CN109439605A - The engineering bacteria and its construction method of raising tyrosine phenol lyase stability and application - Google Patents

The engineering bacteria and its construction method of raising tyrosine phenol lyase stability and application Download PDF

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CN109439605A
CN109439605A CN201810455195.7A CN201810455195A CN109439605A CN 109439605 A CN109439605 A CN 109439605A CN 201810455195 A CN201810455195 A CN 201810455195A CN 109439605 A CN109439605 A CN 109439605A
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tyrosine phenol
engineering bacteria
phenol lyase
construction method
tpl
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储消和
吴黎诚
沈建
生英涛
陈万河
方明山
程跃
赖秧秧
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Zhejiang Lyuchuang Biotechnology Co ltd
Zhejiang University of Technology ZJUT
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Zhejiang Lyuchuang Biotechnology Co ltd
Zhejiang University of Technology ZJUT
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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C12P13/225—Tyrosine; 3,4-Dihydroxyphenylalanine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/99—Other Carbon-Carbon Lyases (1.4.99)
    • C12Y401/99002—Tyrosine phenol-lyase (4.1.99.2)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin

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Abstract

The present invention relates to a kind of engineering bacteria and its construction method, the engineering bacteria and its construction method of specifically a kind of raising tyrosine phenol lyase stability and application belong to gene engineering technology field.The engineering bacteria is identified as escherichia coli, it is named as escherichia coli Trx-TPL (Escherichia coli Trx-TPL), it is stored in China typical culture collection center, preservation address: Wuhan, China, Wuhan University, the deposit date is on November 13rd, 2017, deposit number was CCTCC NO:M2017684.Two tyrosine phenol lyase genes and sulphur hydrogen reduction protein gene are linked together and construct expression plasmid by the present invention, it can effectively improve the stability of the enzyme of expression, especially thermal stability, the time of Synthesis levodopa can be extended in the case where providing related substrates, increase concentration of substrate, simple process, low in cost, yield is high, the application value with industrialized production.

Description

The engineering bacteria and its construction method of raising tyrosine phenol lyase stability and application
Technical field
The present invention relates to a kind of engineering bacteria and its construction method, specifically a kind of tyrosine phenol lyase engineering bacteria and its structure Construction method and application, belong to genetic engineering field.
Background technique
The chemical name of levodopa (3,4-dihydroxyphenyl-L-ananine, abbreviation L-DOPA) is 3,4- bis- Hydroxyphenylalanine, structural formula are as follows:
As a kind of important bioactive substance, L-DOPA is from l-tyrosine to catechol or the biochemical generation of melanin Thank to the important intermediate during approach.
The sixties in last century, external many scholars start the research for being dedicated to microbial enzyme method synthesis L-DOPA.In order to mention High L-DOPA yield and the substrate transformation rate, researchers largely grind to the process of microbial enzyme method synthesis L-DOPA Study carefully.
Tyrosine phenol lyase (Tyrosine phenol lyase, TPL, E.C.4.1.99.2) also known as β-tyrosine Enzyme, the enzyme molecular weight about 200kDa are a tetramer enzymes.TPL enzyme with phosphopyridoxal pyridoxal phosphate (pyridoxal-phosphate, It PLP is) coenzyme, using potassium ion and ammonium ion as confactor, TPL can be catalyzed l-tyrosine and β-elimination reaction generation benzene occurs Phenol, pyruvic acid and ammonia.Since this reaction is reversible, by catechol replace phenol after, can by catechol, pyruvic acid and Ammonia can generate L-DOPA under TPL catalysis.The precursor of levodopa has inhibiting effect to enzyme activity in higher concentration, wherein adjacent Benzenediol and pyruvic acid also result in the irreversible inactivation of enzyme other than having high inhibition effect, and reaction condition is difficult to control, by-product Object is more, the low yield of L-DOPA.
India R.KrishnaveniVandanaRathod et al. converts L- junket ammonia using fungi Acremoniumrutilum Acid to L-3,4 dihydroxyphenylalanine, tyrosinase reaches 1095U/mg than living.But current production capacity is lower, only 0.89g/L.
Pakistani Ikram-U1-Haq et al. carries out ultraviolet mutagenesis to the Aspergillusoryzae for producing tyrosinase, Mutagenic fungi maximum production reaches 1.28g/L.
Egyptian Doaa A.R.MahmoudMagda A.El Bendary utilizes Egyptian halophilic black The tyrosinase of yeast is converted into L-DOPA, and yield is 66 μ g/ml.
When South Korea researcher is converted using tyrosinase, replace go back original reagent using electricity, by DOPA quinone (DOPAquinone) it is reduced to L-3,4 dihydroxyphenylalanine, makes conversion ratio up to 95.9, produces intensity 47.27mg/L*h.
There are also using the bacterium in nature, as Escherichia, Proteus (proteus), Stizolobiumhassjoo and Erwinia (Erwinia) etc., Lai Hecheng L-DOPA, as levodopa enzymatic clarification is summarized Report, TPL Recombinant organism convert 30h, are converted into the L-DOPA of 29.6g/L.For another example, Jang-Young Lee Et al. Clone Origin in the p-hydroxyphenylaceticacid -3- hydroxylase (p- of Escherichia coli W (ATCC11105) Hydroxyphenylacetate3-hydroxylase, PHAH), conversion l-tyrosine is L-DOPA, product accumulation to 10g/L. Although researcher has found that the expression quantity of these recombinant bacterial strains TPL is higher than wild strain, final L-DOPA synthesis capability does not have Be significantly improved even lower than wild strain.This, which may be because, will obtain higher L-DOPA synthesis capability, and bacterial strain is in addition to having Outside TPL high activity, also there are the substrate of comparatively perfect, product to enter and leave the transporting mechanism of cell membrane and press down to substrate catechol The tolerance etc. of enzyme activity processed.On the whole, reaction condition is difficult to control, and stability is poor, and by-product is more, the low yield of L-DOPA.
Summary of the invention
An object of the present invention is to provide a kind of engineering bacteria for improving tyrosine phenol lyase stability；It is accredited as greatly Intestines Escherichia is named as escherichia coli Trx-TPL (Escherichia coli Trx-TPL), is stored in Chinese Typical Representative Culture collection, preservation address: Wuhan, China, Wuhan University, the deposit date is on November 13rd, 2017, deposit number was CCTCC NO:M2017684.
The second object of the present invention is to provide the construction method of the engineering bacteria, by tyrosine phenol lyase gene and sulphur oxygen Reduction protein gene, which links together, constructs expression plasmid, i.e. amalgamation and expression sulphur hydrogen reduction protein and tyrosine phenol lyase Expression plasmid, obtain escherichia coli Trx-TPL.The stabilization of the tyrosine phenol lyase enzyme of the bacterium fermentation unit biomass Property it is high.
The engineering bacteria is full genome synthetic hydroxyphenylaminopropionic acid phenols cracking enzyme gene, is connected into containing sulphur hydrogen reduction protein base On the expression vector of cause, plasmid pTrx-TPL is constructed.
The bacterium of the above operation building improves the stability of enzyme, and when subsequent transformation synthesizes levodopa, enzyme stability is high, conversion Time extends, and production concentration increases.
Preferably, the tyrosine phenol lyase gene source is connected on expression vector in citrobacter freundii, after The enzyme that continued reaches is stablized, and transformation time can be extended, and increases production concentration, effectively reduces production cost.
Preferably, the expression vector uses pET32a.
Preferably, the recipient bacterium uses BL21 (DE3), expression of enzymes is high-efficient, and expresses and stablize, and yield is high, loses It is high to pass stability.
Preferably, construction method specifically includes: (1) the tyrosine phenol lyase full genome segment for obtaining clone is integrated Enter BL21 (DE3) on expression vector pET32a, and by the reformation plasmid conversion after integration, obtains Trx- TPL bacterium.
Above scheme can effectively improve the stability of tyrosine phenol lyase by preferred expression carrier and recipient bacterium.
Above-mentioned obtained engineering bacteria escherichia coli Trx-TPL, under substrate solution existence condition, conversion to be produced Raw L-3,4 dihydroxyphenylalanine.
Concrete operation step includes:
(1) it chooses single colonie to be inoculated into the test tube of the culture medium containing LB, 30-37 DEG C of plus ampicillin (100mg/L), 220rpm cultivates 12-16h, obtains first order seed；
(2) first order seed is inoculated into the shaking flask containing fermentation medium, 30-37 DEG C, 200-250rpm, cultivates 2- 5h adds IPTG to final concentration 0.6mM, 28-32 DEG C, 180-220rpm, cultivates 10-12h；The fermentation medium component is as follows: Tryptone 12g/L, yeast extract 24g/L, glycerol 5g/L, potassium dihydrogen phosphate 2.31g/L, three water dipotassium hydrogen phosphates 16.43g/L；
(3) bacterium is received in centrifugation, obtains thallus；
(4) thallus 35g is added substrate solution, stirs evenly, 25 DEG C, seals concussion reaction；The substrate solution includes 14- The Sodium Pyruvate of 16g/L, the catechol of 10-12g/L, the ammonium chloride of 40-45g/L, the sodium sulfite of 2-5g/L, 1-3g/L EDTA adjusts pH7.5-8.5.
Beneficial effects of the present invention are as follows: the present invention is by two tyrosine phenol lyase genes and sulphur hydrogen reduction protein gene It links together and constructs expression plasmid, can effectively improve the stability of the enzyme of expression, especially thermal stability, can provide Under related substrates, extending the time of Synthesis levodopa, increases concentration of substrate, simple process and low cost, yield is high, Application value with industrialized production.
Specific embodiment
Below by specific embodiment, technical scheme of the present invention will be further explained in detail.
Embodiment 1:
1. BL21 (DE3) is prepared into competent cell
Using TAKARA competent cell reagent preparation box, by specification operation prepares BL21 (DE3) competence.
2. full genome synthetic hydroxyphenylaminopropionic acid phenols cracking enzyme gene segment
According to the sequence that Gene ID:X66978.1 is provided, the tyrosine phenol lyase full genome segment of synthesis source.
3. constructing tyrosinase expression plasmid pTrx-TPL
Tyrosinase full genome segment is subcloned to sulphur hydrogen reduction protein gene segment downstream on pET32a plasmid.
4. expression plasmid pTrx-TPL imports competent cell
1) competence is inserted into 3min in ice-water bath after taking out from -70 degree at once；
2) it takes 1 microlitre of plasmid pTrx-TPL that competence is added in super-clean bench, flicks mixing, be inserted into ice-water bath at once 25min is stood；
3) competence is gently transferred to 42 degree of water-bath heat shock 1.5min, is gently transferred to ice-water bath 5min at once；
4) add 700 microlitres or so of LB, 150rpm, 37 degree, 60min recovery；
5) 3500rpm*3min abandons 600-700 microlitres of supernatant, and remaining bacterium solution pressure-vaccum mixes, and is coated with plus ampicillin (100mg/L) LB plate, 37 DEG C of culture 16h obtain escherichia coli pTrx-TPL.
Example 2:
Fermentation generates tyrosine phenol lyase
LB culture medium: tryptone 10g/L, yeast extract 0.5g/L, sodium chloride 10g/L, pure water.
Fermentation medium: tryptone 12g/L, yeast extract 24g/L, glycerol 5g/L, potassium dihydrogen phosphate 2.31g/L, Three water dipotassium hydrogen phosphate 16.43g/L, pure water.
1) it chooses in single colonie inoculation 4ml LB culture medium test tube, plus ampicillin (100mg/L), 37 DEG C, 220rpm, training 12h is supported, first order seed is obtained；
2) first order seed is inoculated into the shaking flask of the fermentation medium of 100ml, 37 DEG C, 220rpm, is cultivated 4h, is added IPTG extremely Final concentration 1mM, cultivates 12h by 25 DEG C, 220rpm；
3) thallus is received in bacterium solution centrifugation in step (2), places -20 DEG C of refrigerators.
Example 3: tyrosine phenol lyase conversion generates L-3,4 dihydroxyphenylalanine
1) 1L substrate solution: the Sodium Pyruvate of 14g/L, the catechol of 10g/L, the ammonium chloride of 40g/L, 2g/L sulfurous The EDTA of sour sodium, 1g/L adjusts pH8.0；
2) thallus 35g is added 1L substrate solution, stirs evenly, 25 DEG C, seals concussion reaction；
3) per half an hour adds a substrate (Sodium Pyruvate and neck benzenediol of equivalent), controls two kinds of substrate concentration Not higher than 10g/L；
4) when catechol residual concentration to 1g/L hereinafter, stopping reaction, L-3,4 dihydroxyphenylalanine concentration is accumulate to 120g/L or more.
Above-mentioned embodiment is only a preferred solution of the present invention, not the present invention is made in any form Limitation, there are also other variations and modifications on the premise of not exceeding the technical scheme recorded in the claims.

Claims (7)

1. a kind of engineering bacteria for improving tyrosine phenol lyase stability, which is characterized in that it is uncommon that the engineering bacteria is named as large intestine angstrom Salmonella Trx-TPL (Escherichia coli Trx-TPL), is stored in China typical culture collection center, preservation address: Wuhan, China, Wuhan University, the deposit date is on November 13rd, 2017, deposit number was CCTCC NO:M2017684.
2. improving the construction method of the engineering bacteria of tyrosine phenol lyase stability according to claim 1, which is characterized in that The method are as follows: tyrosine phenol lyase gene is connected into sulphur hydrogen reduction protein gene, using pET32a as carrier, constructs plasmid pTrx-TPL。
3. improving the construction method of the engineering bacteria of tyrosine phenol lyase stability according to claim 2, which is characterized in that The tyrosine phenol lyase gene is full genome synthetic hydroxyphenylaminopropionic acid phenols cracking enzyme gene.
4. improving the construction method of the engineering bacteria of tyrosine phenol lyase stability according to claim 2, which is characterized in that The tyrosine phenol lyase gene source is in citrobacter freundii.
5. improving the construction method of the engineering bacteria of tyrosine phenol lyase stability according to claim 2, which is characterized in that Recipient bacterium uses e. coli bl21 (DE3).
6. improving the construction method of the engineering bacteria of tyrosine phenol lyase stability according to claim 5, which is characterized in that Construction method specifically includes: (1) the tyrosine phenol lyase full genome segment that clone obtains being connected into carrier pET32a sulphur oxygen also Former protein gene, and the reformation plasmid conversion after integration is entered into BL21 (DE3), obtain Trx-TPL bacterium.
7. the engineering bacteria for improving tyrosine phenol lyase stability described in a kind of claim 6 is used in substrate solution existence condition Under, conversion generates L-3,4 dihydroxyphenylalanine, which is characterized in that
Concrete operation step includes:
(1) it chooses single colonie to be inoculated into the test tube of the culture medium containing LB, plus ampicillin 100mg/L, is trained by 30-37 DEG C, 220rpm 12-16h is supported, first order seed is obtained；
(2) first order seed is inoculated into the shaking flask containing fermentation medium, 30-37 DEG C, 200-250rpm, cultivates 2-5h, Add IPTG to final concentration 0.6mM, 28-32 DEG C, 180-220rpm, cultivates 10-12h；The fermentation medium component is as follows: pancreas egg White peptone 12g/L, yeast extract 24g/L, glycerol 5g/L, potassium dihydrogen phosphate 2.31g/L, three water dipotassium hydrogen phosphate 16.43g/L；
(3) bacterium is received in centrifugation, obtains thallus；
(4) thallus 35g is added substrate solution, stirs evenly, 25 DEG C, seals concussion reaction；The substrate solution includes 14-16g/L's Sodium Pyruvate, the catechol of 10-12g/L, the ammonium chloride of 40-45g/L, the sodium sulfite of 2-5g/L, 1-3g/L EDTA, adjust pH7.5-8.5。
CN201810455195.7A 2018-05-14 2018-05-14 The engineering bacteria and its construction method of raising tyrosine phenol lyase stability and application Pending CN109439605A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110305805A (en) * 2019-06-24 2019-10-08 浙江工业大学 A kind of recombinant yeast pichia pastoris engineering bacteria and its application in synthesis levodopa
CN110331153A (en) * 2019-06-24 2019-10-15 浙江工业大学 A kind of gram Lyu Wall Salmonella tyrosine phenol lyase mutant and its application

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Publication number Priority date Publication date Assignee Title
US5260198A (en) * 1990-03-31 1993-11-09 Mitsui Toatsu Chemicals, Inc. Cloned tyrosine phenol-lyase gene, recombinant plasmid containing the same and escherichia coli transformed with the same
CN107325996A (en) * 2017-05-03 2017-11-07 浙江绿创生物科技有限公司 A kind of tyrosine phenol lyase engineering bacteria and its construction method and application

Patent Citations (2)

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US5260198A (en) * 1990-03-31 1993-11-09 Mitsui Toatsu Chemicals, Inc. Cloned tyrosine phenol-lyase gene, recombinant plasmid containing the same and escherichia coli transformed with the same
CN107325996A (en) * 2017-05-03 2017-11-07 浙江绿创生物科技有限公司 A kind of tyrosine phenol lyase engineering bacteria and its construction method and application

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110305805A (en) * 2019-06-24 2019-10-08 浙江工业大学 A kind of recombinant yeast pichia pastoris engineering bacteria and its application in synthesis levodopa
CN110331153A (en) * 2019-06-24 2019-10-15 浙江工业大学 A kind of gram Lyu Wall Salmonella tyrosine phenol lyase mutant and its application
CN110331153B (en) * 2019-06-24 2021-04-30 浙江工业大学 Kluyveromyces tyrosol lyase mutant and application thereof
CN110305805B (en) * 2019-06-24 2021-07-13 浙江工业大学 Recombinant pichia pastoris engineering bacteria and application thereof in synthesis of levodopa

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