acrA. And the stlA is further subjected to cell surface display, so that the engineering probiotics become an 'immobilized cell factory' to perform catalytic reaction, and the degradation capability of the probiotics on phenylalanine is further improved.
It should be understood that in the specific operation of constructing the genetically engineered bacteria of the present invention, the sequence of steps A, B, C to I, etc. is not fixed from the front to the back according to the English letter sequence, and they may be operated alternately or reversely, as long as each step can achieve its own function and accomplish the oriented change of host cell genotype.
The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
The addition amount, content and concentration of various substances are referred to herein, wherein the percentage refers to the mass percentage unless otherwise specified.
Examples
Materials and methods
The whole gene synthesis, primer synthesis and sequencing herein were performed by Nanjing Jinruis Biotechnology Ltd.
The molecular biological experiments in the examples include plasmid construction, enzyme digestion, competent cell preparation, transformation, and the like, which are mainly performed with reference to molecular cloning, a guide to experiments (third edition), J. SammBruk, D.W. Lassel (America), Huangpeitang, et al, science publishers, Beijing, 2002). For example, the methods for competent cell transformation and competent cell preparation are described in Chapter 1, 96 of molecular cloning, A laboratory Manual (third edition). The specific experimental conditions can be determined by simple experiments if necessary.
Main culture medium:
LB culture medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride. (20 g/L agar powder was additionally added to the solid medium.)
Original Escherichia coli Nissle1917, plasmid pTargetF (Addgene:62226), pCas (Addgene:62225) and pSU2718 were gifted by the Yangxing project group in the molecular plant science and innovation center of Chinese academy of sciences.
plasmids such as pTargetF plasmid (Addgene:62226), pCas plasmid (Addgene:62225) and pSU2718 are stored by Shanghai Touchen Biotechnology Limited responsibility company, and any unit and person can obtain the plasmid and related plasmids and bacteria for verifying the invention, but the plasmid and related plasmids and bacteria cannot be used for other purposes including development and utilization, scientific research and teaching without permission of the Shanghai Touchen Biotechnology Limited responsibility company.
Construction of surface-displayed PAL engineered probiotics reference is made to the literature of Isabella, V.et al (Development of a synthetic bacterial therapeutic for the human metabolic cleavage phenyl ketonuria. Nat Biotechnol 36, 857-. The genomic modification of probiotic bacteria was carried out using the Crispr-Cas9 method (Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, apple Environ Microbiol,2015) of Jiang Y et al.
The primer sequence information used in the following examples is shown in Table 1.
TABLE 1 primer List used in the examples
Figure BDA0002887476820000071
Figure BDA0002887476820000081
Figure BDA0002887476820000091
Figure BDA0002887476820000101
Figure BDA0002887476820000111
Figure BDA0002887476820000121
In Table 1, "-F" in the name represents the forward direction; "-R" represents reverse.
Example 1: construction of surface display PAL engineering probiotics EcN/pINP-stlA
1.1 pINP-stlA plasmid construction
(1) PCR amplification of 15A fragment, about 1kb, using pSU2718 plasmid as template and 15A-F/Psu-RG as primers; using pPIC9k plasmid as template and Kan-FG/Kan-R (15A) as primer to PCR amplify Kan fragment with about 1 kb; using pUC-inaK plasmid as template (Kisry synthesis) and inaK-F/inaK-R as primer, carrying out PCR amplification to obtain inaK-N fragment of about 600 bp; using pUC-sltA plasmid (Kinry synthesis) as a template and stlA (inaK) -F/stlA-R as primers, and carrying out PCR amplification on a stlA (inaK) fragment with the length of about 1.6 kb; the rrnB fragment, about 400bp, is obtained by PCR amplification with pTrc99a plasmid as template and rrnB-F/rrnB-R as primer.
(2)15A, Kan, inaK-N, stlA (inaK) and rrnB fragments were ligated by the DNA assembly method (DNA assembly Kit purchased from all-over-the-gold), the ligation products were transformed into DH 5. alpha. chemocompetent cells, and the resuscitative solution was applied to LB solid plates containing kanamycin (final concentration 50. mu.g/mL) to obtain transformants containing pINP-stlA plasmid.
1.2 construction of recombinant engineering probiotic EcN/pINP-stlA
(1) Coli Nissle1917(EcN) transformed competent cells were prepared according to the method described in molecular cloning, Experimental guidelines for molecular cloning (third edition).
(2) The pINP-stlA plasmid is transformed into EcN transformed competent cells, and bacterial liquid is coated on an LB solid plate containing kanamycin (with the final concentration of 50 mu g/mL) to obtain EcN/pINP-stlA recombinant engineering probiotics.
Example 2: construction of engineering probiotics TYS009 and TYS009/pINP-stlA for degrading phenylalanine
2.1 pTargetF-malP plasmid construction
Using pTargetF plasmid (Addgene:62226) as a template and malP-N20-F/pTargetF-R as a primer, carrying out PCR amplification on a malP-N20 fragment, carrying out self-ligation after about 2.2kb and DpnI digestion on the PCR fragment, transforming the fragment into an Escherichia coli DH5 alpha chemical competent cell, and carrying out screening on an LB solid plate containing spectinomycin (50 mu g/mL) at 37 ℃ to obtain pTargetF-malP plasmid; methods for preparing chemically transformed competent cells are described in molecular cloning, A laboratory Manual (third edition).
2.2 MalP site knock-in Pj23119-stlA
(1) Preparing an electric conversion section: using E.coli Nissle1917 genome as a template, and using malP-F1/malP-R1 and malP-F2/malP-R2 as primers respectively, and performing PCR amplification to obtain malP-UP and malP-DN fragments respectively with about 600 bp; using pUC-sltA plasmid (Kinry synthesis) as a template and stlA (malP) -F/stlA (malP) -R as primers, and carrying out PCR amplification to obtain a stlA (malP) fragment with about 1.6 kb; PCR-amplifying a Pj23119(malP) fragment with about 100bp by using the pTargetF plasmid as a template and Pj23119(malP) -F/Pj23119(malP) -R as primers; using Pj23119(malP)/stlA (malP) fragment as a template, using Pj23119(malP) -F/stlA (malP) -R as a primer, and performing Overlap PCR amplification to obtain stlA (malP) -2 fragment with about 1.7 kb; using malP-UP/stlA (malP) -2/malP-DN fragment as a template and malP-F1/malP-R2 as primers, and carrying out Overlap PCR amplification to obtain malP, wherein Pj23119-stlA fragment is about 3 kb.
(2) Preparing competent cells: coli Nissle1917, and screening on LB solid plates containing kanamycin (50. mu.g/mL) to obtain EcN/pCas transformants (see molecular cloning, laboratory Manual, third edition). EcN/pCas single colonies were picked up in 4mL LB tubes containing kanamycin (50. mu.g/mL), cultured at 30 ℃ at 220rpm, and grown at OD600At 0.4, induction was performed by adding arabinose to a final concentration of 10mM, and culturing was continued for 1 hour to prepare electroporation competent cells (Multigene differentiation in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al, apple Environ Microbiol, 2015);
(3) electric conversion: the malP: Pj23119-stlA fragment and pTargetF-malP plasmid were electroporated into EcN/pCas competent cells (electroporation conditions: 2.5kV, 200. omega., 25. mu.F), spread on LB plate containing spectinomycin (50. mu.g/ml) and kanamycin (50. mu.g/ml), and cultured overnight at 30 ℃; after a single colony grows out, colony PCR verification is carried out by using a malP-V-F/stlA-V-R primer, and a positive fragment is about 600 bp;
(4) loss of pTargetF-malP plasmid: selecting a colony which is verified as a positive single colony by PCR, inoculating the colony in an LB test tube containing kanamycin (the final concentration is 50 mu g/ml), adding IPTG (isopropyl thiogalactoside) with the final concentration of 1mM, and culturing overnight at 30 ℃; the bacterial liquid in the test tube on the next day is directly streaked on an LB plate containing kanamycin (the final concentration is 50 mu g/ml) and cultured overnight at the temperature of 30 ℃; the following day a single colony was picked and transferred to LB plates containing spectinomycin (final concentration 50. mu.g/ml), and if it did not grow, it showed that the pTargetF-malP plasmid had been lost, giving EcN (malP:: Pj23119-stlA)/pCas strain.
2.3 pTargetF-yicS plasmid construction
Using pTargetF plasmid as a template and yicS-N20-F/pTargetF-R as a primer, carrying out PCR amplification on yicS-N20 fragment, carrying out self-ligation after about 2.2kb and DpnI digestion on the PCR fragment, transforming the PCR fragment into Escherichia coli DH5 alpha chemically competent cells, and screening on an LB solid plate containing spectinomycin (50 mu g/mL) at 37 ℃ to obtain pTargetF-yicS plasmid; methods for preparing chemically transformed competent cells are described in molecular cloning, A laboratory Manual (third edition).
2.4 yicS site knock-in Pj23119-stlA
(1) Preparing an electric conversion section: using E.coli Nissle1917 genome as a template, and using yicS-F1/yicS-R1 and yicS-F2/yicS-R2 as primers respectively, and obtaining yicS-UP and yicS-DN fragments respectively by PCR amplification, wherein the fragments are respectively about 600 bp; using the stlA (malP) -2 fragment as a template and Pj23119(yicS) -F/stlA (malP) -R as primers, and carrying out PCR amplification to obtain a stlA (yicS) fragment with about 1.7 kb; the yicS-UP/stlA (yicS)/yicS-DN fragment is used as a template, yicS-F1/yicS-R2 is used as a primer, and Overlap PCR is carried out to obtain yicS, wherein the yicS fragment Pj23119-stlA is about 3 kb.
(2) Electric conversion: the same procedure as 2.2, the yicS fragment Pj23119-stlA and pTargetF-yicS plasmid were electroporated into EcN (malP:: Pj23119-stlA)/pCas competent cells, plated on LB plates containing spectinomycin (50. mu.g/ml) and kanamycin (50. mu.g/ml), and cultured overnight at 30 ℃; growing a single colony, and carrying out colony PCR verification by using a yicS-V-F/stlA-V-R primer to obtain a positive fragment of about 500 bp;
(3) loss of pTargetF-yicS plasmid: the same procedure as 2.2 gave EcN (malP:: Pj23119-stlA, yicS:: Pj23119stlA)/pCas strain.
2.5 MalE site knock-in apramycin resistance Gene
(1) Amplification of the homologous arm-containing fragment: PCR amplifying an Apr (malE) fragment, about 1.4kb, DpnI digesting the PCR fragment, using pIJ773 plasmid (Gust B, et al, PCR-targeted Streptomyces gene replacement information a protein domain need for biosynthesis of the sesquiterpene soil odor or biosmin. Proc. Natl. Acad. Sci. U.S.A.2003,100: 1541. 1546.) as a template;
(2) electric conversion: the same procedure as 2.2, Apr (malE) fragment was electroporated into EcN (malP:: stlA, yicS:: stlA)/pCas competent cells, plated on LB plates containing apramycin (50. mu.g/ml) and kanamycin (50. mu.g/ml), and cultured overnight at 30 ℃; the single colony is grown out and is subjected to colony PCR verification by using malE-V-F/Apr-V-R primers, the positive fragment is about 800bp, and EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Apr)/pCas strains are obtained.
2.6 pTargetF-Apr plasmid construction
Using pTargetF plasmid as a template and Apr-N20-F/pTargetF-R as a primer, carrying out PCR amplification on an Apr-N20 fragment, carrying out self-ligation after digesting the PCR fragment by about 2.2kb and DpnI, transforming the fragment into an Escherichia coli DH5 alpha chemically competent cell, and screening on an LB solid plate containing spectinomycin (50 mu g/mL) at 37 ℃ to obtain pTargetF-Apr plasmid; methods for preparing chemically transformed competent cells are described in molecular cloning, A laboratory Manual (third edition).
2.7 malE site knock-in Pj23119-stlA
(1) Preparing an electric conversion section: using E.coli Nissle1917 genome as a template, and using malE-F1/malE-R1 and malE-F2/malE-R2 as primers respectively, and performing PCR amplification to obtain malE-UP and malE-DN fragments respectively with about 600 bp; using the stlA (malP) -2 fragment as a template and Pj23119(malE) -F/stlA (malP) -R as primers, and carrying out PCR amplification to obtain a stlA (malE) fragment with about 1.7 kb; MalE-UP/stlA (malE)/malE-DN fragment is used as a template, malE-F1/malE-R2 is used as a primer, and Overlap PCR is carried out to obtain malE, Pj23119-stlA fragment and about 3 kb.
(2) Electric conversion: the same procedure as 2.2, the malE:: Pj23119-stlA fragment and pTargetF-Apr plasmid were electroporated into EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Apr)/pCas competent cells, plated on LB plate containing spectinomycin (50. mu.g/ml) and kanamycin (50. mu.g/ml), and cultured overnight at 30 ℃; after a single colony grows out, colony PCR verification is carried out by using malE-V-F/stlA-V-R primers, and a positive fragment is about 500 bp;
(3) loss of pTargetF-Apr plasmid: the same procedure as 2.2 gave EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA)/pCas strain.
2.8 pTargetF-rhtC plasmid construction
Using pTargetF plasmid as a template and rhtC-N20-F/pTargetF-R as a primer, carrying out PCR amplification on rhtC-N20 fragment, carrying out self-ligation after about 2.2kb and DpnI digestion on the PCR fragment, transforming the fragment into Escherichia coli DH5 alpha chemically competent cells, and screening on an LB solid plate containing spectinomycin (50 mu g/mL) at 37 ℃ to obtain pTargetF-rhtC plasmid; methods for preparing chemically transformed competent cells are described in molecular cloning, A laboratory Manual (third edition).
2.9 construction of pTargetT-Ptac-stlA (rhtC) plasmid
E.coli Nissle1917 genome is taken as a template, rhtC-F1/rhtC-R1 and rhtC-F2/rhtC-R2 are respectively taken as primers, and rhtC-UP and rhtC-DN fragments are respectively obtained by PCR amplification and are respectively about 600 bp; taking p57-tac plasmid as a template and tac-F/tac-R as a primer, and carrying out PCR amplification on a tac fragment with about 1.5 kb; using the stlA (malP) -2 fragment as a template and stlA (rhtC) -F/stlA (rhtC) -R as primers, and carrying out PCR amplification to obtain a stlA (rhtC) fragment with about 1.6 kb; the rhtC-UP, tac, stlA (rhtC) and rhtC-DN fragments were cloned into EcoRI/HindIII sites of pTargetF-rhtC using the DNA assembly method (DNA assembly Kit is purchased from all-type gold) to obtain pTargetT-Ptac-stlA (rhtC) plasmid.
2.10 knock-in of the rhtC site Ptac-stlA
(1) Electric conversion: the same procedure as 2.2, pTargetT-Ptac-stlA (rhtC) plasmid was electroporated into EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA)/pCas competent cells, plated on LB plates containing spectinomycin (50. mu.g/ml) and kanamycin (50. mu.g/ml), and cultured overnight at 30 ℃; the single colony is grown out and is subjected to colony PCR verification by using rhtC-V-F/stlA-V-R primers, and the positive fragment is about 2.5 kb;
(2) loss of pTargetT-Ptac-stlA (rhtC) plasmid: the same procedure as 2.2 gave EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA)/pCas strain.
2.11 pTargetF-exo plasmid construction
Using pTargetF plasmid as a template and exo-N20-F/pTargetF-R as a primer, carrying out PCR amplification on an exo-N20 fragment, carrying out self-ligation after digesting the PCR fragment by about 2.2kb and DpnI, transforming the fragment into an Escherichia coli DH5 alpha chemically competent cell, and screening on an LB solid plate containing spectinomycin (50 mu g/mL) at 37 ℃ to obtain pTargetF-exo plasmid; methods for preparing chemically transformed competent cells are described in molecular cloning, A laboratory Manual (third edition).
2.12 pTargetT-Ptac-stlA (exo) plasmid construction
Using E.coli Nissle1917 genome as a template, respectively using exo-F1/exo-R1 and exo-F2/exo-R2 as primers, and respectively obtaining exo-UP and exo-DN fragments by PCR amplification, wherein the exo-UP and exo-DN fragments are respectively about 600 bp; PCR-amplifying tac (exo) fragment of about 2.1kb with plasmid p57-tac as template and tac (exo) -F/tac-R as primer; using pTargetT-Ptac-stlA (rhtC) plasmid as a template and stlA (rhtC) -F/stlA (exo) -R as primers, and carrying out PCR amplification to obtain a stlA (exo) fragment with about 1.6 kb; the exo-UP, tac (exo), stlA (exo) and exo-DN fragments were cloned into EcoRI/HindIII sites of pTargetF-exo using the DNA assembly method (DNA assembly Kit is purchased from all-purpose gold) to obtain pTargetT-Ptac-stlA (exo) plasmid.
2.13 exo site knock-in Ptac-stlA
(1) Electric conversion: the same procedure as 2.2, pTargetT-Ptac-stlA (exo) plasmid was electrotransformed into EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA)/pCas competent cells, plated on LB plates containing spectinomycin (50. mu.g/ml) and kanamycin (50. mu.g/ml), and cultured overnight at 30 ℃; carrying out colony PCR verification on the single colony by using exo-V-F/stlA-V-R primers, wherein the positive fragment is about 3 kb;
(2) loss of pTargetT-Ptac-stlA (exo) plasmid: the same procedure as 2.2 gave EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA)/pCas strain.
2.14 pTargetF-lacZ plasmid construction
Using pTargetF plasmid as a template and lacZ-N20-F/pTargetF-R as a primer, carrying out PCR amplification on a lacZ-N20 fragment, carrying out self-ligation after digesting the PCR fragment by about 2.2kb and DpnI, transforming the fragment into an Escherichia coli DH5 alpha chemically competent cell, and screening on an LB solid plate containing spectinomycin (50 mu g/mL) at 37 ℃ to obtain pTargetF-lacZ plasmid; methods for preparing chemically transformed competent cells are described in molecular cloning, A laboratory Manual (third edition).
2.15 lacZ site knock-in Pj23119-pheP
(1) Preparing an electric conversion section: taking E.coli Nissle1917 genome as a template, and taking lacZ-F1/lacZ-R1 and lacZ-F2/lacZ-R2 as primers respectively, and carrying out PCR amplification to obtain lacZ-UP and lacZ-DN fragments respectively, wherein the fragments are about 600bp and 700bp respectively; PCR-amplifying a Pj23119(lacZ) fragment with pTargetF plasmid as a template and Pj23119(lacZ) -F/Pj23119(lacZ) -R as primers to obtain about 100 bp; taking E.coli MG1655 genome as a template, taking pheP (lacZ) -F/pheP (lacZ) -R as a primer, and carrying out PCR amplification to obtain a pheP (lacZ) -1 fragment with about 1.4 kb; taking Pj23119(lacZ)/pheP (lacZ) -1 fragment as a template and Pj23119(lacZ) -F/pheP (lacZ) -R as a primer, and carrying out PCR amplification to obtain pheP (lacZ) -2 fragment with about 1.5 kb; using lacZ-UP/pheP (lacZ) -2/lacZ-DN fragment as a template and lacZ-F1/lacZ-R2 as a primer, and carrying out Overlap PCR amplification to obtain lacZ, wherein Pj23119-pheP fragment is about 2.8 kb.
(2) Electric conversion: the same procedure as 2.2, the lacZ fragment Pj23119-pheP and pTargetF-lacZ plasmids were electroporated into EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA)/pCas competent cells, plated on LB plates containing spectinomycin (50. mu.g/ml) and kanamycin (50. mu.g/ml), and incubated overnight at 30 ℃; a single colony is grown, and the lacZ-V-F/pheP-V-R primer is used for carrying out colony PCR verification, and the positive fragment is about 500 bp;
(3) loss of pTargetF-lacZ plasmid: the same procedure as 2.2 gave EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119-pheP)/pCas strain.
2.16 pTargetF-agaI plasmid construction
Using pTargetF plasmid as a template and agaI-N20-F/pTargetF-R as a primer, carrying out PCR amplification on an agaI-N20 fragment, carrying out self-ligation after digesting the PCR fragment by about 2.2kb and DpnI, transforming the fragment into Escherichia coli DH5 alpha chemically competent cells, and screening on an LB solid plate containing spectinomycin (50 mu g/mL) at 37 ℃ to obtain pTargetF-agaI plasmid; methods for preparing chemically transformed competent cells are described in molecular cloning, A laboratory Manual (third edition).
2.17 agaI site knock-in Pj23119-pheP
(1) Preparing an electric conversion section: using E.coli Nissle1917 genome as a template, and using agaI-F1/agaI-R1 and agaI-F2/agaI-R2 as primers respectively, and performing PCR amplification to obtain agaI-UP and agaI-DN fragments respectively, wherein the fragments are about 600bp respectively; taking the pheP (lacZ) -2 fragment as a template and the pheP (agaI) -F/pheP (agaI) -R as primers, and carrying out PCR amplification to obtain a pheP (agaI) fragment with about 1.4 kb; the agaI is obtained by taking agaI-UP/pheP (agaI) -2/agaI-DN fragments as a template and agaI-F1/agaI-R2 as primers through Overlap PCR amplification, wherein Pj23119-pheP fragments are about 2.7 kb.
(2) Electric conversion: the same procedure as 2.2, the agaI fragment Pj23119-pheP and pTargetF-agaI plasmid were electroporated into EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119-pheP)/pCas competent cells, plated on LB containing spectinomycin (50. mu.g/ml) and kanamycin (50. mu.g/ml), and cultured overnight at 30 ℃; after a single colony grows out, colony PCR verification is carried out by utilizing an agaI-V-F/pheP-V-R primer, and a positive fragment is about 500 bp;
(3) loss of pTargetF-agaI plasmid: the same procedure as 2.2 gave EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119-pheP, agaI:: Pj23119-pheP)/pCas strain.
2.18 pTargetF-araBD plasmid construction
Using pTargetF plasmid as a template and araBD-N20-F/pTargetF-R as a primer, carrying out PCR amplification on an araBD-N20 fragment, carrying out self-ligation after about 2.2kb and DpnI digestion on the PCR fragment, transforming the PCR fragment into an Escherichia coli DH5 alpha chemically competent cell, and screening on an LB solid plate containing spectinomycin (50 mu g/mL) at 37 ℃ to obtain pTargetF-araBD plasmid; methods for preparing chemically transformed competent cells are described in molecular cloning, A laboratory Manual (third edition).
2.19 pTargetT-Para-pma (araBD) plasmid construction
Using E.coli Nissle1917 genome as a template, and using araBD-F1/araBD-R1 and araBD-F2/araBD-R2 as primers respectively, and performing PCR amplification to obtain araBD-UP and araBD-DN fragments respectively with about 600 bp; PCR amplification using pUC-pma plasmid (Kinry synthesis) as template and pma (araBD) -F/pma (araBD) -R as primers gave a pma (araBD) fragment, about 1.6 kb; the araBD-UP, araBD-DN, and stlA (araBD) fragments were cloned into EcoRI/HindIII sites of pTargetF-araBD using the DNA assembly method (DNA assembly Kit is available from all-type gold) to obtain pTargetT-Para-pma (araBD) plasmid.
2.20 the araBD site knock-in Para-pma
(1) Electric conversion: the same procedure as 2.2, pTargetT-Para-pma (araBD) plasmid was electroporated into EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119-pheP, agaI:: Pj23119-pheP)/pCas competent cells, plated on LB plate containing spectinomycin (50. mu.g/ml) and kanamycin (50. mu.g/ml), and cultured overnight at 30 ℃; a single colony is grown out, colony PCR verification is carried out by utilizing araBD-V-F/pma-V-R primers, and a positive fragment is about 1.6 kb;
(2) loss of pTargetT-Para-pma (araBD) plasmid: the same procedure as 2.2 gave EcN (malP:: stlA, yicS:: stlA, malE:: stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: pheP, agaI:: pheP, araBD:: Para-pma)/pCas strain.
2.21 pTargetF-dapA plasmid construction
Using pTargetF plasmid as a template and dapA-N20-F/pTargetF-R as a primer, carrying out PCR amplification on a dapA-N20 fragment, carrying out self-ligation after digesting the PCR fragment by about 2.2kb and DpnI, transforming the fragment into Escherichia coli DH5 alpha chemically competent cells, and carrying out screening on an LB solid plate containing spectinomycin (50 mu g/mL) at 37 ℃ to obtain pTargetF-dapA plasmid; methods for preparing chemically transformed competent cells are described in molecular cloning, A laboratory Manual (third edition).
2.22 pTargetT-dapA plasmid construction
Using E.coli Nissle1917 genome as a template, and dapA-F1/dapA-R1 and dapA-F2/dapA-R2 as primers respectively, and performing PCR amplification to obtain dapA-UP fragments and dapA-DN fragments which are respectively about 600 bp; the dapA-UP and dapA-DN fragments were cloned into EcoRI/HindIII sites of pTargetF-dapA by the DNA assembly method (DNA assembly Kit is purchased from all-purpose gold) to obtain pTargetT-dapA plasmid.
2.23 dapA Gene knockout
(1) Electric conversion: the same procedure as 2.2, pTargetT-dapA plasmid was electrotransformed into EcN (malP:: stlA, yicS:: stlA, malE:: stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: pheP, agaI:: pheP, araBD:: Para-pma)/pCas competent cells, plated on LB plates containing spectinomycin (50. mu.g/mL), kanamycin (50. mu.g/mL) and diaminopimelic acid (100. mu.g/mL), and incubated overnight at 30 ℃; the single colony is grown out, colony PCR verification is carried out by using dapA-V-F/dapA-V-R primers, and the positive fragment is about 1 kb;
(2) loss of pTargetT-dapA plasmid: the same procedure as 2.2 gave EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119-pheP, agaI:: Pj23119-pheP, araBD:: Para-pma,. DELTA.dapA)/pCas strains.
(3) Loss of the pCas plasmid: selecting a colony with positive loss of pTargetT-dapA plasmid, inoculating the colony in an LB test tube, and culturing overnight at 37 ℃; streaking the secondary day bacterium liquid on LB plate, culturing at 37 deg.c overnight; the next day a single colony was picked and transferred to LB plates containing kanamycin (final concentration 50. mu.g/mL) and diaminopimelic acid (100. mu.g/mL) indicating loss of pCas plasmid if it could not grow, giving EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119-pheP, agaI:: Pj23119-pheP, araBD:: Para-pma,. DELTA.dapA) strain, designated TYS 009.
2.24 construction of recombinant engineered probiotic TYS009/pINP-stlA
(1) Engineering probiotics TYS009 chemotropic cells were prepared, and the preparation method of chemotropic cells was referred to "molecular cloning Experimental guidelines" (third edition).
(2) The pINP-stlA plasmid was transformed into TYS009 chemocompetent cells, and the bacterial solution was spread on LB solid plate containing kanamycin (final concentration 50. mu.g/mL) to obtain TYS009/pINP-stlA recombinant engineered probiotic bacteria.
Example 3: construction of arginine pathway enhanced engineering probiotic TYS010
3.1 knock-in apramycin resistance Gene at the argA site
(1) Amplification of the homologous arm-containing fragment: PCR amplifying an Apr (argA) fragment, about 1.4kb, and DpnI digesting the PCR fragment, using pIJ773 plasmid (Gust B, et al, PCR-targeted Streptomyces gene replacement information a protein domain need for biosynthesis of the sesquiterpene soil odor or biosmin. Proc. Natl. Acad. Sci. U.S.A.2003,100: 1541. supplement 1546.) as a template and Apr (argA) -F/Apr (argA) -R as a primer;
(2) electric conversion: the same procedure as 2.2, the Apr (argA) fragment was electroporated into EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119-pheP, agaI:: Pj23119-pheP, araBD:: Para-pma,. DELTA.dapA)/pCas competent cells plated on LB plates containing apramycin (50. mu.g/mL), kanamycin (50. mu.g/mL) and diaminopimelic acid (100. mu.g/mL) overnight at 30 ℃; a single colony was grown and confirmed by colony PCR using argA-V-F/argA-V-R primers, and the positive fragment was about 2.6kb, yielding EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119-pheP, agaI:: Pj23119-pheP, araBD:: Para-pma,. DELTA.dada, argpA: Apr)/pCas strains.
3.2 construction of argA site-directed mutagenesis (Y19C)
(1) Preparing an electric conversion section: taking E.coli Nissle1917 genome as a template, and argAmu-F1/argAmu-R1 and argAmu-F2/argAmu-R2 as primers respectively, and performing PCR amplification to obtain argAmu-1 fragments and argAmu-2 fragments which are respectively about 500 bp; the fragments argAmu-1 and argAmu-2 were used as templates, and argAmu-F1/argAmu-R2 was used as primers, and the fragments argA (i.e., argAmut (Y19C)) were obtained by Overlap PCR amplification to about 1 kb.
(2) Electric conversion: the same procedure as 2.2, electro-transformation of the argA fragment and pTargetF-Apr plasmid into EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119-pheP, agaI:: Pj23119-pheP, araBD:: Para-pma,. DELTA.dapA, argA:: Apr)/pCas competent cells, plating on LB plates containing spectinomycin (50. mu.g/mL), kanamycin (50. mu.g/mL) and diaminopimelic acid (100. mu.g/mL), overnight culture at 30 ℃; after single colony grows out, colony PCR verification is carried out by utilizing argA-V-F/argA-V-R primers, and a positive fragment is about 1.1 kb;
(3) loss of pTargetF-Apr plasmid: the same procedure as 2.2 gave EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119-pheP, agaI:: Pj23119-pheP, araBD:: Para-pma,. DELTA.dapA, argA)/pCas strains.
3.3 pTargetF-argR plasmid construction
Using pTargetF plasmid as a template and argR-N20-F/pTargetF-R as a primer, carrying out PCR amplification on an argR-N20 fragment, carrying out self-ligation after about 2.2kb of the PCR fragment is digested by DpnI, transforming the fragment into Escherichia coli DH5 alpha chemically competent cells, and screening on an LB solid plate containing spectinomycin (50 mu g/mL) at 37 ℃ to obtain pTargetF-argR plasmid; methods for preparing chemically transformed competent cells are described in molecular cloning, A laboratory Manual (third edition).
3.4 argR Gene knockout
(1) Preparing an electric conversion section: using E.coli Nissle1917 genome as a template, and argR-aL-F/argR-aL-R, argR-aR-F/argR-aR-R as primers, respectively, and performing PCR amplification to obtain argR-KO1 and argR-KO2 fragments which are respectively about 500 bp; the argR-KO fragment was obtained by performing an Overlap PCR amplification using argR-KO1 and argR-KO2 fragments as templates and argR-aL-F/argR-aR as primers, and having about 1 kb.
(2) Electric conversion: the procedure as in 2.2, electro-transformation of the argR-KO fragment and pTargetF-argR plasmid into EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119-pheP, agaI:: Pj23119-pheP, araBD:: Para-pma, Δ dapA, argA)/pCas competent cells, plated on LB plates containing spectinomycin (50 μ g/mL), kanamycin (50 μ g/mL) and diaminopimelic acid (100 μ g/mL) overnight at 30 ℃; after single colony grows out, colony PCR verification is carried out by using argR-aL-F/argR-aR-R primers, and a positive fragment is about 1 kb;
(3) loss of pTargetF-argR plasmid: the same procedure as 2.2 gave EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119-pheP, agaI:: Pj23119-pheP, araBD:: Para-pma, Δ dapA, argA, Δ argR)/pCas strains.
(4) Loss of the pCas plasmid: the same procedure as 2.23 gave EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119-pheP, agaI:: Pj23119-pheP, araBD:: Para-pma, Δ dapA, argA, Δ argR) strain, which was named TYS 010.
Example 4: construction of efflux pump enhanced engineering probiotic TYS011
4.1 construction of pTargetF-lacZ2 plasmid
Using pTargetF plasmid as a template and lacZ2-N20-F/pTargetF-R as a primer, carrying out PCR amplification on lacZ2-N20 fragment, carrying out self-ligation after about 2.2kb and DpnI digestion on the PCR fragment, transforming the fragment into Escherichia coli DH5 alpha chemical competent cells, and carrying out screening on an LB solid plate containing spectinomycin (50 mu g/mL) at 37 ℃ to obtain pTargetF-lacZ2 plasmid; methods for preparing chemically transformed competent cells are described in molecular cloning, A laboratory Manual (third edition).
4.2 lacZ site knock-in Pj23119-acrA
(1) Preparing an electric conversion section: taking E.coli Nissle1917 genome as a template and lacZ2-L-F/lacZ2-L-R, lacZ2-R-F/lacZ2-R-R as primers respectively, and carrying out PCR amplification to obtain lacZ2-UP and lacZ2-DN fragments which are respectively about 600bp and 700 bp; taking E.coli Nissle1917 genome as a template and acrA-F/acrA-R as a primer, and carrying out PCR amplification to obtain an acrA-1 fragment of about 1.2 kb; using the acrA-1 fragment as a template and acrA-F2/acrA-R as a primer, and carrying out PCR amplification to obtain a Pj23119-acrA fragment with about 1.3 kb; using lacZ2-UP/Pj23119-acrA/lacZ2-DN fragment as template and lacZ2-L-F/lacZ2-R-R as primer, the Overlap PCR amplification obtains lacZ2, Pj23119-acrA fragment, about 2.6 kb.
(2) Electric conversion: the procedure as 2.2, lacZ 2:Pj23119-acrA fragment and pTargetF-lacZ2 plasmid were electroporated into EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119-pheP, agaI:: Pj23119-pheP, araBD:: Para-pma, DeltadapA, argA,. DELTA.arggR)/pCas competent cells, plated on LB containing spectinomycin (50. mu.g/mL), kanamycin (50. mu.g/mL) and diaminopimelic acid (100. mu.g/mL), cultured overnight at 30 ℃; the single colony is grown out and is subjected to colony PCR verification by using a lacZ2-V-F/acrA-V-R primer, and the positive fragment is about 800 bp;
(3) loss of pTargetF-lacZ2 plasmid: the same procedure as 2.2 gave EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119-pheP, agaI:: Pj23119-pheP, araBD:: Para-pma, Δ dapA, argA, Δ argR, lacZ:: Pj23119-acrA)/pCas strains.
(4) Loss of the pCas plasmid: the same procedure as 2.23 gave EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119-pheP, agaI:: Pj23119-pheP, araBD:: Para-pma, Δ dapA, argA, Δ argR, lacZ:: Pj23119-acrA) strain, which was named TYS 011.
Example 5: construction of RBS optimized engineering probiotics TYS012 and TYS012/pINP-stlA
5.1 construction of pTargetF-agaI2 plasmid
Using pTargetF plasmid as a template and agaI2-N20-F/pTargetF-R as a primer, carrying out PCR amplification on an agaI2-N20 fragment, carrying out self-ligation after about 2.2kb and DpnI digestion on the PCR fragment, transforming the PCR fragment into Escherichia coli DH5 alpha chemically competent cells, and carrying out screening on an LB solid plate containing spectinomycin (50 mu g/mL) at 37 ℃ to obtain pTargetF-agaI2 plasmid; methods for preparing chemically transformed competent cells are described in molecular cloning, A laboratory Manual (third edition).
5.2 agaI Gene knockout
(1) Preparing an electric conversion section: by taking a TYS010 genome as a template and agaIH-F/agaIH-R, phePH-F/pheP-seq-R as primers, carrying out PCR amplification to respectively obtain agaIKO-UP and agaIKO-DN fragments of about 500bp and 200bp respectively; the agaIKO-UP and agaIKO-DN fragments are taken as templates, agaIH-F/pheP-seq-R is taken as a primer, and the agaI-KO fragment is obtained by the Overlap PCR amplification, and is about 700 bp.
(2) Electric conversion: the procedure was as in 2.2, the agaI-KO fragment and pTargetF-agaI2 plasmid were electroporated into EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119-pheP, agaI:: Pj23119-pheP, araBD:: Para-pma, dapA, argA:,. DELTA.argR, lacZ:: Pj23119-acrA)/pCas, plated on LB plates containing spectinomycin (50. mu.g/mL), kanamycin (50. mu.g/mL) and diaminopimelic acid (100. mu.g/mL) overnight at 30 ℃; growing a single colony, and carrying out colony PCR verification by using an agaIH/pheP-seq-R primer to obtain a positive fragment of about 700 bp;
(3) loss of pTargetF-agaI2 plasmid: the same procedure as 2.2 gave EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119-pheP, araBD:: Para-pma,. DELTA.dapA, argA,. DELTA.argR, lacZ:: Pj23119-acrA,. DELTA.agaI)/pCas strains.
5.3 insertion of the agaI site into RBS enhanced Pj23119H-pheP
(1) Preparing an electric conversion section: by taking a TYS010 genome as a template and agaIH-F/23119H (agaI) -R, pheP (23119H) -F/pheP-seq-R as primers, carrying out PCR amplification to obtain agaIH-UP and phePH-DN fragments respectively of about 800bp and 300 bp; the agaIH-UP and phePH-DN fragments are taken as templates, the agaIH-F/pheP-seq-R is taken as a primer, and the Overlap PCR is carried out to obtain the agaI fragment Pj23119H-pheP fragment with about 1.1 kb.
(2) Electric conversion: the same procedure as 2.2, the agaI fragment Pj23119H-pheP fragment and pTargetF-Apr plasmid were electroporated into EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119-pheP, araBD:: Para-pma,. DELTA.dapA, argA,. DELTA.argR, lacZ:: Pj23119-acrA,. DELTA.agiI)/pCas competent cells, plated on kanamycin-containing spectinomycin (50. mu.g/mL), 50. mu.g/mL) and diaminopimelic acid (100. mu.g/mL) on LB plates overnight at 30 ℃; carrying out colony PCR verification on the grown single colony by using an agaIH/pheP-seq-R primer, wherein the positive fragment is about 900 bp;
(3) loss of pTargetF-Apr plasmid: the same procedure as 2.2 gave EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119-pheP, agaI:: Pj231 23119H-pheP, araBD:: Para-pma, delta dapA, argA:, delta argR, lacZ:: Pj23119-acrA)/pCas strain.
5.4 construction of pTargetF-lacZ3 plasmid
Using pTargetF plasmid as a template and lacZ3-N20-F/pTargetF-R as a primer, carrying out PCR amplification on lacZ3-N20 fragment, carrying out self-ligation after about 2.2kb and DpnI digestion on the PCR fragment, transforming the fragment into Escherichia coli DH5 alpha chemical competent cells, and carrying out screening on an LB solid plate containing spectinomycin (50 mu g/mL) at 37 ℃ to obtain pTargetF-lacZ3 plasmid; methods for preparing chemically transformed competent cells are described in molecular cloning, A laboratory Manual (third edition).
5.5 lacZ site knock-in RBS enhanced Pj23119H-pheP
(1) Preparing an electric conversion section: taking TYS010 genome as a template and lacZH-F/23119H (agaI) -R as a primer, and carrying out PCR amplification to obtain lacZH-UP (about 700 bp); taking lacZH-UP and phePH-DN fragments as templates, taking lacZH-F/pheP-seq-R as primers, and carrying out Overlap PCR amplification to obtain lacZ, Pj23119H-pheP fragments and about 1 kb.
(2) Electric conversion: the same procedure as 2.2, electro-transformation of the lacZ: Pj23119H-pheP fragment and pTargetF-lacZ3 plasmid into EcN (malP: Pj23119-stlA, yicS: Pj23119-stlA, malE: Pj23119-stlA, rthC: Ptac-stlA, exo: Ptac-stlA, lacZ: Pj23119-pheP, agaI: Pj23119H-pheP, araBD: Para-pma, DeltapA, argA, arggR, lacZ: Pj 23119-acrA)/pCaI cells, plated on agar competent cells containing spectinomycin (50. mu.g/mL), kanamycin (50. mu.g/mL) and diaminopimelic acid (100. mu.g/mL) overnight at 30 ℃ for overnight culture; a single colony is grown out, and colony PCR verification is carried out by using a lacZH-F/pheP-seq-R primer, and a positive fragment is about 1 kb;
(3) loss of pTargetF-lacZ3 plasmid: the same procedure as 2.2 gave EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119H-pheP, agaI:: Pj23119H-pheP, araBD:: Para-pma, Δ dapA, argA:, Δ gR, lacZ:: Pj23119-acrA)/pCas strains.
5.6 pTargetF-malE plasmid construction
Using pTargetF plasmid as a template and malE-N20-F/pTargetF-R as a primer, carrying out PCR amplification on a malE-N20 fragment, carrying out self-ligation after digesting the PCR fragment by about 2.2kb and DpnI, transforming the fragment into an Escherichia coli DH5 alpha chemical competent cell, and screening on an LB solid plate containing spectinomycin (50 mu g/mL) at 37 ℃ to obtain pTargetF-malE plasmid; methods for preparing chemically transformed competent cells are described in molecular cloning, A laboratory Manual (third edition).
5.7 malE Gene knockout
(1) Preparing an electric conversion section: respectively obtaining malEKO-UP fragments and malEKO-DN fragments which are respectively about 300bp and 100bp by PCR amplification by taking a TYS010 genome as a template and malE-LH-F/malE-LH-R, stlAH-F/stlA-seq-R as primers; the malEKO-UP and malEKO-DN fragments are taken as templates, and the malE-LH-F/stlA-seq-R is taken as a primer, so that the malE-KO fragment with about 400bp is obtained by the Overlap PCR amplification.
(2) Electric conversion: the procedure as in 2.2, electro-transformation of the malE-KO fragment and pTargetF-malE plasmid into EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119H-pheP, agaI:: Pj23119H-pheP, araBD:: Para-pma, DeltadapA, argA,. DELTA.argR, lacZ:: Pj23119-acrA)/pCaS, plated on plates containing spectinomycin (50. mu.g/mL), kanamycin (50. mu.g/mL) and diaminopimelic acid (100. mu.g/mL) overnight at 30 ℃; after a single colony grows out, colony PCR verification is carried out by using a malE-LH-F/stlA-seq-R primer, and a positive fragment is about 400 bp;
(3) loss of pTargetF-malE plasmid: the same procedure as 2.2 gave EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA,. DELTA.malE, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119H-pheP, agaI:: Pj23119H-pheP, araBD:: Para-pma,. DELTA.dapA, argA:,. DELTA.ar, lacZ:: Pj23119-acrA)/pCas strain.
5.8 malE site knockout RBS enhanced Pj23119H-stlA
(1) Preparing an electric conversion section: taking a TYS010 genome as a template, respectively taking malE-LH-F/23119H (malE) -R, stlA (23119H) -F/stlA-seq-R as primers, and respectively obtaining malEH-UP and stlAH-DN with about 900bp and 100bp by PCR amplification; the malEH-UP and stlAH-DN fragments are taken as templates, and the malE-LH-F/stlA-seq-R is taken as a primer, and the Overlap PCR is carried out to obtain the malE, Pj23119H-stlA fragment and about 1 kb.
(2) Electric conversion: the procedure was as in 2.2, electro-transforming the malE:: Pj23119H-stlA fragment and pTargetF-Apr plasmid into EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA,. DELTA.malE, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119H-pheP, agaI:: Pj23119H-pheP, araBD:: Para-pma, Δ dapA, argA,. DELTA.argR, lacZ:: Pj23119-acrA)/pCas, plated on LB plates containing spectinomycin (50. mu.g/mL), kanamycin (50. mu.g/mL) and diaminopimelic acid (100. mu.g/mL) overnight at 30 ℃; after a single colony grows out, colony PCR verification is carried out by using malE-LH-F/stlA-seq-R primers, and a positive fragment is about 1 kb;
(3) loss of pTargetF-Apr plasmid: the same procedure as 2.2 gave EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119H-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119H-pheP, agaI:: Pj23119H-pheP, araBD:: Para-pma, Δ dapA, argA,. DELTA.argR, lacZ: 23119:: acrA)/pCas strains.
5.9 pTargetF-malP2 plasmid construction
Using pTargetF plasmid as a template and malP2-N20-F/pTargetF-R as a primer, carrying out PCR amplification on a malP2-N20 fragment, carrying out self-ligation after about 2.2kb and DpnI digestion on the PCR fragment, transforming the fragment into Escherichia coli DH5 alpha chemically competent cells, and carrying out screening on an LB solid plate containing spectinomycin (50 mu g/mL) at 37 ℃ to obtain pTargetF-malP2 plasmid; methods for preparing chemically transformed competent cells are described in molecular cloning, A laboratory Manual (third edition).
5.10 malP Gene knockout
(1) Preparing an electric conversion section: taking TYS010 genome as a template and malP-LH-F/malP-LH-R as a primer, and carrying out PCR amplification to obtain a malPKO-UP fragment of about 400 bp; using malPKO-UP and malEKO-DN fragments as templates, using malP-LH-F/stlA-seq-R as primers, and performing Overlap PCR amplification to obtain a malP-KO fragment of about 500 bp.
(2) Electric conversion: the procedure was as in 2.2, electro-transforming the malP-KO fragment and pTargetF-malP2 plasmid into EcN (malP:: Pj23119-stlA, yicS:: Pj23119-stlA, malE:: Pj23119H-stlA, rthC:: Ptac-stlA, exo:: Ptj-stlA, lacZ:: Pj23119H-pheP, agaI:: Pj23119H-pheP, araBD:: Para-pma, Δ dapA, argA,. DELTA.argR, lacZ:: Pj23119-acrA)/pCas, plated in competent cells, plated on LB plates containing spectinomycin (50. mu.g/mL), kanamycin (50. mu.g/mL) and diaminopimelic acid (100. mu.g/mL) overnight at 30 ℃; after a single colony grows out, colony PCR verification is carried out by using a malP-LH-F/stlA-seq-R primer, and a positive fragment is about 500 bp;
(3) loss of pTargetF-malP plasmid: the same procedure as 2.2 gave EcN (Δ malP, yicS:: Pj23119-stlA, malE:: Pj23119H-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119H-pheP, agaI:: Pj23119H-pheP, araBD:: Para-pma, Δ dapA, argA:, Δ argR, lacZ:: Pj23119-acrA)/pCas strains.
5.11 malP site knock-in RBS enhanced Pj23119H-stlA
(1) Preparing an electric conversion section: using TYS010 genome as template, malP-LH-F/23119H (malE) -R as primer, PCR amplifying to obtain malPH-UP, about 700 bp; the malP fragment Pj23119H-stlA fragment is obtained by performing PCR amplification on malPH-UP and stlAH-DN by taking malP-LH-F/stlA-seq-R as a primer, wherein the length of the malP fragment is about 800 bp.
(2) Electric conversion: the same procedure as 2.2, electro-transforming the malP: (Pj 23119H-stlA fragment) and pTargetF-Apr plasmid into EcN (Δ malP, yicS:: Pj23119-stlA, malE:: Pj23119H-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119H-pheP, agaI:: Pj23119H-pheP, araBD:: Paraa-pma, Δ daa, argA, Δ argR, lacZ:: Pj23119-acrA)/pCas competent cells, plated on LB containing spectinomycin (50 μ g/mL), kanamycin (50 μ g/mL) and diaminopimelic acid (100 μ g/mL), overnight at 30 ℃; after single colony grows out, colony PCR verification is carried out by using malP-LH-F/stlA-seq-R primers, and a positive fragment is about 800 bp.
(3) Loss of pTargetF-Apr plasmid: the same procedure as 2.2 gave EcN (malP:: Pj23119H-stlA, yicS:: Pj23119-stlA, malE:: Pj23119H-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119H-pheP, agaI:: Pj23119H-pheP, araBD:: Para-pma, delta pA, argA:, delta argR, lacZ:: Pj23119-acrA)/pCas strains.
5.12 pTargetF-yicS2 plasmid construction
Using pTargetF plasmid as a template and yicS2-N20-F/pTargetF-R as a primer, carrying out PCR amplification on yicS2-N20 fragment, carrying out self-ligation after about 2.2kb and DpnI digestion of the PCR fragment, transforming the fragment into Escherichia coli DH5 alpha chemically competent cells, and carrying out screening on an LB solid plate containing spectinomycin (50 mu g/mL) at 37 ℃ to obtain pTargetF-yicS2 plasmid; methods for preparing chemically transformed competent cells are described in molecular cloning, A laboratory Manual (third edition).
5.13 yicS Gene knockout
(1) Preparing an electric conversion section: using TYS010 genome as template, using yicS-LH-F/yicS-LH-R as primer, PCR amplifying to obtain yicSKO-UP fragment, about 400 bp; the yicSKO-UP and malEKO-DN fragments are taken as templates, yicS-LH-F/stlA-seq-R is taken as a primer, and the yicS-KO fragment is obtained by the Overlap PCR amplification, and is about 500 bp.
(2) Electric conversion: the procedure was as in 2.2, the yicS-KO fragment and pTargetF-yicS2 plasmid were electroporated into EcN (malP:: Pj23119H-stlA, yicS:: Pj23119-stlA, malE:: Pj23119H-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119H-pheP, agaI:: Pj23119H-pheP, araBD:: Para-pma, Δ dapA, argA,. DELTA.arggR, lacZ:: Pj23119-acrA)/pCas competent cells plated on plates containing spectinomycin (50. mu.g/mL), kanamycin (50. mu.g/mL) and diaminopimelic acid (100. mu.g/mL) overnight at 30 ℃ and LB; growing a single colony, and carrying out colony PCR verification by using a yicS-LH-F/stlA-seq-R primer to obtain a positive fragment of about 500 bp;
(3) loss of pTargetF-yicS2 plasmid: the same procedure as 2.2 gave EcN (malP:: Pj23119H-stlA,. DELTA.yics, malE:: Pj23119H-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119H-pheP, agaI:: Pj23119H-pheP, araBD:: Para-pma,. DELTA.dapA, argA,. DELTA.argR, lacZ:: Pj23119-acrA)/pCas strains.
5.14 yicS site knock-in RBS enhanced Pj23119H-stlA
(1) Preparing an electric conversion section: using TYS010 genome as template, using yicS-LH-F/23119H (male) R as primer, PCR amplifying to obtain yicSH-UP, about 800 bp; the yicSH-UP and stlAH-DN fragments are taken as templates, yicS-LH-F/stlA-seq-R is taken as a primer, and the Overlap PCR is carried out to obtain yicS, Pj23119H-stlA fragments and about 900 bp.
(2) Electric conversion: the same procedure as 2.2, electroconverting yicS:: Pj23119H-stlA fragment and pTargetF-Apr plasmid into EcN (malP:: Pj23119H-stlA,. DELTA.yics, malE:: Pj23119H-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119H-pheP, agaI:: Pj23119H-pheP, araBD:: Paraa-pma,. DELTA.dada, argA,. DELTA.argR, lacZ:: Pj23119-acrA)/pCas, plated in competent cells containing spectinomycin (50. mu.g/mL), kanamycin (50. mu.g/mL) and diaminopimelic acid (100. mu.g/mL), overnight at 30 ℃; and a single colony is grown out, colony PCR verification is carried out by using a yicS-LH-F/stlA-seq-R primer, and a positive fragment is about 900 bp.
(3) Loss of pTargetF-Apr plasmid: the same procedure as 2.2 gave EcN (malP:: Pj23119H-stlA, yicS:: Pj23119H-stlA, malE:: Pj23119H-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119H-pheP, agaI:: Pj23119H-pheP, araBD:: Para-pma, delta dapA, argA:, delta argR, lacZ:: Pj23119-acrA)/pCas strains.
5.15 construction of pTargetF-lacZ4 plasmid
Using pTargetF plasmid as a template and lacZ4-N20-F/pTargetF-R as a primer, carrying out PCR amplification on lacZ4-N20 fragment, carrying out self-ligation after about 2.2kb and DpnI digestion on the PCR fragment, transforming the fragment into Escherichia coli DH5 alpha chemical competent cells, and carrying out screening on an LB solid plate containing spectinomycin (50 mu g/mL) at 37 ℃ to obtain pTargetF-lacZ4 plasmid; methods for preparing chemically transformed competent cells are described in molecular cloning, A laboratory Manual (third edition).
5.16 lacZ site knock-in RBS enhanced Pj23119H-acrA
(1) Preparing an electric conversion section: taking TYS010 genome as a template, and lacZ4-LH-F/lacZ4-LH-R, acrA (23119H) -F/acrA-V-R as primers respectively, and carrying out PCR amplification to obtain lacZ4H-UP-1 and arcAH-DN fragments of about 330bp and 500bp respectively; using lacZ4H-UP-1 fragment as a template, using lacZ4-LH-F/23119H (acrA) -R as a primer, and carrying out OVlap PCR amplification to obtain lacZ4H-UP fragment with about 360 bp; the lacZ is obtained by taking lacZ4H-UP and arcAH-DN fragments as templates and lacZ4-LH-F/acrA-V-R as primers through Overlap PCR amplification, wherein the lacZ is Pj23119H-acrA fragment and is about 900 bp.
(2) Electric conversion: the same procedure as 2.2, electro-transformation of the lacZ:: Pj23119H-acrA fragment and pTargetF-lacZ4 plasmid into EcN (malP:: Pj23119H-stlA, yicS:: Pj23119H-stlA, malE:: Pj23119H-stlA, rthC:: Ptj-stlA, exo:: Ptac-stlA, lacZ:: Pj23119H-pheP, agaI:: Pj23119H-pheP, araBD:: Para-pma, Δ dapA, argA:, Δ argR, lacZ: Pj 19-acrA)/pCas competent cells, plated on plates containing spectinomycin (50. mu.g/mL), kanamycin (50. mu.g/mL) and diamino diacid (100. mu.g/g/mL) overnight at 30 ℃ for culture; the single colony is grown out and is verified by colony PCR by using a lacZ4-LH-F/acrA-V-R primer, and the positive fragment is about 900 bp.
(3) Loss of pTargetF-lacZ4 plasmid: the same procedure as 2.2 gave EcN (malP:: Pj23119H-stlA, yicS:: Pj23119H-stlA, malE:: Pj23119H-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119H-pheP, agaI:: Pj23119H-pheP, araBD:: Para-pma, delta dapA, argA:, delta argR, lacZ:: Pj23119H-acrA)/pCas strains.
(4) Loss of the pCas plasmid: the same procedure as 2.23 gave EcN (malP:: Pj23119H-stlA, yicS:: Pj23119H-stlA, malE:: Pj23119H-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119H-pheP, agaI:: Pj23119H-pheP, araBD:: Para-pma, Δ dapA, argA:, Δ argR, lacZ:: Pj23119H-acrA) strain, named TYS 012.
5.17 construction of recombinant engineered probiotic TYS012/pINP-stlA
(1) The preparation method of engineering probiotics TYS012 transformed competent cells refers to the molecular cloning experimental guidelines (third edition).
(2) pINP-stlA plasmid is transformed into TYS012 transformed competent cells, and bacterial liquid is coated on an LB solid plate containing kanamycin (the final concentration is 50 mu g/mL) to obtain TYS012/pINP-stlA recombinant engineering probiotics.
Example 6: integration of surface display PAL Module into TYS012 genome
6.1 dapA locus knock-in surface display PAL expression Module
(1) Construction of pTargetF-dapA2 plasmid: using pTargetF plasmid as a template and dapA2-N20-F/pTargetF-R as a primer, carrying out PCR amplification on a dapA2-N20 fragment, carrying out self-ligation after about 2.2kb and DpnI digestion on the PCR fragment, transforming the fragment into Escherichia coli DH5 alpha chemical competent cells, and carrying out screening on an LB solid plate containing spectinomycin (50 mu g/mL) at 37 ℃ to obtain pTargetF-dapA2 plasmid; methods for preparing chemically transformed competent cells are described in molecular cloning, A laboratory Manual (third edition).
(2) Preparing an electric conversion section: using EcN genome as a template and dapA2-L-F/23119-R and dapA2-R-F2/dapA2-R-R as primers respectively, and carrying out PCR amplification to obtain dapA2(inaK) -UP and dapA2(inaK) -DN fragments of about 600bp and 500bp respectively; taking pINP-stlA plasmid as a template and Pj23119-F/INP-R as a primer, and carrying out PCR amplification to obtain an INP-stlA fragment with about 2.6 kb; using dapA2(inaK) -UP/INP-stlA/dapA2(inaK) -DN fragment as a template and dapA2-L-F/dapA2-R-R as primers, Overlap PCR was performed to obtain dapA:, inaK-stlA fragment, about 3.6 kb.
(3) Electric conversion: the same procedure as 2.2, electro-transformation of the dapA:: inaK-stlA fragment and pTargetF-dapA2 plasmid into EcN (malP:: Pj23119H-stlA, yicS:: Pj23119H-stlA, malE:: Pj23119H-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119H-pheP, agaI:: Pj23119H-pheP, araBD:: ParapA-pma, Deltadada, argA, DeltaargR:: Pj 23119H-ac)/pCas status cells, plated on a sensory plate containing spectinomycin (50. mu.g/mL), kanamycin (50. mu.g/mL) and diamino diacid (100. mu.g/g/mL) overnight at 30 ℃ for culture; a single colony was grown and confirmed by colony PCR using dapA2-L-F/stlA-seq-R primers, and the positive fragment was about 1.2 kb.
(4) Loss of pTargetF-dapA2 plasmid: the same procedure as 2.2 gave EcN (malP:: Pj23119H-stlA, yicS:: Pj23119H-stlA, malE:: Pj23119H-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119H-pheP, agaI:: Pj23119H-pheP, araBD:: Para-pma, dapA:: inaK-stlA, argA,. DELTA.argR, lacZ:: Pj23119H-acrA)/pCas strains.
(5) Loss of the pCas plasmid: the same procedure as 2.23 gave EcN (malP:: Pj23119H-stlA, yicS:: Pj23119H-stlA, malE:: Pj23119H-stlA, rthC:: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119H-pheP, agaI:: Pj23119H-pheP, araA:: Para-pma, dapA:: inaK-stlA, argA,. DELTA.argR, lacZ:: Pj23119H-acrA) strain, which was named TYS 013.
Example 7: in vitro comparison of the Effect of engineering probiotics on the degradation of phenylalanine
7.1 engineering Probiotics in vitro Activity
(1) Original strains E.coli Nissle1917(EcN), engineered strains EcN/pINP-stlA, TYS009/pINP-stlA, TYS010, TYS011, TYS012/pINP-stlA and TYS013 were inoculated into LB medium (except EcN and EcN/pINP-stlA, diaminopimelic acid was added to the final concentration of 100. mu.g/mL for the other strains), and cultured overnight at 37 ℃ and 250 rpm. The cells were inoculated into 30mL of LB medium (except for EcN and EcN/pINP-stlA, diaminopimelic acid was added to the cells to a final concentration of 100. mu.g/mL) at 1% inoculum size, and the cells were cultured at 37 ℃ and 250rpm for 1.5 hours, and after 1.5 hours, they were further cultured with 10mM arabinose and 1mM IPTG for 3 hours.
(2) Centrifuging at 4000rpm to collect thallus, suspending thallus in M9 culture medium (containing 0.5% glucose), and collecting the thallus concentrate OD600Adjusted to 1.0. 0.4mL of each of the cells was centrifuged at 4500rpm for 10min in a 2mL centrifuge tube, the supernatant was removed, and the cells were suspended in a common tube with 5mL of an assay buffer (M9 medium, 5g/L glucose, 50mM MOPS, 4mM L-phenylalanine) and incubated at 37 ℃ and 250rpm for 3 hours with a positioning time of 0h, followed by 1mL of a sample. 13000rpm, centrifuging for 10min, collecting supernatant sample and performing HPLCAnd (6) measuring.
7.2 HPLC detection method of phenylalanine, phenylpyruvic acid and trans-cinnamic acid
Column ZORBAX SB-C18 column (150 mm. times.4.6 mm, 5 μm); the column temperature is 40 ℃; taking 1.5% acetic acid and acetonitrile as mobile phases, and carrying out gradient elution; an ultraviolet detector is adopted, the detection wavelength is 260nm, and the sample injection amount is 5 mu L.
The results of in vitro detection of phenylalanine degradation by each strain are shown in FIG. 1.
As can be seen from figure 1, the engineered probiotics have the capacity of obviously degrading phenylalanine to generate trans-cinnamic acid. Compared with a TYS010 strain, the enhanced efflux pump acrA strain TYS011 has the advantage that the generation amount of trans-cinnamic acid is improved by 3.1%; compared with a TYS011 strain, the strain TYS012 optimized by stlA, pheP and acrA gene RBS has the advantage that the generation amount of trans-cinnamic acid is improved by 16.8 percent; compared with a TYS012 strain, the generation amount of trans-cinnamic acid is improved by 16.5% when the surface display stlA plasmid is transferred into the TYS012 strain; compared with the strain TYS012, the surface shows that the generation amount of trans-cinnamic acid of the stlA integration strain TYS013 is improved by 15.7%.
In conclusion, the engineering probiotics TYS013 for enhancing the efflux pump gene acrA and displaying PAL on the surface, which is constructed by the invention, has obvious degradation capability on phenylalanine in vitro, is expected to be applied in vivo and is used for treating phenylketonuria.
Sequence listing
<110> Shanghai Toudan Cheng Biotechnology Limited responsibility company
<120> engineering probiotics with surface display phenylalanine ammonia lyase
<130> SHPI2010685
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tatggaatca atacaggatt tggagggaat gccaatttag ttgtgccatt tgagaaaatc 240
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aaaccttgta ttaaagcgtc acaatttact atgttacttt ctgtttgcaa aggttggtct 360
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gcacgagcat tatgtggtat cggcaaagtt tattatatgg gcgcagaaat tgacgctgct 540
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aaactattta aagcctcaat ttctgcgatt gcccttgctg ttgaagcatt acttgcatct 720
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gcaagtgcat tgcgtaattt attggcaggt tcaacgcagg ttaatctatt atctggggtt 840
aaagaacaag ccaataaagc ttgtcgtcat caagaaatta cccaactaaa tgatacctta 900
caggaagttt attcaattcg ctgtgcacca caagtattag gtatagtgcc agaatcttta 960
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gatccagaaa atggcgatgt tctacacggt ggaaatttta tggggcaata tgtcgcccga 1080
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ggcatgtatc aaggttttaa aggcgtccaa ctttctcaaa ccgctttagt tgctgcaatt 1260
cgccatgatt gtgctgcatc aggtattcat accctcgcca cagaacaata caatcaagat 1320
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agtgaaattg cgcctgaaac tgctaaattt taccatgcag tacgcgaaat cagttctcct 1500
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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
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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
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<210> 3
<211> 1422
<212> DNA
<213> Proteus mirabilis HI4320
<400> 3
atgaacattt caaggagaaa gctactttta ggtgttggtg ctgcgggcgt tttagcaggt 60
ggtgcggctt tagttccaat ggttcgccgt gacggcaaat ttgtggaagc taaatcaaga 120
gcatcatttg ttgaaggtac gcaaggggct cttcctaaag aagcagatgt agtgattatt 180
ggtgccggta ttcaagggat catgaccgct attaaccttg ctgaacgtgg tatgagtgtc 240
actatcttag aaaagggtca gattgccggt gagcaatcag gccgtgcata cagccaaatt 300
attagttacc aaacatcgcc agaaatcttc ccattacacc attatgggaa aatattatgg 360
cgtggcatga atgagaaaat tggtgcggat accagttatc gtactcaagg tcgtgtagaa 420
gcgctggcag atgaaaaagc attagataaa gctcaagcgt ggatcaaaac agctaaagaa 480
gcggcaggtt ttgatacacc attaaatact cgcatcatta aaggtgaaga gctatcaaat 540
cgcttagtcg gtgctcaaac gccatggact gttgctgcat ttgaagaaga ttcaggctct 600
gttgatcctg aaacaggcac acctgcactc gctcgttatg ccaaacaaat cggtgtgaaa 660
atttatacca actgtgcagt aagaggtatt gaaactgcgg gtggtaaaat ctctgatgtg 720
gtgagtgaga aaggggcgat taaaacgtct caagttgtac tcgctggggg tatctggtcg 780
cgtttattta tgggcaatat gggtattgat atcccaacgc tcaatgtata tctatcacaa 840
caacgtgtct caggggttcc tggtgcacca cgtggtaatg tgcatttacc taatggtatt 900
catttccgcg aacaagcgga tggtacttat gccgttgcac cacgtatctt tacgagttca 960
atagtcaaag atagcttcct gctagggcct aaatttatgc acttattagg tggcggagag 1020
ttaccgttgg aattctctat tggtgaagat ctatttaatt catttaaaat gccgacctct 1080
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tcagaagttg ttgaacgttg gggtgccgtt gtgagtccaa catttgatga attacctatc 1260
atttctgagg tcaaagaata cccaggctta gtgattaaca cggcaacagt gtggggtatg 1320
acagaaggcc cggcagcggg tgaagtgacc gctgatattg tcatgggcaa gaaacctgtt 1380
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atgaacaaaa acagagggtt tacgcctctg gcggtcgttc tgatgctctc aggcagctta 60
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ggcgtagtaa cagtcaaaac tgaacctctg cagatcacaa ccgagcttcc gggtcgcacc 180
agtgcctacc ggatcgcaga agttcgtcct caagttagcg ggattatcct gaagcgtaat 240
ttcaaagaag gtagcgacat cgaagcaggt gtctctctct atcagattga tcctgcgacc 300
tatcaggcgg catacgacag tgcgaaaggt gatctggcga aagcccaggc tgcagccaat 360
atcgcgcaat tgacggtgaa tcgttatcag aaattgctcg gtactcagta catcagtaag 420
caagagtacg atcaggctct ggctgatgcg caacaggcga atgctgcggt aactgcggcg 480
aaagctgccg ttgaaactgc gcgaatcaat ctggcttaca ccaaagttac ctctccgatt 540
agtggtcgca ttggtaagtc aaacgtgacg gaaggcgcat tggtacagaa cggtcaggcg 600
actgcgctgg caaccgtgca gcaacttgat ccgatctacg ttgatgtgac ccagtccagc 660
aacgacttcc tgcgcctgaa acaggaactg gcgaatggca cgctgaaaca agagaacggc 720
aaagccaaag tgtcgctgat caccagtgac ggcattaagt tcccgcagga cggtacgctg 780
gaattctctg acgttaccgt tgatcagacc actgggtcta tcaccctacg cgctatcttc 840
ccgaacccgg atcacactct gctgccgggt atgttcgtgc gtgcacgtct ggaagaaggg 900
cttaatccaa acgctatttt agtcccgcaa cagggcgtaa cccgtacgcc gcgtggcgat 960
gccaccgtac tggtggttgg cgcggatgac aaagtggaaa cccgtccgat cgttgcaagc 1020
caggctatcg gcgataagtg gctggtgaca gaaggtctga aagcaggcga tcgcgtagta 1080
ataagtgggc tgcagaaagt gcgtcctggt gtccaggtaa aagcacaaga agttaccgct 1140
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<210> 5
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<212> DNA
<213> Artificial sequence ()
<400> 5
ctcgcgagaa ttaagaagaa aggaggtttt tttt 34
<210> 6
<211> 43
<212> DNA
<213> Artificial sequence ()
<400> 6
ggagttatct ctcccgggtc acaatattaa ggaggtttta ttt 43
<210> 7
<211> 35
<212> DNA
<213> Artificial sequence ()
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gaggctaaca ggcacattca ataaggaggt ttttt 35

Claims (10)

1. An engineering probiotic, which is a derivative of escherichia coli Nissle1917, and is characterized by comprising surface-displayed L-phenylalanine ammonia lyase.
2. The engineered probiotic of claim 1, obtained by plasmid expression or genomic integration using escherichia coli Nissle1917 as a substrate; or
Based on the Escherichia coli Nissle1917 engineering bacteria which have phenylalanine ammonia lyase in cells, the surface display phenylalanine ammonia lyase is obtained by genetic engineering transformation.
3. The engineered probiotic bacteria of claim 2, wherein said engineered bacterium of escherichia coli Nissle1917 having a phenylalanine ammonia lyase incorporated therein is constructed by the following steps: an exogenous L-phenylalanine ammonia lyase gene stlA, an exogenous L-phenylalanine transport protein gene pheP and an exogenous L-amino acid deaminase gene pma are integrated on a genome of Escherichia coli Nissle 1917.
4. The engineered probiotic according to claim 3, characterized in that an endogenous efflux pump gene acrA;
and/or the gene argR is knocked out and/or a mutation of the gene argA (Y19C) occurs;
and/or the RBS sequence of the stlA, pheP and acrA genes is optimized.
5. The engineered probiotic of claim 4, wherein the nucleotide sequence of the L-phenylalanine ammonia lyase gene stlA is SEQ ID NO 1; the nucleotide sequence of the L-phenylalanine transport protein gene pheP is SEQ ID NO. 2; the nucleotide sequence of the L-amino acid deaminase gene pma is SEQ ID NO 3; the nucleotide sequence of the efflux pump gene acrA is SEQ ID NO. 4.
6. The engineered probiotic of claim 4, wherein the RBS sequence of the stlA gene is changed to SEQ ID NO 5; the RBS sequence of the pheP gene is changed into SEQ ID NO 6; the RBS sequence of the acrA gene was changed to SEQ ID NO 7.
7. The engineered probiotic of claim 1, wherein the genotype of the strain is: EcN (malP:: Pj23119H-stlA, yicS:: Pj23119H-stlA, malE:: Pj23119H-stlA, rthC: Ptac-stlA, exo:: Ptac-stlA, lacZ:: Pj23119H-pheP, agaI:: Pj23119H-pheP, araBD:: Para-pma, dapA:: inaK-stlA, argA,. DELTA.argR, lacZ:: Pj 23119H-acrA).
8. A method of constructing engineered probiotics according to any one of claims 1 to 7, comprising the steps of:
A. taking engineering probiotics EcN/pINP-stlA containing surface display stlA plasmid pINP-stlA as chassis bacteria, knocking in L-phenylalanine ammonia-lyase gene stlA at more than one of malP site, yicS site, malE site, rhtC site and exo site of genome respectively, and obtaining stlA gene integration strain;
B. knocking in an L-phenylalanine endogenous protein gene pheP at more than one of lacZ sites and agaI sites of a genome of the stlA gene integrated strain obtained in the step A to obtain a stlA + pheP integrated strain;
C. knocking in an L-amino acid deaminase gene pma at an araBD locus of a genome of the stlA + pheP integrated strain obtained in the step B to obtain the stlA + pheP + pma integrated strain;
D. knocking out a dihydrodipicolinate synthase gene dapA in the genome of the stlA + pheP + pma integrated strain obtained in the step C to obtain a stlA + pheP + pma delta dapA strain;
E. for the stlA + pheP + pma Δ dapA strain obtained in step D, mutation was performed (Y19C) on the argA site of its genome to give a stlA + pheP + pma Δ dapA argA strain;
F. knocking out argR in the genome of the stlA + pheP + pma delta dapA argA strain obtained in the step E to obtain the stlA + pheP + pma delta dapA argA delta argR strain.
9. The method of claim 8, further comprising the steps of:
G. knocking in an efflux pump gene acrA at a lacZ site of the genome of the stlA + pheP + pma delta dapA argA delta argR strain obtained in the step F to obtain the stlA + pheP + pma + acrA delta dapA argA delta argR strain;
H. for the stlA + pheP + pma + acrA delta dapA argA delta argR strain obtained in the step G, the RBS sequence of the stlA gene in the genome is changed into SEQ ID NO 5, the RBS sequence of the pheP gene is changed into SEQ ID NO 6, and the RBS sequence of the acrA gene is changed into SEQ ID NO 7, so as to obtain an RBS optimized strain;
I. and (D) knocking in the dapA locus of the genome of the RBS optimized strain obtained in the step (H) to display the stlA gene on the surface to obtain the engineering probiotics with the surface displayed phenylalanine ammonia lyase.
10. Use of an engineered probiotic of any one of claims 1-7 in the manufacture of a medicament for the treatment of phenylketonuria.
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CN113969292A (en) * 2021-11-15 2022-01-25 山东第一医科大学(山东省医学科学院) Engineering probiotics for treating phenylketonuria and construction method and application thereof
WO2022148362A1 (en) * 2021-01-07 2022-07-14 上海陶宇晟生物技术有限责任公司 Engineering probiotic having surface display phenylalanine ammonia-lyase
WO2023165521A1 (en) * 2022-03-02 2023-09-07 和度生物技术(上海)有限公司 Engineered microorganism for treating hyperphenylalaninemia and use thereof

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CN113969292A (en) * 2021-11-15 2022-01-25 山东第一医科大学(山东省医学科学院) Engineering probiotics for treating phenylketonuria and construction method and application thereof
CN113969292B (en) * 2021-11-15 2023-07-07 山东第一医科大学(山东省医学科学院) Engineering probiotics for treating phenylketonuria and construction method and application thereof
WO2023165521A1 (en) * 2022-03-02 2023-09-07 和度生物技术(上海)有限公司 Engineered microorganism for treating hyperphenylalaninemia and use thereof

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