CN105886450A

CN105886450A - Trosine phenol lyase engineering bacteria as well as construction method thereof and application thereof

Info

Publication number: CN105886450A
Application number: CN201610287965.2A
Authority: CN
Inventors: 储消和; 吴黎诚; 余炜; 方明山; 徐顺清; 周卫国; 张拥军
Original assignee: Zhejiang Lyuchuang Biological Technology Co Ltd; Zhejiang Wild Wind Pharmaceutical Co Ltd
Current assignee: Zhejiang Lvchuang Biotechnology Co ltd; Zhejiang University of Technology ZJUT; Zhejiang Wild Wind Pharmaceutical Co Ltd
Priority date: 2016-05-04
Filing date: 2016-05-04
Publication date: 2016-08-24
Anticipated expiration: 2036-05-04
Also published as: CN105886450B

Abstract

The invention relates to trosine phenol lyase engineering bacteria as well as a construction method thereof and application thereof. A tyrosine phenol lyase gene is constructed to express plasmids; chaperonin is introduced to express the plasmids to obtain the trosine phenol lyase engineering bacteria which are identified as escherichia coli FPLF8 (Escherichia coli HPLF8) preserved in China Center for Type Culture Collection on February 19, 2016, with a preservation number being CCTCCNO:M 2016065. The engineering bacteria can be synthesized into levodopa by fermentation and conversion, and are efficiently expressed; the expressed product is stable and high in activity, and can be synthesized into levodopa by conversion in the presence of related substrates; and the process is simple, the cost is low, the yield is high, emission of three wastes is a little, and the application value for industrial production is achieved.

Description

A kind of tyrosine phenol lyase engineering bacteria and construction method thereof and application

Technical field

The present invention relates to a kind of engineering bacteria and construction method thereof, specifically a kind of tyrosine phenol lyase engineering bacteria and structure thereof Construction method and application, belong to genetic engineering field.

Background technology

The chemical name of levodopa (3,4-dihydroxyphenyl-L-ananine, be called for short L-DOPA) is 3,4-bis- Hydroxyphenylalanine, its structural formula is:

As a kind of important bioactive substance, L-DOPA is from TYR to catechol or melanic biochemical metabolism way Important intermediate during footpath.

The sixties in last century, external many scholars start to be devoted to the research of microbial enzyme method synthesis L-DOPA.In order to carry High L-DOPA yield and substrate conversion efficiency, the process of microbial enzyme method synthesis L-DOPA has been carried out grinding in a large number by researchers Study carefully.

Tyrosine phenol lyase (Tyrosine phenol lyase, TPL, E.C.4.1.99.2), has another name called β-tyrosine Enzyme, with pyridoxal 5-phosphate (pyridoxal-phosphate, PLP) for coenzyme, with potassium ion and ammonium ion as cofactor, TPL Can be catalyzed TYR occurs β-elimination reaction to generate phenol, acetone acid and ammonia.Owing to this reaction is reversible, by neighbour's benzene After diphenol replaces phenol, can be generated L-DOPA under TPL is catalyzed by catechol, acetone acid and ammonia.The precursor of levodopa Living enzyme when higher concentration and have inhibitory action, wherein catechol and acetone acid are in addition to having high inhibition effect, also can lead Causing the irreversible inactivation of enzyme, reaction condition is difficult to control to, and by-product is many, and the productivity of L-DOPA is low.

Also some are had to utilize the antibacterial in nature, asEscherichia、Proteus(proteus),StizolobiumhassjooWithErwinia(Erwinia) etc., synthesize L-DOPA, as levodopa enzymatic clarification is summarized Report, TPL Recombinant organism converts 30h, is converted into the L-DOPA of 29.6g/L.And for example, Jang-Young Lee Et al. Clone Origin in the p-hydroxyphenylaceticacid-3-hydroxylase (p-of Escherichia coli W (ATCC11105) Hydroxyphenylacetate 3-hydroxylase, PHAH), conversion TYR is L-DOPA, product accumulation to 10g/ L, this art applications patent US5837504.Although researcher finds that the expression of these recombinant bacterial strains TPL is higher than wild mushroom Strain, but final L-DOPA synthesis capability does not significantly improve even below wild strain.This is probably due to obtain higher L-DOPA synthesis capability, bacterial strain in addition to possessing TPL high activity, the substrate of comparatively perfect to be had, product come in and go out cell membrane Transporting mechanism and to substrate catechol inhibitory enzyme live tolerance etc..On the whole, reaction condition is difficult to control to, stable Property is poor, and by-product is many, and the productivity of L-DOPA is low.

Summary of the invention

An object of the present invention is to provide a kind of engineering bacteria；It is accredited as colon bacillus, named E Bacterium FPLF8(Escherichia coli FPLF8), it is stored in China typical culture collection center, preservation address: Wuhan is big Learning Luo Jia Shan, Wuchang, preservation date is on February 19th, 2016, and deposit number is CCTCCNO:M 2016065.

An object of the present invention is to provide the construction method of described engineering bacteria, by gene constructed for tyrosine phenol lyase table Reach plasmid, be simultaneously directed chaperone expression plasmid, obtain colon bacillus FPLF8.

The three of the purpose of the present invention are to provide the application of described engineering bacteria, utilize this engineering bacteria, can be by fermentation, conversion Synthesis levodopa, productivity is high, by-product is few, and good stability, quality better, three waste discharge is few.

In order to achieve the above object, the present invention adopts the following technical scheme that

Described engineering bacteria is to be connected on expression vector by tyrosine phenol lyase gene, construction expression plasmid pFPL, then will express Plasmid pFPL imports in recipient bacterium escherichia coli and obtains.

As preferably, in the escherichia coli being imported with expression plasmid pFPL, import chaperone expression plasmid again, it is thus achieved that Colon bacillus FPLF8.Import chaperone expression plasmid, can play promote colon bacillus FPLF8 enzyme can Dissolubility is expressed, thus the enzyme improving unit thalline is lived, and the stablizing of fermentation enzyme active unit, subsequent transformation synthesis levodopa Time, throwing bacterium amount is few, and reaction quickly, keeps high yield, subsequent transformation synthesis levodopa to stablize.

Further, described chaperone expression plasmid is pG-KJE8, and effect becomes apparent from, colon bacillus FPLF8 mesh Expression of enzymes more preferably.

As preferably, described tyrosine phenol lyase gene source, in citrobacter freundii, is connected on expression vector and melts Conjunction is good, and follow-up expression is stable, expression product more efficiently, more single-minded synthesis levodopa, by-product is few, to follow-up point Bring the biggest convenience from purification, and simplify separating step, effectively reduce production cost.

As preferably, described expression vector uses pET24a, and more preferable with genes of interest segment composition, follow-up expression is more steady Fixed.

As preferably, described recipient bacterium escherichia coli use BL21(DE3), expression of enzymes efficiency is high, and expresses stable, produces Rate is high, and hereditary stability is high.

As preferably, construction method specifically includes: the tyrosine phenol lyase full genome fragment that clone is obtained by (1) is integrated Enter on expression vector pET24a, and will integrate after reformation Plastid transformation enter BL21(DE3), it is thus achieved that FPL bacterium；(2) by companion Protein expressing plasmid imports in FPL bacterium, it is thus achieved that FPLF8 bacterium.

Such scheme passes through preferred expression carrier and recipient bacterium, can be effectively improved the expression efficiency of genes of interest fragment, Expression stability, by chaperone expression plasmid, improves engineering bacterium expression and produces tyrosine phenol lyase activity and stablize Property.

Engineering bacteria colon bacillus FPLF8 obtained by above-mentioned, under substrate solution existence condition, converts and produces Raw L-3,4 dihydroxyphenylalanine.

Concrete operation step includes:

(1) single colony inoculation is chosen in the test tube containing LB culture medium, Jia Kana mycin (100mg/L), chloromycetin (25mg/L), 35-37 DEG C, 220rpm, cultivates 12-16h, obtains first order seed；

(2) described first order seed is inoculated in the shaking flask containing fermentation medium, 35-37 DEG C, 200-250rpm, cultivates 2-5h, Add IPTG to final concentration 1-1.5mM, 23-27 DEG C, 180-220rpm, cultivates 10-12h；Described fermentation medium component is as follows: pancreas Peptone 12g/L, yeast extract 24g/L, glycerol 5g/L, potassium dihydrogen phosphate 2.31 g/L, three water dipotassium hydrogen phosphates 16.43 g/L；

(3) centrifugal receipts bacterium, obtains thalline；

(4) thalline 60g, adds substrate solution, stirs evenly, 25 DEG C, seals concussion reaction；Described 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, the EDTA of 1-3g/L, adjust pH7.5-8.5；

As preferably, in step (4) during reaction, repeatedly add substrate catechol and Sodium Pyruvate, control catechol dense Degree is less than 10g/L.

Beneficial effects of the present invention is as follows: the present invention obtains engineering bacteria, expresses efficiently, and expression product height stable, active can To provide under related substrates, Synthesis levodopa, technique is simple, with low cost, and productivity is high, and three waste discharge is few simultaneously, There is the using value of industrialized production.

Detailed description of the invention

Below in conjunction with embodiment, the present invention is illustrated further.

Embodiment 1:

1. by BL21(DE3) it is prepared as competent cell

Using TAKARA competent cell to prepare test kit, by specification operates, and prepares BL21(DE3) competence.

2. full genome synthetic hydroxyphenylaminopropionic acid phenols cracking enzyme genetic fragment

The sequence provided according to Gene ID:X66978.1, the tyrosine phenol lyase full genome fragment in synthesis source.

3. build tryrosinase expression plasmid pFPL

By tryrosinase full genome fragment sub-clone to pET24a plasmid, restriction enzyme site BamHI, XhoI.

4. expression plasmid pFPL imports competent cell

1) competence inserts 3min ice-water bath after-70 degree take out at once；

2) the plasmid pFPL taking 1 microlitre in super-clean bench adds competence, flicks mixing, inserts ice-water bath 25min at once, stands；

3) competence is transferred to gently 42 degree of water-bath heat shock 1.5min, is transferred to ice-water bath 5min the most gently；

4) adding the LB, 150rpm, 37 degree of 700 microliters, 60min recovers；

5) 3500rpm*3min, abandons supernatant 600-700 microlitre, residue bacterium solution pressure-vaccum mixing, is coated with plus ampicillin (100mg/ L) LB flat board, cultivates 16h for 37 DEG C, it is thus achieved that colon bacillus FPLF8.

Embodiment 2:

1. by BL21(DE3) it is prepared as competent cell

3. build tryrosinase expression plasmid pFPL

4. expression plasmid pFPL imports competent cell

6) competence inserts 3min ice-water bath after-70 degree take out at once；

7) the plasmid pFPL taking 1 microlitre in super-clean bench adds competence, flicks mixing, inserts ice-water bath 25min at once, stands；

8) competence is transferred to gently 42 degree of water-bath heat shock 1.5min, is transferred to ice-water bath 5min the most gently；

9) adding the LB, 150rpm, 37 degree of 700 microliters, 60min recovers；

10) 3500rpm*3min, abandons supernatant 600-700 microlitre, residue bacterium solution pressure-vaccum mixing, is coated with plus ampicillin (100mg/L) LB flat board, cultivates 16h for 37 DEG C, it is thus achieved that FPL bacterium；

Prepared by 5.FPL bacterium competence

6. chaperone expression plasmid imports FPL competent cell

1) competence inserts 3min ice-water bath after-70 degree take out at once；

2) the chaperone expression plasmid pG-KJE8 taking 1 microlitre in super-clean bench adds competence, flicks mixing, inserts ice at once Water-bath 25min, stands；

4) adding the LB, 150rpm, 37 degree of 700 microliters, 60min recovers；

5) 3500rpm*3min, abandons supernatant 600-700 microlitre, residue bacterium solution pressure-vaccum mixing, is coated with Jia Kana mycin (100mg/ L), chloromycetin (25mg/L) LB flat board, 37 DEG C cultivate 16h, it is thus achieved that FPLF8 bacterium.

Embodiment 3:

Being with the difference of embodiment 2, chaperone expression plasmid selects pKJE7.

Embodiment 4:

1. by BL21(DE3) it is prepared as competent cell

3. build tryrosinase expression plasmid pFPL

4. expression plasmid pFPL imports competent cell

11) competence inserts 3min ice-water bath after-70 degree take out at once；

12) the plasmid pFPL taking 1 microlitre in super-clean bench adds competence, flicks mixing, inserts ice-water bath 25min at once, quiet Put；

13) competence is transferred to gently 42 degree of water-bath heat shock 1.5min, is transferred to ice-water bath 5min the most gently；

14) adding the LB, 150rpm, 37 degree of 700 microliters, 60min recovers；

15) 3500rpm*3min, abandons supernatant 600-700 microlitre, residue bacterium solution pressure-vaccum mixing, is coated with plus ampicillin (100mg/L) LB flat board, cultivates 16h for 37 DEG C, it is thus achieved that FPL bacterium；

Prepared by 5.FPL bacterium competence

6. chaperone plasmid imports FPL competent cell

6) competence inserts 3min ice-water bath after-70 degree take out at once；

7) the chaperone expression plasmid pG-KJE8 taking 1 microlitre in super-clean bench adds competence, flicks mixing, inserts ice at once Water-bath 25min, stands；

9) adding the LB, 150rpm, 37 degree of 700 microliters, 60min recovers；

10) 3500rpm*3min, abandons supernatant 600-700 microlitre, residue bacterium solution pressure-vaccum mixing, is coated with Jia Kana mycin (100mg/ L), chloromycetin (25mg/L) LB flat board, 37 DEG C cultivate 16h, it is thus achieved that FPLF8 bacterium；

7. fermentation produces tyrosine phenol lyase

LB culture medium: tryptone 10g/L, yeast extract 0.5 g/L, sodium chloride 10g/L, pure water.

Fermentation medium: tryptone 12g/L, yeast extract 24g/L, glycerol 5g/L, potassium dihydrogen phosphate 2.31 g/L, Three water dipotassium hydrogen phosphate 16.43 g/L, pure water.

1) choose in single colony inoculation 4ml LB culture medium test tube, Jia Kana mycin (100mg/L), chloromycetin (25mg/L), 37 DEG C, 220rpm, cultivates 12h, obtains first order seed；

2) in the shaking flask of the fermentation medium that first order seed is inoculated into 100ml, 37 DEG C, 220rpm, cultivates 4h, adds IPTG to the denseest Degree 1mM, 25 DEG C, 220rpm, cultivates 12h；

3) in step (2), bacterium solution is centrifugal receives thalline, places-20 DEG C of refrigerators.

8. tyrosine phenol lyase converts and produces L-3,4 dihydroxyphenylalanine

1) Sodium Pyruvate of 1L substrate solution: 14g/L, the catechol of 10g/L, the ammonium chloride of 40g/L, the sulfurous acid of 2g/L Sodium, the EDTA of 1g/L, adjust PH8.0；

2) thalline 60g, adds 1L substrate solution, stirs evenly, 25 DEG C, seals concussion reaction；

3) when catechol residual concentration to below 1g/L, stopped reaction, L-3,4 dihydroxyphenylalanine concentration is accumulate to more than 85g/L.

Embodiment 5:

It is with the difference of embodiment 4:

1. fermentation produces tyrosine phenol lyase

4) single colony inoculation 4ml LB test tube is chosen, Jia Kana mycin (100mg/L), chloromycetin (25mg/L), 35 DEG C, 200rpm, Cultivate 14h, obtain first order seed；

5) the TB shaking flask of one-level test tube seed inoculation 100ml, 35 DEG C, 200rpm, cultivate 2h, add IPTG to final concentration 1.5mM, 23 DEG C, 180rpm, cultivates 10h；Fermentation medium: tryptone 12g/L, yeast extract 24g/L, glycerol 5g/L, biphosphate Potassium 2.31 g/L, three water dipotassium hydrogen phosphate 16.43 g/L.

6) centrifugal receipts bacterium, places-20 DEG C of refrigerators.

2. tyrosine phenol lyase converts and produces L-3,4 dihydroxyphenylalanine

4) Sodium Pyruvate of 1L substrate solution: 15g/L, the catechol of 11g/L, the ammonium chloride of 43g/L, the sulfurous acid of 3g/L Sodium, the EDTA of 2g/L, adjust PH7.5；

5) thalline 60g, PLP-100mg, adds 1L substrate solution, stirs evenly, 25 DEG C, seals concussion reaction；

6) conversion process adds substrate catechol and Sodium Pyruvate, keeps catechol concentration 8g/L；

7) L-3,4 dihydroxyphenylalanine is accumulate to 65g/L, stops increasing substrate, as catechol residual concentration to below 1g/L, stopped reaction, L- DOPA concentration is accumulate to more than 85g/L.

Embodiment 6:

It is with the difference of embodiment 4:

Under substrate solution existence condition, the concrete operation step of the generation L-3,4 dihydroxyphenylalanine that ferments, converts includes:

(1) single colony inoculation is chosen in the test tube containing LB culture medium, Jia Kana mycin (100mg/L), chloromycetin (25mg/L), 36 DEG C, 250rpm, cultivates 16h, obtains first order seed；

(2) described first order seed is inoculated in the shaking flask containing fermentation medium, 36 DEG C, 250rpm, cultivates 2-5h, add IPTG extremely Final concentration 1mM, 27 DEG C, 220rpm, cultivates 11h；Described fermentation medium component is as follows: tryptone 12g/L, yeast extract 24g/L, glycerol 5g/L, potassium dihydrogen phosphate 2.31 g/L, three water dipotassium hydrogen phosphate 16.43 g/L；

(3) in step (2), bacterium solution is centrifugal collects thalline；

(4) thalline 60g, adds substrate solution, stirs evenly, 25 DEG C, seals concussion reaction；Described substrate solution includes the third of 16g/L Keto acid sodium, the catechol of 12g/L, the ammonium chloride of 45g/L, the sodium sulfite of 5g/L, the EDTA of 3g/L, adjust pH8.5；

(5) when catechol residual concentration to below 0.5g/L, stopped reaction.

Claims

1. a tyrosine phenol lyase engineering bacteria, entitled colon bacillus FPLF8(Escherichia coli FPLF8), being stored in China typical culture collection center, preservation date is on February 19th, 2016, and deposit number is CCTCCNO:M 2016065.

A kind of tyrosine phenol lyase engineering bacteria, it is characterised in that described engineering bacteria is by cheese ammonia Acid phenols cracking enzyme gene is connected on expression vector, construction expression plasmid pFPL, then expression plasmid pFPL is imported recipient bacterium large intestine Bacillus obtains.

A kind of tyrosine phenol lyase engineering bacteria, it is characterised in that to being imported with expression plasmid The escherichia coli of pFPL import chaperone expression plasmid again, it is thus achieved that colon bacillus FPLF8.

The construction method of a kind of tyrosine phenol lyase engineering bacteria, it is characterised in that described companion Protein expressing plasmid is pG-KJE8.

A kind of tyrosine phenol lyase engineering bacteria, it is characterised in that described tyrosine phenol lyase Gene source is in citrobacter freundii.

A kind of tyrosine phenol lyase engineering bacteria, it is characterised in that described expression vector uses pET24a。

A kind of tyrosine phenol lyase engineering bacteria, it is characterised in that described recipient bacterium escherichia coli Use BL21(DE3).

The construction method of a kind of tyrosine phenol lyase engineering bacteria the most as claimed in claim 1, it is characterised in that including: (1) will The tyrosine phenol lyase full genome fragment that obtains of clone is integrated on expression vector pET24a, and the reformation plasmid after integrating Convert and enter BL21(DE3), it is thus achieved that FPL bacterium；(2) chaperone expression plasmid is imported in FPL bacterium, it is thus achieved that FPLF8 bacterium.

The application of a kind of tyrosine phenol lyase engineering bacteria the most as claimed in claim 1, it is characterised in that at substrate solution Under existence condition, ferment, convert generation L-3,4 dihydroxyphenylalanine.

The application of a kind of tyrosine phenol lyase engineering bacteria, it is characterised in that at substrate solution Under existence condition, ferment, convert produce L-3,4 dihydroxyphenylalanine concrete operation step include:

(1) single colony inoculation is chosen in the test tube containing LB culture medium, Jia Kana mycin (100mg/L), chloromycetin (25mg/L), 35-37 DEG C, 200-250rpm, cultivates 12-16h, obtains first order seed；

(2) described first order seed is inoculated in the shaking flask containing fermentation medium, 35-37 DEG C, 200-250rpm, cultivates 2-5h, Add IPTG to final concentration 1-1.5mM, 23-27 DEG C, 180-220rpm, cultivates 10-12h；

(3) in step (2), bacterium solution is centrifugal collects thalline；

(5) when catechol residual concentration to below 1g/L, stopped reaction.