CN117106813A - Recombinant expression vector for producing L-tyrosine, recombinant engineering bacterium and construction method thereof - Google Patents
Recombinant expression vector for producing L-tyrosine, recombinant engineering bacterium and construction method thereof Download PDFInfo
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- CN117106813A CN117106813A CN202311017069.0A CN202311017069A CN117106813A CN 117106813 A CN117106813 A CN 117106813A CN 202311017069 A CN202311017069 A CN 202311017069A CN 117106813 A CN117106813 A CN 117106813A
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- Prior art keywords
- tyrosine
- phenol
- recombinant
- culture
- expression vector
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- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 title claims abstract description 105
- 229960004441 tyrosine Drugs 0.000 title claims abstract description 54
- 241000894006 Bacteria Species 0.000 title claims abstract description 50
- 239000013604 expression vector Substances 0.000 title claims abstract description 27
- 238000003259 recombinant expression Methods 0.000 title claims abstract description 24
- 238000010276 construction Methods 0.000 title claims abstract description 9
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- 108091000100 Tyrosine Phenol-Lyase Proteins 0.000 claims abstract description 52
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- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 claims description 54
- 239000013612 plasmid Substances 0.000 claims description 33
- 229940107700 pyruvic acid Drugs 0.000 claims description 27
- 241000588724 Escherichia coli Species 0.000 claims description 16
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims description 16
- 238000006911 enzymatic reaction Methods 0.000 claims description 15
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- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 8
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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Abstract
The invention provides a recombinant expression vector for producing L-tyrosine, recombinant engineering bacteria and a construction method thereof, wherein the recombinant vector comprises a tyrosine phenol lyase coding gene and a display element coding gene, and the display element coding gene is a bacterial self-transport protein gene. The recombinant engineering bacteria comprise the recombinant expression vector. The recombinant engineering bacterium provided by the invention can be used for expressing enzyme for producing L-tyrosine on the surface of a host cell, directly contacts with a substrate and a product, avoids the barrier effect of cell walls and/or cell membranes on mass transfer of the substrate and the product in the whole cell catalysis process, has better tolerance to phenol, can reduce the phenol content in a conversion product and a filtrate, and has good industrial development and application prospects.
Description
Technical Field
The present invention belongs to the field of gene engineering and biotransformation technology. In particular, the invention relates to a recombinant expression vector for producing L-tyrosine, recombinant engineering bacteria and a construction method thereof.
Background
Tyrosine (Tyr) with chemical name of 2-amino-3-p-hydroxyphenyl propionic acid, which is aromatic polar alpha-amino acid containing phenolic hydroxyl group, and its molecular formula is C 9 H 11 NO 3 The relative molecular weight is 181.18900, and the molecular weight is soluble in acid and alkali and insoluble in diethyl ether and ethanol, and the structural formula is as follows:
l-tyrosine is one of important amino acids required for synthesizing proteins in animals, plays an important role in growth and metabolism of humans and animals, and is widely used in the fields of foods, feeds, medicines and cosmetics. In the food field, L-tyrosine is an important food additive; in the medical field, L-tyrosine is often used as a nutritional supplement, an essential amino acid for phenylketonuria patients, and a raw material for preparing medical products such as L-dopa, p-hydroxy cinnamic acid, p-hydroxystyrene and the like; in the cosmetic field, the L-tyrosine salt product can effectively relieve melanin.
The existing methods for preparing L-tyrosine mainly comprise hydrolysis method, fermentation method and synthesis method. Protein hydrolysis extraction is one of the main methods for producing L-tyrosine, and natural protein resources are utilized to extract L-tyrosine through steps of hydrolysis, decoloration, refining and the like. The extraction method has the defects that the basic components of the protein contain various natural amino acids, and the separation of high-purity L-tyrosine from the basic components is difficult, so that the yield of a target product is low; the product of the chemical synthesis method is racemized DL-tyrosine, and L-tyrosine is obtained by further resolution, so that the defects of complicated steps, high energy consumption, high requirement on equipment and the like exist. The direct fermentation method has the problems of long fermentation period, low sugar acid conversion rate and high production cost due to the process requirement, and does not have market competitiveness.
The enzyme conversion method is an important way for synthesizing L-tyrosine gradually due to the advantages of strong specificity, high catalytic efficiency, mild reaction conditions, short reaction period, easy separation of products and the like. The enzymes used for synthesizing L-tyrosine at present mainly comprise tyrosine phenol lyase, tyrosinase, aminotransferase and the like. The enzymatic synthesis of tyrosine takes pyruvic acid, phenol and ammonia as raw materials; or serine and phenol are used as raw materials; or glycine, formaldehyde and phenol are used as raw materials. More studied Tyrosinase (TPL) with higher enzyme activity is mainly derived from the microorganisms erwinia herbicola (Erwinia herbicola), citric acid bacterium intermedia (Citrobacter intermedius), citric acid bacterium freundii (Citrobacter freundii), thermophilic bacteria (Symbiobacterium toebii) and the like.
Phenol is an important raw material for synthesizing L-tyrosine, but since it is three types of carcinogens, phenol residue needs to be strictly controlled in the production process of L-tyrosine. However, as the product L-tyrosine is continuously separated out in the enzyme conversion process, the situation that phenol and L-tyrosine are bonded and coated exists, so that the phenol content in the converted crude product is high, and the phenol needs to be removed in the refining process to ensure that the phenol residue of the finished product is qualified.
In 2018, feng Zhibin et al co-expressed the tyrosine phenol lyase gene derived from Erwinia herbicola and the signal peptide gene, transformed competent cells of escherichia coli, constructed signal peptide-mediated extracellular secretion engineering bacteria of the tyrosine phenol lyase, and improved the enzyme release amount by more than 50% and the total enzyme activity by 36% compared with the common expression.
CN105886450a discloses an engineering bacterium for intracellular expression of tyrosine phenol lyase, a construction method and application thereof, which overcomes the defects of low protein expression level and easy production of inclusion bodies, and coexpresses target protein and chaperone protein, thereby improving protein expression level and enzyme activity.
CN105969819a discloses a process for producing L-tyrosine, which comprises adding beta-tyrosinase, phenol and ammonium chloride in the late stage of pyruvic acid fermentation, converting the generated pyruvic acid into L-tyrosine to obtain fermentation liquor containing L-tyrosine, and then carrying out acid dissolution, activated carbon decolorization, ceramic membrane filtration and security filter filtration, and then carrying out alkali precipitation on the clear liquid to obtain L-tyrosine. The method has certain toxicity to the raw material phenol added in the later stage of pyruvic acid fermentation, has higher control requirement to the reaction process, and the phenol supplementing rate is less than or equal to 17.5 g/L.h.
CN114560783a discloses an extraction method of L-tyrosine, which comprises the steps of alkaline dissolving the conversion solution, filtering by a ceramic membrane, neutralizing and centrifuging by hydrochloric acid, decolorizing by active carbon after acid dissolution again, neutralizing by ammonia water, centrifuging and collecting the refined product. The yield of the finished product is 90.6-91.2%. The method requires secondary recrystallization to obtain the finished product.
CN109929889a discloses a preparation method of L-tyrosine, which takes L-alanine and phenol as raw materials, takes D-amino acid oxidase, alanine racemase, catalase and tyrosine phenol lyase as catalysts, takes oxygen-containing gas as an oxidant, and synthesizes L-tyrosine by multi-enzyme coupling. The method needs to use four enzymes as catalysts at the same time, and the reaction process needs to consider the optimal temperature, pH value and other parameters of various enzymes to influence the refining and purification of the later-stage products.
In summary, the existing technology for producing L-tyrosine by using an enzymatic conversion method has the following problems:
(1) The recombinant expressed bacteria intracellular enzyme needs to be separated and purified after the cell is broken, so that the defect of complicated production steps is caused;
(2) The whole cell of the recombinant bacterium containing intracellular enzyme is influenced by mass transfer resistance of cell walls and/or cell membranes to substrates and products during catalysis, and the apparent catalytic activity is insufficient;
(3) There is a problem in that the enzyme has poor tolerance to phenol;
(4) The conversion product and the filtrate have higher phenol content.
Based on this, there is a need for new genetically engineered strains with improved tolerance to phenol for the enzymatic conversion of L-tyrosine production, which both ensure the conversion of L-tyrosine and reduce the phenol content in the product.
Disclosure of Invention
Aiming at the defects of the prior art, the inventor designs a recombinant expression vector, and constructs a recombinant engineering bacterium with the surface displaying expressed tyrosine phenol lyase by using the recombinant expression vector, thereby providing a high-efficiency and low-toxicity process path for L-tyrosine production.
In one aspect, the invention provides a recombinant expression vector comprising a tyrosine phenol lyase encoding gene and a display motif encoding gene, wherein the display motif encoding gene is a bacterial autotransporter gene.
According to some embodiments of the invention, the display motif encoding gene of the invention is the E.coli (K-12) transferrin Ag43 gene; preferably, the nucleotide sequence of the displayed motif encoding gene according to the present invention is as shown in SEQ ID NO.2, or is a nucleotide sequence having more than 70%, for example 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the nucleotide sequence shown in SEQ ID NO.2 and retaining the autotransporter activity of its expression product.
According to some embodiments of the invention, the tyrosine phenol lyase encoding gene of the invention is the tyrosine phenol lyase gene of erwinia herbicola (Erwinia herbicola); preferably, the nucleotide sequence of the gene encoding the tyrosine phenol lyase is shown as SEQ ID NO.1, or a nucleotide sequence which has more than 70%, such as 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the nucleotide sequence shown as SEQ ID NO.1 and maintains the activity of the tyrosine phenol lyase of the expression product thereof.
According to some embodiments of the invention, the starting vector of the recombinant expression vector of the invention is selected from any one of pET plasmid and pBAD plasmid or a combination thereof; preferably, the pET plasmid is a pET28a plasmid and/or a pET3a plasmid; preferably, the pBAD plasmid is selected from any one of pBAD HisA, pBAD30, pGEX 2T, pXMJ19 and pecxk99e or the combination thereof; more preferably, the starting vector of the recombinant expression vector of the present invention is pET28a plasmid.
In another aspect, the invention provides a recombinant engineering bacterium comprising the recombinant expression vector of the invention.
According to some embodiments of the invention, the host cell of the recombinant engineering bacterium is a prokaryotic cell; preferably, the host cell is E.coli; more preferably, the host cell is E.coli BL21 (DE 3).
In still another aspect, the present invention provides a method for constructing the recombinant engineering bacteria, which includes the following steps:
the recombinant expression vector is introduced into host cells to obtain the recombinant engineering bacteria.
According to some embodiments of the invention, the tyrosine phenol lyase encoding gene and the display motif encoding gene are ligated in tandem into a starting vector to obtain the recombinant expression vector.
Specifically, a display motif coding gene Ag43 with a nucleotide sequence shown as SEQ ID NO.2 and a tyrosine phenol lyase coding gene with a nucleotide sequence shown as SEQ ID NO.1 are connected in series into an initial vector to obtain the recombinant expression vector, and the screened recombinant expression vector is introduced into a host cell to obtain recombinant engineering bacteria with the surface displaying and expressing the tyrosine phenol lyase. The method comprises designing primers according to coding gene sequences of tyrosine phenol lyase and coding gene sequences of display motif coding gene Ag43, amplifying target genes by PCR, double-enzyme cutting two target genes by using restriction enzymes, or connecting two target genes together and enzyme cutting by overlapping PCR, connecting with corresponding enzyme-cut vectors, transforming, extracting plasmids, identifying positive recombinants by PCR or enzyme cutting, and sequencing to verify positive recombinants.
In yet another aspect, the present invention provides a method for preparing L-tyrosine, comprising the steps of:
the recombinant engineering bacteria of the invention are adopted to carry out biological conversion by taking pyruvic acid and phenol as raw materials, and the L-tyrosine is obtained.
According to some embodiments of the present invention, the preparation method of L-tyrosine according to the present invention comprises the steps of:
(1) Inoculating the recombinant engineering bacteria into a culture medium for culture to obtain seed liquid;
(2) Transferring the seed liquid obtained in the step (1) into a fresh culture medium for culture, optionally adding an inducer for inducing the expression of the tyrosine phenol lyase, and collecting recombinant engineering bacteria;
(3) Adding the recombinant engineering bacteria collected in the step (2) into a substrate containing pyruvic acid and phenol to perform enzymatic reaction to obtain L-tyrosine.
According to some embodiments of the invention, in step 1 of the preparation method, the medium is LB liquid medium; preferably, the temperature of the culture is 36-37 ℃; preferably, the culture is shaking culture at a rotation speed of 180rpm to 220 rpm; preferably, the time of the cultivation is 10 to 16 hours.
According to some embodiments of the invention, in step 2 of the preparation method, the seed liquid is inoculated in an amount of 1% to 5% by volume; preferably, when the culture is carried out until the OD600 of the culture solution is 0.8 to 1.0, an inducer for inducing the expression of the tyrosine phenol lyase is optionally added; preferably, the culture medium is LB liquid culture medium; preferably, the temperature of the culture is 25-37 ℃; preferably, the culture is shaking culture at a rotation speed of 160rpm to 220 rpm; preferably, the time of the cultivation is 12 to 20 hours.
The choice of inducer according to the present invention is not restricted but can be selected according to the conventional knowledge of those skilled in the art. For example, if the host cell is E.coli, its expression vector is pET28a, it can be induced with β -D-thiogalactoside (IPTG); for another example, if the host cell selects lactic acid bacteria, the expression vector is pMG36e, and expression is achieved without induction.
According to some embodiments of the invention, in step 3 of the preparation method, the method further comprises a step of extracting and refining the obtained L-tyrosine.
According to some embodiments of the invention, in step 3 of the preparation method, the mass ratio of the recombinant engineering bacteria, pyruvic acid and phenol is 0.05-1: 0.95 to 1.4:1, a step of; preferably, the mass ratio of the recombinant engineering bacteria, pyruvic acid and phenol is 0.084:1.011:1.
according to some embodiments of the invention, in step 3 of the preparation method, further comprising the step of adding a substrate during the enzymatic reaction; preferably, the mass ratio of pyruvic acid to phenol in the added substrate is 0.25-0.6: 1, a step of; preferably, the mass ratio of pyruvic acid to phenol in the added substrate is 0.36:1.
according to some embodiments of the invention, in step 3 of the preparation method, the temperature of the enzymatic reaction is 20-55 ℃, preferably 44 ℃; preferably, the time of the enzymatic reaction is 7 to 15 hours, preferably 10 hours; preferably, the pH of the enzymatic reaction is between 6 and 10, preferably 8.45; preferably, the stirring speed of the enzymatic reaction is 20 to 200rpm, preferably 50rpm.
According to some embodiments of the invention, in step 3 of the preparation method, the substrate further comprises cetyltrimethylammonium bromide (CTAB) and/or pyridoxal phosphate (PLP).
According to some embodiments of the invention, the method of preparation comprises the steps of:
(1) Inoculating the recombinant engineering bacteria into an LB liquid culture medium, and carrying out shaking culture for 10-12h at 37 ℃ and 180rpm to obtain seed liquid;
(2) Transferring the seed liquid obtained in the step (1) into a fresh LB liquid culture medium with an inoculum size of 2%, carrying out shaking culture at 37 ℃ and 180rpm, adding an inducer for inducing expression of the tyrosine phenol lyase when the OD600 = 0.8-1.0 of the culture liquid, and then carrying out continuous shaking culture at 25 ℃ and 180rpm for 14-16 hours to collect recombinant engineering bacteria;
(3) Adding the recombinant engineering bacteria collected in the step (2) to a substrate containing pyruvic acid and phenol and having a pH of 8.45, and carrying out enzymatic reaction in the presence of cetyltrimethylammonium bromide (CTAB) and/or pyridoxal phosphate (PLP) under stirring at 44 ℃ and 50rpm to obtain L-tyrosine, wherein the substrate containing pyruvic acid and phenol is supplemented and the phenol concentration in the reaction system is controlled to be less than or equal to 8g/L.
Compared with the prior art, the invention has at least one of the following beneficial effects:
1. the recombinant engineering bacterium provided by the invention can enable the enzyme to be expressed on the surface of a host cell, and the recombinant engineering bacterium is directly contacted with a substrate and a product, so that the blocking effect of cell walls and/or cell membranes on the mass transfer of the substrate and the product in the whole cell recombination catalysis process is avoided;
2. the enzyme is displayed and expressed on the surface of the recombinant engineering bacteria, so that the substrate can be contacted with the enzyme to complete conversion without entering a bacterial cell, the aim of improving the apparent activity of the recombinant bacteria is fulfilled, meanwhile, the tolerance to phenol is also good, meanwhile, the phenol content in conversion products and filtrate can be reduced, and the method has good industrial development and application prospects;
3. the recombinant expression vector, the construction method of the recombinant engineering bacteria and the like have the advantages of low technical cost, simplicity, convenience and easiness in operation.
Drawings
Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a diagram showing the cleavage assay of the recombinant plasmid in example 1;
FIG. 2 is a protein expression map of example 2;
FIG. 3 is a graph comparing the phenol resistance of example 3.
Detailed Description
The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention.
The experimental methods used in the following examples, unless otherwise specified, are all routine in the art. The experimental materials used in the following examples were purchased from biochemical reagent sales companies, where not specified, and were: plasmid pET28a was purchased from Wuhan vast, biotechnology Co., ltd; restriction enzymes such as EcoR I and HindIII, competent cells of Escherichia coli DH5 alpha and BL21 (DE 3), DNA markers, a plasmid extraction kit, a DNA gel recovery and purification kit and T4 ligase are all purchased from Takara Bao bioengineering (Dalian) Co., ltd; DNA polymerase (Q5 High-Fidelity DNAPolymerase) was purchased from Gene Inc.; kanamycin sulfate and BCA protein assay kits were purchased from Biosharp corporation; the chemical reagents are all analytical pure of Chinese medicine. The plasmid extraction operation steps refer to the specification of a plasmid small extraction kit; the DNA gel recovery operation steps refer to the specification of a DNA gel recovery kit; the DNA fragment ligation procedure is described in the specification of T4 ligase; protein content procedure reference BCA protein content assay kit instructions. The PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the plasmid or DNA template suppliers. Can be adjusted if necessary by simple tests.
In the following examples, LB medium composition (g/L): tryptone 10, yeast extract 5, sodium chloride 10, pH nature (LB solid medium is added with 20g/L agar powder), and sterilizing at 121deg.C for 20min. Fermentation medium (g/L): peptone 12, yeast extract 8, disodium hydrogen phosphate 15, potassium dihydrogen phosphate 3, sodium chloride 0.5, ammonium chloride 1, dipotassium hydrogen phosphate 9, ferric ammonium citrate 0.3, citric acid 2, glycerol 10, magnesium sulfate heptahydrate 0.5, defoamer 0.4ml/L, and sterilizing at 121deg.C for 20min.
EXAMPLE 1 construction of recombinant plasmid for intracellular expression and surface display of tyrosine phenol lyase
According to NCBI database and patent publication information, referring to sequence information (SEQ ID No: 1) of tyrosine phenol lyase gene TPL derived from Erwinia herbicola (Erwinia herbicola), performing codon optimization on the gene sequence according to the codon preference rule of Escherichia coli, designing and adding EcoR I and Hind III cleavage sites at two ends of the gene, and then sending to Jin Kairui Biotechnology limited company for artificial synthesis.
The EcoR I/Hind III is used for respectively double-enzyme cutting plasmid pET28a and synthesized tyrosine phenol lyase gene fragment, and after agarose electrophoresis, rubber tapping is recovered. The recovered plasmid large fragment and the digested and recovered tyrosine phenol lyase gene are subjected to enzyme digestion in a molar ratio of 1:3, adding ligase to join overnight, and transforming the competent cells of the escherichia coli DH5 a. After randomly picking monoclonal and PCR identifying positive recombinants, extracting plasmids, identifying by EcoR I/Hind III double enzyme digestion, and carrying out sequencing analysis to prove that the plasmid pET28a-TPL for intracellular expression of the tyrosine phenol lyase is successfully constructed.
The reaction system is as follows: plasmid or tyrosine phenol lyase gene fragment 1. Mu.g, 10 Xbuffer 5. Mu.l, ecoR I1. Mu.l, hind III 1. Mu.l, and water was added to 50. Mu.l. And enzyme cutting at 37 ℃ for 5 hours. The nucleotide fragment of 6.5kb was recovered by 1% agarose gel electrophoresis and a DNA gel recovery purification kit.
The T4 DNA ligase is utilized to connect the TPL gene DNA fragment and the pET28a vector, and the connection system is as follows: pET28a 0.5. Mu.l, solution I5. Mu.l, TPL gene DNA fragment 4.5. Mu.l. The ligation was carried out overnight at 16 ℃.
The ligation product was transformed into E.coli DH 5. Alpha. Competent cells by heat shock transformation, the transformation product was spread on LB plate containing 50. Mu.g/ml kanamycin sulfate, cultured overnight at 37℃and positive transformants were selected and sent to Jin Kairui Biotechnology Co., ltd. For sequencing, and the correct transformants were sequenced, namely plasmid vector pET28a-TPL containing the tyrosine phenol lyase gene.
According to NCBI database and patent publication information, referring to sequence information (SEQ ID No: 2) of the autotransporter Ag43 gene from the Escherichia coli K-12MG1655 genome, performing codon optimization on the gene sequence according to the codon preference rule of Escherichia coli, designing and adding EcoR I and Hind III restriction enzyme sites at two ends of the gene, and then sending to Jin Kairui Biotechnology limited company for artificial synthesis.
The synthesized Ag43 gene fragment is cut by EcoR I/HindIII double enzyme, agarose electrophoresis is carried out, and rubber tapping is carried out for recovery. The recovered plasmid large fragment and the target gene TPL and Ag43 fragment recovered by enzyme digestion are mixed according to the molar ratio of 1:3:3, adding ligase to join overnight, and transforming the competent cells of the escherichia coli DH5 a. After randomly picking up a monoclonal and identifying a positive recombinant by PCR, extracting plasmids, and identifying by using EcoR I/Hind III double digestion, the result is shown in a figure 1, wherein lanes 1-4 are the results of double digestion of the monoclonal 1-4 recombinant plasmids respectively, and M is DNAMarkIV standard molecular weight. FIG. 1 shows that the corresponding positions of the target gene of TPL of about 1400bp and the corresponding positions of the vector pET28a-Ag43 of about 6000bp are provided with bands after double enzyme digestion, which indicates that the surface display protein gene Ag43 and the TPL gene are successfully constructed on the expression vector pET28a to obtain the recombinant plasmid pET28-Ag43-TPL, and sequencing analysis proves that the recombinant plasmid pET28-Ag43-TPL of the surface display tyrosine phenol lyase is successfully constructed.
EXAMPLE 2 expression of E.coli recombinant intracellular TPL enzyme and recombinant surface display TPL enzyme
E.coli BL21 (DE 3) competent cells were transformed with the plasmids pET28a-TPL and pET28a-Ag43-TPL constructed in example 1, respectively, and plated on a dish of LB solid medium containing 50. Mu.g/mL kanamycin, and cultured upside down at 37℃overnight. The monoclonal cells were individually picked up and inoculated into LB liquid medium containing 50. Mu.g/mL kanamycin, and cultured in a constant temperature shaker at 37℃and 180rpm for 10-12 hours. Inoculating the culture solution into fresh LB culture medium containing 50 μg/mL kanamycin at 2%, shake culturing in shaking table at 37deg.C and 180rpm until OD600 reaches 0.6-0.8, adding 0.2mM final concentration IPTG, and shake culturing at 25deg.C and 180rpm for 14-16 hr to induce expression of tyrosine phenol lyase.
The fermentation broth was centrifuged at 10000r/min for 15min at 4℃to collect the bacterial pellet, and the pellet was washed and suspended with buffer (20 mM potassium phosphate buffer, pH 7.0), sonicated, and examined for protein expression by centrifugation, and the results of protein electrophoresis were shown in FIG. 2, wherein column 1 was the protein standard, column 2 was the pre-induction protein, column 3 was the post-induction whole cell protein, column 4 was the post-induction-disruption precipitated protein, and column 5 was the post-induction-disruption supernatant protein. From the figure, it can be seen that the protein is expressed well and is present in soluble form in the supernatant.
Example 3 intracellular expression of tyrosol lyase and analysis of apparent enzyme Activity and phenol tolerance of recombinant engineering bacteria expressing tyrosol lyase on surface display
1. Comparison of apparent enzyme Activity
The reaction system: 45g/L ammonium acetate, 6g/L phenol, 10g/L sodium pyruvate, 2g/L sodium sulfite, 1g/L EDTA, pH 8.0,0.5% wet cells, at 37 ℃ for 20min, adding an equal volume of hydrochloric acid with pH of 1-2 to terminate the reaction, and determining the content of L-tyrosine by HPLC.
The enzyme activity is defined as the amount of enzyme required to convert 1. Mu. Mol of L-tyrosine to 1 enzyme activity unit U per L of min at a pH of 8.0 and a temperature of 37 ℃.
According to the measurement, the apparent enzyme activity of the recombinant engineering bacteria with the surface displaying expressed tyrosine phenol lyase is 276U/g, and the apparent enzyme activity of the recombinant engineering bacteria with the intracellular expressed tyrosine phenol lyase under the same culture condition is 100U/g, which is improved by 176 percent compared with the recombinant engineering bacteria with the intracellular expressed tyrosine phenol lyase.
2. Comparison of tolerance to phenol:
preparing 1-15g/L phenol water solutions with different concentrations, respectively taking 5g of centrifugal thalli from fermentation liquor with expressed tyrosine phenol lyase and centrifugal thalli from fermentation liquor with expressed intracellular tyrosine phenol lyase, placing the thalli into 500mL of prepared phenol solution, stirring at 44 ℃ for 1h, centrifuging to obtain thalli, and detecting the enzyme activity. The results are shown in table 1 below and in fig. 3:
table 1 comparison table of phenol tolerance
As can be seen from table 1 and fig. 3 above: at phenol concentrations below 3g/L, the surface display expression is comparable to the tolerance of intracellular expressed tyrosine phenol lyase to phenol; however, when the phenol concentration is higher than 3g/L, the intracellular expressed tyrosine phenol lyase has obviously poorer tolerance to phenol, and when the phenol concentration reaches 15g/L, only 2.5 percent of the enzyme activity can be maintained, and the surface-displayed expressed tyrosine phenol lyase can still maintain 25.60 percent of the enzyme activity, so that the surface-displayed tyrosine phenol lyase has better tolerance to phenol.
EXAMPLE 4 surface display expression of tyrosine phenol lyase bacteria to convert phenol and pyruvate to tyrosine
Preparing a base material: adding 1.5L of pyruvic acid with the content of 60g/L into a four-neck flask, adding 8g/L of phenol, adjusting the pH to 8.45 by ammonia water, and heating to 44 ℃;
enzyme liquid treatment: taking 500mL of a pyruvic acid solution with the content of 60g/L, and adjusting the pH value to 8.45 by ammonia water; adding 5g/L of surface display expression tyrosine phenol lyase thallus, 1g/L of CTAB and 40mg/L of PLP according to the total reaction system, and fully stirring for 30min;
and (3) material supplementing preparation: taking 1L of pyruvic acid with the content of 60g/L, adding 166g/L of phenol, and stirring and mixing uniformly;
enzymatic conversion: adding the treated enzyme solution into the base material, setting the rotating speed to 50rpm, feeding the fed-batch, controlling the concentration of phenol in the conversion system to be less than or equal to 8g/L, and keeping the product in a small grain size particle state. Enzymatic reaction is carried out for 10 hours at 44 ℃, and the mol ratio of pyruvic acid to phenol in a reaction system is 1.08:1, the total volume after the reaction was 3250mL. Sampling alkali dissolution, detecting the concentration of L-tyrosine in the conversion solution to be 101.7g/L through HPLC, wherein the molar conversion rate of phenol is 96.5%, and collecting 1157g of an L-tyrosine crude product through suction filtration, wherein the phenol content is 50ppm;
extracting and refining: adding 3460mL of 0.5M sulfuric acid into a four-neck flask, stirring and heating to 60 ℃, adding the converted crude product into sulfuric acid for dissolution, adding 60g of diatomite for heat preservation for 30min, filtering and sterilizing, keeping the filtrate volume at 4500mL, adding 45g of 1% active carbon, keeping the temperature and stirring for 1h at 60 ℃, passing the filtrate through a 0.22 mu M microporous filter membrane, slowly dripping 3M sodium hydroxide solution purified by the 0.22 mu M microporous filter membrane into the filtrate, stopping dripping alkaline solution when white turbidity appears in the system, keeping the temperature and culturing for 1h, continuing dripping alkaline solution to pH5.66, completely precipitating the product L-tyrosine, filtering and filtering a filter cake by using 1.5L of pure water preheated to 50-60 ℃, filtering and filtering the filter cake by using 1.5L of 95% (volume) ethanol slurry preheated to 50 ℃ for 30min, filtering and filtering the filter cake by using 1.5L of pure water preheated to 50-60 ℃, and drying the wet product in vacuum for 12h at 60 ℃ to obtain L-320 g, wherein the yield is 96%, and the light transmittance of the product is 97% and the residual phenol is not detected.
Comparative example 5 intracellular expression of tyrosine phenol lyase thallus to convert phenol and pyruvic acid to prepare tyrosine
Preparing a base material: adding 1.5L of a pyruvic acid solution with the content of 60g/L into a four-neck flask, adding 2g/L of phenol, adjusting the pH to 8.45 by ammonia water, and heating to 44 ℃;
enzyme liquid treatment: taking 500mL of pyruvic acid with the content of 60g/L, and adjusting the pH value to 8.45 by ammonia water; 15g/L of intracellular expression tyrosine phenol lyase thallus, 1g/L of CTAB and 40mg/L of PLP are added according to the total reaction system, and the mixture is fully stirred for 30min;
and (3) material supplementing preparation: taking 1L of pyruvic acid with the content of 60g/L, adding 151g/L of phenol, and stirring and mixing uniformly;
enzymatic conversion: adding the treated enzyme solution into the base material, setting the rotating speed to 50rpm, feeding the fed-batch, controlling the concentration of phenol in the conversion system to be less than or equal to 2g/L, and keeping the product in a small grain size particle state. Enzymatic reaction is carried out for 10 hours at 44 ℃, and the mol ratio of pyruvic acid to phenol in a reaction system is 1.2:1, the total volume after the reaction was 3190mL. Sampling alkali dissolution, detecting the concentration of L-tyrosine in the conversion solution to be 89.3g/L through HPLC, wherein the molar conversion rate of phenol is 92.4%, and collecting 887.5g of an L-tyrosine crude product through suction filtration, wherein the phenol content is 432ppm;
extracting and refining: adding 3000mL of 0.5M sulfuric acid into a four-neck flask, stirring and heating to 60 ℃, adding the converted crude product into sulfuric acid for dissolution, adding 60g of diatomite for heat preservation for 30min, filtering and sterilizing, keeping the filtrate volume at 3970mL, adding 40g of active carbon with the volume of 1%, keeping the temperature and stirring for 1h at 60 ℃, passing the filtrate through a 0.22 mu M microporous filter membrane, slowly dropwise adding 3M sodium hydroxide solution purified by the 0.22 mu M microporous filter membrane into the filtrate, stopping dropwise adding alkaline solution when white turbidity appears in the system, keeping the temperature and growing crystals for 1h, continuing dropwise adding alkaline solution to pH5.66, completely precipitating the product L-tyrosine, filtering and filtering a filter cake by using 1.5L of pure water preheated to 50-60 ℃ for 30min, filtering and filtering the filter cake by using 1.5L of 95% (volume) ethanol slurry preheated to 50 ℃ for 30min, filtering and filtering the filter cake by using 1.5L of pure water preheated to 50-60 ℃, and drying the wet product in vacuum for 12h at 60 ℃ to obtain L-tyrosine g, the finished product with the light transmittance of 94%, and the light transmittance of 95 ppm of phenol residue is 95.25 ppm.
In conclusion, the surface display expression tyrosine phenol lyase constructed by the invention has the enzyme activity obviously higher than that of intracellular expression tyrosine phenol lyase, the tolerance of L-tyrosine produced by adopting the enzyme conversion to phenol is improved, the conversion rate of tyrosine can reach 96.5%, the phenol content in the finished product is not detected, and the invention has industrial development and application prospects.
While only examples of the embodiments of the present invention have been described above, it will be understood by those skilled in the art that the foregoing is illustrative only and the scope of the invention is defined by the appended claims. Various changes and modifications to these embodiments may be made by those skilled in the art without departing from the principles and spirit of the invention, but such changes and modifications should not be construed as falling within the scope of the invention.
Claims (10)
1. A recombinant expression vector comprising a tyrosine phenol lyase encoding gene and a display motif encoding gene, wherein the display motif encoding gene is a bacterial autotransporter gene.
2. The recombinant expression vector of claim 1, wherein the display motif encoding gene is the escherichia coli (K-12) autotransporter Ag43 gene; preferably, the nucleotide sequence of the display motif encoding gene is as shown in SEQ ID No.2, or is a nucleotide sequence which has more than 70%, e.g. 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the nucleotide sequence shown in SEQ ID No.2 and retains the autotransporter activity of its expression product;
preferably, the tyrosine phenol lyase encoding gene is the tyrosine phenol lyase gene of erwinia herbicola (Erwinia herbicola); preferably, the nucleotide sequence of the tyrosine phenol lyase encoding gene is shown as SEQ ID NO.1, or is a nucleotide sequence which has more than 70%, for example 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the nucleotide sequence shown as SEQ ID NO.1 and retains the tyrosine phenol lyase activity of the expression product thereof.
3. The recombinant expression vector according to claim 1 or 2, wherein the starting vector of the recombinant expression vector is selected from any one of pET plasmid and pBAD plasmid or a combination thereof; preferably, the pET plasmid is a pET28a plasmid and/or a pET3a plasmid; preferably, the pBAD plasmid is selected from any one of pBAD HisA, pBAD30, pGEX 2T, pXMJ19 and pecxk99e or the combination thereof; more preferably, the starting vector of the recombinant expression vector is a pET28a plasmid.
4. A recombinant engineering bacterium comprising the recombinant expression vector according to any one of claims 1 to 3.
5. The recombinant engineering bacterium of claim 4, wherein the host cell of the recombinant engineering bacterium is a prokaryotic cell; preferably, the host cell is E.coli; more preferably, the host cell is E.coli BL21 (DE 3).
6. A method of constructing the recombinant engineering bacterium according to claim 4 or 5, comprising the steps of:
introducing the recombinant expression vector according to any one of claims 1 to 3 into a host cell to obtain the recombinant engineering bacterium.
7. The construction method according to claim 6, wherein the recombinant expression vector is obtained by ligating the tyrosine phenol lyase encoding gene and the display motif encoding gene in series into a starting vector.
8. A method for preparing L-tyrosine, comprising the steps of:
the L-tyrosine is obtained by biotransformation of pyruvic acid and phenol as raw materials by using the recombinant engineering bacteria according to claim 4 or 5 or the recombinant engineering bacteria constructed according to the construction method according to claim 6 or 7.
9. The preparation method according to claim 8, comprising the steps of:
(1) Inoculating the recombinant engineering bacteria into a culture medium for culture to obtain seed liquid;
(2) Transferring the seed liquid obtained in the step (1) into a fresh culture medium for culture, optionally adding an inducer for inducing the expression of the tyrosine phenol lyase, and collecting recombinant engineering bacteria;
(3) Adding the recombinant engineering bacteria collected in the step (2) into a substrate containing pyruvic acid and phenol to perform enzymatic reaction to obtain L-tyrosine.
10. The production method according to claim 8 or 9, wherein in step 1, the medium is an LB liquid medium; preferably, the temperature of the culture is 36-37 ℃; preferably, the culture is shaking culture at a rotation speed of 180rpm to 220 rpm; preferably, the time of the culture is 10-16 hours;
preferably, in the step 2, the seed liquid is inoculated in an amount of 1 to 5% by volume; preferably, when the culture is carried out until the OD600 of the culture solution is 0.8 to 1.0, an inducer for inducing the expression of the tyrosine phenol lyase is optionally added; preferably, the culture medium is LB liquid culture medium; preferably, the temperature of the culture is 25-37 ℃; preferably, the culture is shaking culture at a rotation speed of 160rpm to 220 rpm; preferably, the time of the culture is 12-20 hours;
preferably, in step 3, the method further comprises a step of extracting and refining the obtained L-tyrosine;
preferably, in the step 3, the mass ratio of the recombinant engineering bacteria, the pyruvic acid and the phenol is 0.05-1: 0.95 to 1.4:1, a step of; preferably, the mass ratio of the recombinant engineering bacteria, pyruvic acid and phenol is 0.084:1.011:1, a step of;
preferably, in step 3, a step of supplementing the substrate during the enzymatic reaction is further included; preferably, the mass ratio of pyruvic acid to phenol in the added substrate is 0.25-0.6: 1, a step of; preferably, the mass ratio of pyruvic acid to phenol in the added substrate is 0.36:1, a step of;
preferably, in step 3, the temperature of the enzymatic reaction is 20-55 ℃, preferably 44 ℃; preferably, the time of the enzymatic reaction is 7 to 15 hours, preferably 10 hours; preferably, the pH of the enzymatic reaction is between 6 and 10, preferably 8.45; preferably, the stirring speed of the enzymatic reaction is 20-200 rpm, preferably 50rpm;
preferably, in step 3, the substrate further comprises cetyltrimethylammonium bromide (CTAB) and/or pyridoxal phosphate (PLP).
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| CN118903084A (en) * | 2024-09-23 | 2024-11-08 | 安徽农业大学 | Alpha-amylase synergist and application thereof |
| CN119684143A (en) * | 2024-12-24 | 2025-03-25 | 河北远大九孚生物科技有限公司 | L-tyrosine crystal and its preparation method and use |
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| CN118903084A (en) * | 2024-09-23 | 2024-11-08 | 安徽农业大学 | Alpha-amylase synergist and application thereof |
| CN119684143A (en) * | 2024-12-24 | 2025-03-25 | 河北远大九孚生物科技有限公司 | L-tyrosine crystal and its preparation method and use |
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