CN106754846A - A kind of Fusobacterium nucleatum tyrosine phenol lyase mutant, gene, carrier, engineering bacteria and its application - Google Patents
A kind of Fusobacterium nucleatum tyrosine phenol lyase mutant, gene, carrier, engineering bacteria and its application Download PDFInfo
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Abstract
The invention discloses a kind of Fusobacterium nucleatum tyrosine phenol lyase mutant, gene, carrier, engineering bacteria and its application in levodopa is synthesized, Fusobacterium nucleatum TPL mutant provided by the present invention compared with wild type, with more excellent catalytic performance.TPL mutant synthesis levodopa cumulative concentration is up to more than 140g/L, and 17%~25% is improve compared with wild type, and optical purity is more than 99.5%；The conversion ratio of substrate catechol reaches more than 99.8%, and 15% 20% are improve compared with wild type.
Description
(1) technical field
The present invention relates to the tyrosine phenols cracking that one kind derives from Fusobacterium nucleatum (Fusobacteriumnucleatum)
Enzyme (Tyrosine Phenol Lyase, TPL) mutant, encoding gene and its in levodopa (L-DOPA) is catalyzed and synthesized
Application.
(2) background technology
Levodopa (β -3,4- dihydroxyphenyls-α-alanine, 3,4-dihydroxylphenylalanine-L-
Alanine, L-DOPA) it is bioactivator important in organism.It can reach central nervous system by blood-brain barrier
System, in vivo in the presence of decarboxylase, is converted into dopamine, and then play the effect for the treatment of parkinsonism.With the modern times
The aggravation of increase and the aging population of social life pressure, Parkinsonian's a few days benefit is raised, and levodopa is used as handkerchief
The choice drug of the gloomy syndrome treatment of gold, its demand is continuously increased.
The main method that tradition prepares levodopa includes that extraction and chemical method are asymmetric from plant such as lamb's-quarters beans, cat beans
Synthesis etc..Wherein, levodopa is extracted from plant to be limited by raw material sources, and extraction step is numerous and diverse, yield poorly, far
The market demand can not be met；And chemical dissymmetric synthesis are due to severe reaction conditions, conversion ratio and stereoselectivity is low, environment
Unfriendly the problems such as, it is restricted in the industrial production.Living things catalysis is because its reaction condition is gentle, processing efficient, High level of stereoselectivity,
The advantages of region and chemo-selective, it has also become the important method of levodopa industrialized production.
The biocatalyst of enzymatic clarification levodopa mainly includes tyrosinase, amino-acylase, p- hydroxyl phenylacetic acids
3- hydroxylases, tyrosine phenol lyase etc..The enzyme activity of tyrosinase catalysis TYR synthesis levodopa is relatively low, and due to junket
Propylhomoserin enzyme has diphenol oxidation activity simultaneously, and levodopa can be aoxidized further generation accessory substance DOPA quinone.Amino-acylase
Kinetic Resolution N- acetyl -3- (3,4- dimethoxy phenyl) alanine hydrolysis 3- (3,4- dimethoxy phenyl) alanine, its
Methoxyl group synthesizes levodopa after being deprotected through HBr.The process route is long, and uses and produce toxic gas MeBr, and safety is hidden
Suffer from big.During p- hydroxyl phenylacetic acid 3- hydroxylase catalysis TYR synthesis levodopa, co-factor NADH is needed to participate in catalytic reaction,
Production cost is high.Compared with above-mentioned enzyme method technique, tyrosine phenol lyase (TPL) technique is with catechol, pyruvic acid and ammonia
Substrate, is formed by C-C keys and levodopa is synthesized, and Atom economy is high.Because solubility of the levodopa in water is low,
Constantly separated out in biocatalytic reaction, be conducive to the reversible reaction to be carried out to product generation direction, reach conversion ratio higher,
Thus there is important commercial development prospect.
TPL is widely present in various microorganisms, and the TPL sources reported at present for levodopa synthesis include Freund
Citric acid fungus (Citrobacterfreundii), the raw Erwinia (Erwiniaherbicola) of grass and Thermophilic Bacteria
(Symbiobacterium sp.) etc..
The natural substrate of one of substrate of levodopa catechol not enzyme is catalyzed and synthesized due to TPL, its catalysis effect
Rate has much room for improvement.On the other hand, catechol is strong protein denaturant, and under high strength industrial production environment, high concentration is adjacent
Benzenediol can produce inhibitory action to TPL, or even make its irreversible inactivation.Therefore, new vigor high and catechol high is developed
The TPL of tolerance is of great significance to realizing that the industrialized production of levodopa has.
(3) content of the invention
The tyrosine phenol lyase that Fusobacterium nucleatum is originated is transformed the present invention seeks to pass through orthogenesis means,
A kind of mutant protein is provided, protein vigor is improved, with catechol, pyruvic acid and ammonia as substrate, efficient catalytic synthesis is left-handed
DOPA.
The technical solution adopted by the present invention is：
The present invention provides a kind of Fusobacterium nucleatum tyrosine phenol lyase mutant (i.e. TPL mutant), the mutant
It is that amino acid sequence shown in SEQ ID NO.2 the 84th and 129 is carried out into the double mutation of fallibility PCR to obtain.It is specific described double
Mutation is that the glutamic acid (E) of the 84th is sported into lysine (K), while being different bright ammonia by threonine mutation (T) of the 129th
Sour (I), the variant amino acid sequence is shown in SEQ ID NO.4.It is any in amino acid sequence shown in SEQ ID NO.4
Amino acid is by lacking, inserting or replace one or several amino acid and with TPL activity, still fall within protection model of the invention
Enclose.
From Fusobacterium nucleatum, (F.nucleatum, CGMCC 1.2526 is protected the present invention purchased from Chinese industrial microorganism fungus kind
Hide administrative center) its genome is extracted for template, the successful clone gene (Fn-TPL) of coding TPL is converted in Escherichia coli
The expression of the gene has been carried out after (Escherichia coli).Plasmid containing Fn-TPL is further extracted from Escherichia coli, is made
Random mutation is carried out to TPL genes with many wheel fallibility PCR methods, after being connected to expression vector, Escherichia coli is equally expressed in, led to
The mutant that screening acquisition vigor is improved is crossed, can be used in the industrialized production of levodopa.
The present invention is mutated to the TPL of SEQ ID NO.1 codings, can be using conventional molecular modification means.Preferentially,
Expanded by fallibility PCR, change Mg in PCR system2+Concentration, and addition Mn2+, to obtain mutant DNA sequences SEQ ID
NO.3, its amino acid sequence is SEQ ID NO.4.
The present invention also provides a kind of encoding gene of the Fusobacterium nucleatum TPL mutant, the encoding gene nucleotides
Sequence is shown in SEQ ID NO.3.
Preparation is converted the invention further relates to a kind of recombinant vector of encoding gene structure and by the recombinant vector
Recombination engineering bacteria.
The nucleotide sequence of TPL mutant of the invention can be connected to various loads by the present invention by this area conventional method
It is built-up on body.Recombinant vector of the invention is not limited, as long as it can be in protokaryon and/or the various hosts of eukaryotic
It is kept to replicate or autonomous replication in cell, the carrier can be the conventional various carriers in this area, such as various plasmids, bacteriophage
Or viral vectors etc., preferably pET-28a.It is preferred that recombinant expression carrier of the invention can be obtained by following methods：To obtain
Wild type TPL and mutated gene product be connected with carrier pET-28a, build TPL mutators of the invention recombination expression
Plasmid pET28a-FnTPL and pET28a-FnTPLM.
The host cell for introducing the DNA of coding TPL mutant of the invention is not limited, as long as being it establishes weight
Group expression system, meet recombinant expression carrier can stablize self-replacation and entrained TPL mutators of the invention can be with
Effective expression.Such as Escherichia coli, bacillus subtilis, saccharomycete, actinomyces, Aspergillus, and zooblast and high plant
Thing cell.Preferably Escherichia coli, more preferably E. coli BL21 (DE3) of the invention.By recombinant plasmid pET28a-
FnTPL and pET28a-FnTPLM are converted into E.coli BL21 (DE3), and acquisition engineering bacteria E.coli BL21 (DE3)/
PET28a-FnTPL and E.coli BL21 (DE3)/pET28a-FnTPLM.
The preparation of present invention restructuring TPL mutant includes culture recombinant expression transformants of the invention, and induction is recombinated
TPL mutains.Wherein, the culture medium used by described culture recombinant expression transformants can be that this area can give birth to transformant
Grow and produce the culture medium of TPL of the invention, preferably LB culture mediums：Peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L,
Solvent is water, pH 7.2.Cultural method and condition of culture are not particularly limited, as long as enabling transformant to grow and producing TPL
.It is preferred that following methods：Restructuring E.coli BL21 (DE3)/pET28a-FnTPLM of the present invention is seeded to containing 50 μ
In the LB culture mediums of g/ml kanamycins, 37 DEG C of cultures to optical density OD600When reaching 0.5~0.7, final concentration of 0.1~
Under the induction of 1.0mM isopropyl-beta D-thios galactopyranoside (IPTG), you can high efficient expression TPL mutation egg of the invention
In vain.
The present invention also provides a kind of application of Fusobacterium nucleatum TPL mutant in levodopa is synthesized, specific institute
State and be using the wet thallus obtained with the fermented culture of the recombination engineering bacteria of the mutant code genes of TPL containing Fusobacterium nucleatum
Catalyst, with catechol, Sodium Pyruvate and ammonium acetate as substrate, with sodium sulfite and EDTANa2It is auxiliary agent, with phosphoric acid pyrrole
Aldehyde (Pyridoxal 5 '-phosphate, PLP) of trembling is coenzyme, is reaction with the buffer solution of pH5.0~9.0 (preferably pH8.0)
Medium constitutes transformation system, and conversion reaction is carried out under the conditions of 5~30 DEG C of temperature (preferably 15 DEG C), after reaction terminates, will react
Liquid is isolated and purified, and obtains levodopa.
Further, in the transformation system, when initial, catechol adds 5~50g/L of final concentration (preferably 5g/L), third
Ketone acid sodium adds 5~50g/L of final concentration (preferably 8g/L), ammonium acetate to add 5~100g/L of final concentration (preferably 50g/L), sulfurous
Sour sodium adds 0.5~10g/L of final concentration (preferably 1g/L), EDTANa2Add 0.5~10g/L of final concentration (preferably 2g/L), phosphorus
Sour pyridoxal adds 0.05~5mM of final concentration (preferably 1mM), and wet thallus consumption is 2~50g/L (preferably 20g/L).
Further, feed supplement is carried out to catechol, Sodium Pyruvate and ammonium acetate during the conversion reaction, every 0.5
~4h feed supplements once, the wherein each 0.1~10g/L of feed supplement of catechol (preferably 5g/L), each feed supplement 0.1 of Sodium Pyruvate~
10g/L (preferably 5g/L), each 1~20g/L of feed supplement of ammonium acetate (preferably 3.5g/L).
The TPL mutant that the present invention is provided can be catalyzed in the form of resolvase, immobilised enzymes and restructuring free cell
Synthesis levodopa.
Further, the catalyst is prepared as follows：
(1) inclined-plane culture：The recombination engineering bacteria of the mutant code genes of TPL containing Fusobacterium nucleatum is seeded to containing 50
The slant medium of μ g/ml kanamycins, 8~16h is cultivated at 37 DEG C, obtains inclined-plane thalline；The slant medium final concentration
Constitute and be：Peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, 1.5% agar, solvent is deionized water, pH
7.0.Use 50 μ g/ml kanamycins of preceding addition.
(2) seed culture：Inclined-plane thalline is seeded to seed culture medium, 8~10h is cultivated at 37 DEG C, obtain seed liquor；Institute
Stating seed culture medium final concentration composition is：Peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, that is mould for 50 μ g/ml cards
Element, solvent is deionized water, pH 7.0.
(3) fermented and cultured：Seed liquor is seeded to the inoculum concentration of volumetric concentration 3% aseptic equipped with 3L fermentation mediums
5L mechanical agitations ventilation general-purpose type fermentation tank in, sterilized final concentration of 15g/L lactose is directly appended in fermentation tank
In Fiber differentiation at 28 DEG C.Wet thallus are collected after tank is put after 6~8h of culture.The fermentation medium final concentration is constituted：Albumen
Peptone 15g/L, dusty yeast 12g/L, NaCl10g/L, glycerine 15g/L, (NH4)2SO45g/L, KH2PO41.36g/L, K2HPO4·
3H2O 2.28g/L, MgSO4·7H2O 0.375g/L, solvent is deionized water, pH.
Compared with prior art, beneficial effects of the present invention are embodied in：Fusobacterium nucleatum TPL mutation provided by the present invention
Body compared with wild type, with more excellent catalytic performance.TPL mutant synthesis levodopa cumulative concentration is up to 140g/L
More than, 17%~25% is improve compared with wild type, optical purity is more than 99.5%；The conversion ratio of substrate catechol reaches
More than 99.8%, 15%-20% is improve compared with wild type.
(4) illustrate
Fig. 1 is the reaction process for synthesizing levodopa containing wild type and mutant TPL whole-cell catalytics.
(5) specific embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
The acquisition of the TPL genes of embodiment 1
Fusobacterium nucleatum (F.nucleatum subsp.CGMCC 1.2526, purchased from China are extracted with DNA extraction kit
Research for Industrial Microbial Germ preservation administrative center) complete genome DNA, with the DNA as template, sense primer (5 '
GCTGAGGATCCATGAGATTTGAAGATTATCCAGC3’) and anti-sense primer (5 '
GCATCCTCGAGTTATTTTTTTATTCCAAATCTAGC3’) carry out pcr amplification reaction to act on primer.PCR reaction systems are each
Component addition (the μ L of cumulative volume 50)：5 × PrimeSTARTM HS DNA polymerase Buffer 10 μ L, 10mMdNTP
Mixture (each 2.5mM of dATP, dCTP, dGTP and dTTP) 4 μ L, concentration is 50 μM of sense primer, each 1 μ L of anti-sense primer, base
Because of 1 μ L, PrimeSTARTM HS DNA polymerase of group DNA 0.5 μ L, the μ L of seedless sour water 32.5.PCR reaction conditions are：
95 DEG C of 1min of predegeneration, subsequently into 95 DEG C of 10s of temperature cycles, 56 DEG C of 90s, 72 DEG C of 1min, totally 30 circulations, last 72 DEG C are prolonged
10min is stretched, final temperature is 4 DEG C.Sequencing analysis result shows that the nucleotide sequence length obtained through said process amplification is
1383bp (its nucleotide sequence such as SEQ ID NO：Shown in 1), one complete ORFs of the sequential coding, the ammonia of coding
Base acid sequence such as SEQ ID NO：Shown in 2.
The fallibility PCR of embodiment 2 builds TPL mutated libraries
TPL genes with the acquisition of embodiment 1 are expanded as template by fallibility PCR, obtain mutant nucleotide sequence.Amplimer is
(5’GCTGAGGATCCATGAGATTTGAAGATTATCCAGC3’) and (5 '
GCATCCTCGAGTTATTTTTTTATTCCAAATCTAGC3’)。
Amplification system is：50 μ l reaction systems：
10xTaq polymerase buffer：5μL；
Mg2+(25mM)：2-8μL；
Mm2+(25mM):2-8μL；
10mMdNTP mixture (each 2.5mM of dATP, dCTP, dGTP and dTTP) 4 μ；
Concentration is 50 μM of sense primer, each 1 μ L of anti-sense primer,
DNA profiling：1μL；
Taq archaeal dna polymerases：10U；
System is supplied with distilled water.
PCR reaction conditions are：95 DEG C of 1min of predegeneration, subsequently into 95 DEG C of 10s of temperature cycles, 56 DEG C of 90s, 72 DEG C
1min, totally 30 circulations, last 72 DEG C of extensions 10min, final temperature is 4 DEG C.PCR primer is through 1% agarose gel electrophoresis point
Analysis and gel extraction, double digestion is carried out by BamHI/XhoI, with the same pET28a connections for carrying out digestion treatment, connection liquid electric shock
Transformed E .coli BL21 (DE3) competent cell, LB flat board of the coating containing kanamycins (50 μ g/ml), 37 DEG C of overnight incubations,
Obtain the mutated library of TPL.
The screening of the TPL mutated libraries of embodiment 3
The screening of TPL mutated libraries is with (Choi, et al., Kor.J.Microbiol.2006,34 described in document:58-
62).With the enzyme before mutation as reference, primary dcreening operation obtains the positive colony that vigor is improved, further by liquid chromatogram measuring.Pass through
After first round mutation, active highest mutant is obtained, sequencing shows that the mutant for obtaining is E84K.Extract mutant containing E84K
Plasmid be template, carry out the second wheel fallibility PCR, such as embodiment 2, convert in after Escherichia coli, then the sieve for carrying out mutated library
Choosing, obtains vigor compared with the mutant that E84K is improved, and shows that the mutant for obtaining is double-mutant E84K/T129I, its ammonia through sequencing
Base acid sequence such as SEQ ID No.4, nucleotides sequence is classified as SEQ ID No.3.
The induced expression of the wild type of embodiment 4 and mutant TPL engineering bacterias
The engineering bacteria of wild type (SEQ ID No.1) and mutant TPL (SEQ ID No.3) recombinant plasmid will be contained respectively
E.coli BL21 (DE3)/pET28a-FnTPL and E.coli BL21 (DE3)/pET28a-FnTPLM is seeded to containing 50 μ g/mL
In the LB fluid nutrient mediums of kanamycins, 37 DEG C of overnight incubations, then it is inoculated into containing 50 μ g/mL cards that is mould with 1% (v/v) inoculum concentration
In the 50mL LB culture mediums of element, 37 DEG C, 200rpm cultivated to cell concentration OD600To 0.6 or so, final concentration of 0.1mM is added
IPTG, after 28 DEG C of 6~8h of Fiber differentiation, 4 DEG C, 8000rpm centrifugation 10min collect wet thallus, it is standby in -80 DEG C of storages.
The TPL of embodiment 5 is mutated the preparation of body catalyst
(1) inclined-plane culture：By the recombination engineering bacteria E.coli of the mutant code genes of TPL containing Fusobacterium nucleatum
BL21 (DE3)/pET28a-FnTPLM is seeded to the slant medium containing 50 μ g/ml kanamycins, and 16h is cultivated at 37 DEG C, obtains
Inclined-plane thalline；The slant medium quality final concentration is constituted：Peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/
L, 1.5% agar, solvent is deionized water, and pH 7.0 uses 50 μ g/ml kanamycins of preceding addition.
(2) seed culture：Inclined-plane thalline is seeded to seed culture medium, 8~10h is cultivated at 37 DEG C, obtain seed liquor；Institute
Stating seed culture medium final concentration composition is：Peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, that is mould for 50 μ g/ml cards
Element, solvent is deionized water, pH 7.0.
(3) fermented and cultured：Seed liquor is seeded to the inoculum concentration of volumetric concentration 3% aseptic equipped with 3L fermentation mediums
5L mechanical agitations ventilation general-purpose type fermentation tank in, sterilized final concentration of 15g/L lactose is directly appended in fermentation tank
In Fiber differentiation at 28 DEG C.Wet thallus are collected after tank is put after 6~8h of culture.The fermentation medium final concentration is constituted：Albumen
Peptone 15g/L, dusty yeast 12g/L, NaCl10g/L, glycerine 15g/L, (NH4)2SO45g/L, KH2PO41.36g/L, K2HPO4·
3H2O 2.28g/L, MgSO4·7H2O 0.375g/L, solvent is deionized water, pH natures.
The wild type of embodiment 6 and mutant TPL catalyze and synthesize levodopa
With mutants which had E.coli BL21 (DE3)/pET28a-FnTPLM and starting strain that embodiment 4 is obtained
E.coli BL21 (DE3)/pET28a-FnTPL cells determine the ability for catalyzing and synthesizing levodopa respectively.
(1) catechol 5g/L, Sodium Pyruvate 5g/L, ammonium acetate 5g/L, sodium sulfite are added in 500ml reaction systems
0.5g/L, EDTANa20.5g/L, phosphopyridoxal pyridoxal phosphate (PLP) 0.05mM, ammoniacal liquor adjust pH to 8.0, TPL prepared by embodiment 5 to dash forward
Variant recombinant cell concentration 5g/L (weight in wet base)；Reacted under the conditions of 15 DEG C of temperature.Course of reaction is carried out by the way of feed supplement,
Every 0.5h feed supplements once, the wherein each feed supplement 0.5g/L of catechol, each feed supplement 0.8g/L of Sodium Pyruvate, ammonium acetate is each
Feed supplement 2g/L converts 24h.Reaction is detected that testing conditions are as follows with high performance liquid chromatography after terminating：
Mobile phase:A:B=9:1(A:0.02M KH2PO4-6M HCl, pH=2.6；B:Methyl alcohol)
Chromatographic column:C18(Welchrom 4.6*250mm)
Detection wavelength:280nm
Column temperature:34℃
Sample size:10μL
Flow velocity:1ml/min
TPL mutants which hads cell catalysis synthesize levodopa, and target product concentration reaches 20g/L, conversion ratio up to 45%,
Yield 39%, levodopa optical purity is more than 99.5%；Wild type TPL strain cells catalyze and synthesize levodopa, and target is produced
Thing concentration reaches 15g/L, and up to 36%, up to 29%, levodopa optical purity is more than 99.5% to yield to conversion ratio.
(2) catechol 5g/L, Sodium Pyruvate 8g/L, ammonium acetate 50g/L, sulfurous acid are added in 500ml reaction systems
Sodium 1g/L, EDTANa22g/L, phosphopyridoxal pyridoxal phosphate (PLP) 1mM, ammoniacal liquor adjust pH to 8.0, TPL mutant prepared by embodiment 5
Recombinant cell concentration 20g/L (weight in wet base)；Reacted under the conditions of 15 DEG C of temperature.Course of reaction is carried out by the way of feed supplement, often
Every 1h feed supplements once, the wherein each feed supplement 5g/L of catechol, each feed supplement 5g/L of Sodium Pyruvate, each feed supplement of ammonium acetate
3.5g/L converts 17h.TPL mutants which hads cell catalysis synthesize levodopa, and target product concentration reaches 140g/L (Fig. 1),
Up to 99.8%, yield is higher than 91% to conversion ratio, and levodopa optical purity is more than 99.5%；And wild type TPL strain cells are urged
It is combined to levodopa, target product concentration reaches 120g/L (Fig. 1), up to 85%, yield is up to 78%, levodopa light for conversion ratio
Learn purity and be more than 99.5%.
(3) Catechol 2 0g/L, Sodium Pyruvate 20g/L, ammonium acetate 60g/L, sulfurous are added in 500ml reaction systems
Sour sodium 5g/L, EDTANa25g/L, phosphopyridoxal pyridoxal phosphate (PLP) 3.5mM, ammoniacal liquor adjust pH to 8.0, TPL mutant recombinant cells dense
Degree 35g/L (weight in wet base)；Reacted under the conditions of 15 DEG C of temperature.Course of reaction is carried out by the way of feed supplement, every 2h feed supplements one
It is secondary, the wherein each feed supplement 8g/L of catechol, each feed supplement 8g/L of Sodium Pyruvate, each feed supplement 15g/L conversions 20h of ammonium acetate.
TPL mutants which hads cell catalysis synthesize levodopa, and target product concentration reaches 58g/L, and up to 41%, yield reaches conversion ratio
35%, levodopa optical purity is more than 99.5%；And wild-type strain cell catalysis synthesis levodopa, target product concentration
45g/L is reached, up to 35%, up to 27%, levodopa optical purity is more than 99.5% to yield to conversion ratio.
(4) catechol 50g/L, Sodium Pyruvate 50g/L, ammonium acetate 100g/L, sulfurous are added in 500ml reaction systems
Sour sodium 10g/L, EDTANa210g/L, phosphopyridoxal pyridoxal phosphate (PLP) 5mM, ammoniacal liquor adjust pH to 8.0, TPL prepared by embodiment 5 to dash forward
Variant recombinant cell concentration 50g/L (weight in wet base)；Reacted under the conditions of 15 DEG C of temperature.Course of reaction is entered by the way of feed supplement
OK, every 3h feed supplements once, the wherein each feed supplement 10g/L of catechol, each feed supplement 10g/L of Sodium Pyruvate, ammonium acetate is each
Feed supplement 20g/L converts 9h.TPL mutants which hads cell catalysis synthesize levodopa, and target product concentration reaches 45g/L, convert
Up to 35%, up to 28%, levodopa optical purity is more than 99.5% to yield to rate；And the synthesis of wild-type strain cell catalysis is left-handed more
Bar, target product concentration reaches 30g/L, and up to 25%, up to 19%, levodopa optical purity is more than 99.5% to yield to conversion ratio.
The present invention is not specifically limited text.The present invention can make in the range of claims are summarized
Various changes, these change within the scope of the present invention.
SEQUENCE LISTING
<110>Zhejiang Polytechnical University
<120>A kind of Fusobacterium nucleatum tyrosine phenol lyase mutant, gene, carrier, engineering bacteria and its application
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1383
<212> DNA
<213> Fusobacteriumnucleatum
<400> 1
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attgacttaa ataaattaca aaaattaata gatgaagttg gagcagaaaa cattgcttat 540
gtttgtttag ctgtaactgt aaaccttgct ggtggacaac cagtttctat gaaaaatatg 600
aaagcagtta gagaactaac taaaaaacat ggaataaaag ttttctatga tgcaactaga 660
tgtgttgaaa atgcttactt cattaaagaa caagaagaag gatatcaaga taaaactata 720
aaggaaatag tgcatgaaat gtttagctat gctgatggat gtactatgag tggtaaaaaa 780
gattgtcttg ttaatatagg tggattttta tgtatgaatg atgaagattt attcttagct 840
gcaaaagaaa tagttgttgt ttatgaaggt atgccatctt atggtggact tgctggtaga 900
gatatggaag ctatggcaat agggttaaga gaatctttac aatatgaata cattagacat 960
agaattttac aagttagata cttaggagaa aaattaaaag aagctggtgt acctatactt 1020
gaaccagttg gaggacatgc tgtattccta gatgctagaa gattctgtcc tcatatccca 1080
caagaagaat tcccagctca agctcttgca gcagctatct atgttgaatg tggtgtaaga 1140
actatggaaa gaggaataat ttctgctggt agagatgtaa aaactggtga aaaccataaa 1200
cctaaactag aaactgttag agttactatt ccaagaagag tttatactta taaacatatg 1260
gatgtagtag cagaaggtat aatcaaatta tataaacata aagaagatat aaaaccatta 1320
gaatttgtat atgaaccaaa acaattaaga ttctttacag ctagatttgg aataaaaaaa 1380
taa 1383
<210> 2
<211> 460
<212> PRT
<213> Fusobacteriumnucleatum
<400> 2
Met Arg Phe Glu Asp Tyr Pro Ala Glu Pro Phe Arg Ile Lys Ser Val
1 5 10 15
Glu Thr Val Lys Met Ile Asp Lys Ala Ala Arg Glu Glu Val Ile Lys
20 25 30
Lys Ala Gly Tyr Asn Thr Phe Leu Ile Asn Ser Glu Asp Val Tyr Ile
35 40 45
Asp Leu Leu Thr Asp Ser Gly Thr Asn Ala Met Ser Asp Lys Gln Trp
50 55 60
Gly Gly Leu Met Gln Gly Asp Glu Ala Tyr Ala Gly Ser Arg Asn Phe
65 70 75 80
Phe His Leu Glu Glu Thr Val Lys Glu Ile Phe Gly Phe Lys His Ile
85 90 95
Val Pro Thr His Gln Gly Arg Gly Ala Glu Asn Ile Leu Ser Gln Ile
100 105 110
Ala Ile Lys Pro Gly Gln Tyr Val Pro Gly Asn Met Tyr Phe Thr Thr
115 120 125
Thr Arg Tyr His Gln Glu Arg Asn Gly Gly Ile Phe Lys Asp Ile Ile
130 135 140
Arg Asp Glu Ala His Asp Ala Thr Leu Asn Val Pro Phe Lys Gly Asp
145 150 155 160
Ile Asp Leu Asn Lys Leu Gln Lys Leu Ile Asp Glu Val Gly Ala Glu
165 170 175
Asn Ile Ala Tyr Val Cys Leu Ala Val Thr Val Asn Leu Ala Gly Gly
180 185 190
Gln Pro Val Ser Met Lys Asn Met Lys Ala Val Arg Glu Leu Thr Lys
195 200 205
Lys His Gly Ile Lys Val Phe Tyr Asp Ala Thr Arg Cys Val Glu Asn
210 215 220
Ala Tyr Phe Ile Lys Glu Gln Glu Glu Gly Tyr Gln Asp Lys Thr Ile
225 230 235 240
Lys Glu Ile Val His Glu Met Phe Ser Tyr Ala Asp Gly Cys Thr Met
245 250 255
Ser Gly Lys Lys Asp Cys Leu Val Asn Ile Gly Gly Phe Leu Cys Met
260 265 270
Asn Asp Glu Asp Leu Phe Leu Ala Ala Lys Glu Ile Val Val Val Tyr
275 280 285
Glu Gly Met Pro Ser Tyr Gly Gly Leu Ala Gly Arg Asp Met Glu Ala
290 295 300
Met Ala Ile Gly Leu Arg Glu Ser Leu Gln Tyr Glu Tyr Ile Arg His
305 310 315 320
Arg Ile Leu Gln Val Arg Tyr Leu Gly Glu Lys Leu Lys Glu Ala Gly
325 330 335
Val Pro Ile Leu Glu Pro Val Gly Gly His Ala Val Phe Leu Asp Ala
340 345 350
Arg Arg Phe Cys Pro His Ile Pro Gln Glu Glu Phe Pro Ala Gln Ala
355 360 365
Leu Ala Ala Ala Ile Tyr Val Glu Cys Gly Val Arg Thr Met Glu Arg
370 375 380
Gly Ile Ile Ser Ala Gly Arg Asp Val Lys Thr Gly Glu Asn His Lys
385 390 395 400
Pro Lys Leu Glu Thr Val Arg Val Thr Ile Pro Arg Arg Val Tyr Thr
405 410 415
Tyr Lys His Met Asp Val Val Ala Glu Gly Ile Ile Lys Leu Tyr Lys
420 425 430
His Lys Glu Asp Ile Lys Pro Leu Glu Phe Val Tyr Glu Pro Lys Gln
435 440 445
Leu Arg Phe Phe Thr Ala Arg Phe Gly Ile Lys Lys
450 455 460
<210> 3
<211> 1383
<212> DNA
<213> unknown
<220>
<223>Artificial sequence
<400> 3
atgagatttg aagattatcc agcagagcca tttagaatta aaagtgtaga aactgttaaa 60
atgattgata aggcagcaag agaagaagta attaaaaaag caggatataa tactttctta 120
attaactctg aagatgttta cattgattta ttaactgata gtggaactaa tgctatgagt 180
gataaacaat ggggtggatt aatgcaaggt gatgaagctt atgcaggaag tagaaatttc 240
ttccacttag aaaaaactgt aaaagaaata tttgggttta aacatatagt tcctactcac 300
caaggaagag gagcagaaaa tattttatct caaatagcta taaaacctgg acaatatgtt 360
cctggaaata tgtattttac aactattaga tatcaccaag aaagaaatgg tggaatattt 420
aaagatatta tcagagatga ggcacatgat gctactctta atgttccttt caaaggagat 480
attgacttaa ataaattaca aaaattaata gatgaagttg gagcagaaaa cattgcttat 540
gtttgtttag ctgtaactgt aaaccttgct ggtggacaac cagtttctat gaaaaatatg 600
aaagcagtta gagaactaac taaaaaacat ggaataaaag ttttctatga tgcaactaga 660
tgtgttgaaa atgcttactt cattaaagaa caagaagaag gatatcaaga taaaactata 720
aaggaaatag tgcatgaaat gtttagctat gctgatggat gtactatgag tggtaaaaaa 780
gattgtcttg ttaatatagg tggattttta tgtatgaatg atgaagattt attcttagct 840
gcaaaagaaa tagttgttgt ttatgaaggt atgccatctt atggtggact tgctggtaga 900
gatatggaag ctatggcaat agggttaaga gaatctttac aatatgaata cattagacat 960
agaattttac aagttagata cttaggagaa aaattaaaag aagctggtgt acctatactt 1020
gaaccagttg gaggacatgc tgtattccta gatgctagaa gattctgtcc tcatatccca 1080
caagaagaat tcccagctca agctcttgca gcagctatct atgttgaatg tggtgtaaga 1140
actatggaaa gaggaataat ttctgctggt agagatgtaa aaactggtga aaaccataaa 1200
cctaaactag aaactgttag agttactatt ccaagaagag tttatactta taaacatatg 1260
gatgtagtag cagaaggtat aatcaaatta tataaacata aagaagatat aaaaccatta 1320
gaatttgtat atgaaccaaa acaattaaga ttctttacag ctagatttgg aataaaaaaa 1380
taa 1383
<210> 4
<211> 460
<212> PRT
<213> unknown
<220>
<223>Artificial sequence
<400> 4
Met Arg Phe Glu Asp Tyr Pro Ala Glu Pro Phe Arg Ile Lys Ser Val
1 5 10 15
Glu Thr Val Lys Met Ile Asp Lys Ala Ala Arg Glu Glu Val Ile Lys
20 25 30
Lys Ala Gly Tyr Asn Thr Phe Leu Ile Asn Ser Glu Asp Val Tyr Ile
35 40 45
Asp Leu Leu Thr Asp Ser Gly Thr Asn Ala Met Ser Asp Lys Gln Trp
50 55 60
Gly Gly Leu Met Gln Gly Asp Glu Ala Tyr Ala Gly Ser Arg Asn Phe
65 70 75 80
Phe His Leu Lys Glu Thr Val Lys Glu Ile Phe Gly Phe Lys His Ile
85 90 95
Val Pro Thr His Gln Gly Arg Gly Ala Glu Asn Ile Leu Ser Gln Ile
100 105 110
Ala Ile Lys Pro Gly Gln Tyr Val Pro Gly Asn Met Tyr Phe Thr Thr
115 120 125
Ile Arg Tyr His Gln Glu Arg Asn Gly Gly Ile Phe Lys Asp Ile Ile
130 135 140
Arg Asp Glu Ala His Asp Ala Thr Leu Asn Val Pro Phe Lys Gly Asp
145 150 155 160
Ile Asp Leu Asn Lys Leu Gln Lys Leu Ile Asp Glu Val Gly Ala Glu
165 170 175
Asn Ile Ala Tyr Val Cys Leu Ala Val Thr Val Asn Leu Ala Gly Gly
180 185 190
Gln Pro Val Ser Met Lys Asn Met Lys Ala Val Arg Glu Leu Thr Lys
195 200 205
Lys His Gly Ile Lys Val Phe Tyr Asp Ala Thr Arg Cys Val Glu Asn
210 215 220
Ala Tyr Phe Ile Lys Glu Gln Glu Glu Gly Tyr Gln Asp Lys Thr Ile
225 230 235 240
Lys Glu Ile Val His Glu Met Phe Ser Tyr Ala Asp Gly Cys Thr Met
245 250 255
Ser Gly Lys Lys Asp Cys Leu Val Asn Ile Gly Gly Phe Leu Cys Met
260 265 270
Asn Asp Glu Asp Leu Phe Leu Ala Ala Lys Glu Ile Val Val Val Tyr
275 280 285
Glu Gly Met Pro Ser Tyr Gly Gly Leu Ala Gly Arg Asp Met Glu Ala
290 295 300
Met Ala Ile Gly Leu Arg Glu Ser Leu Gln Tyr Glu Tyr Ile Arg His
305 310 315 320
Arg Ile Leu Gln Val Arg Tyr Leu Gly Glu Lys Leu Lys Glu Ala Gly
325 330 335
Val Pro Ile Leu Glu Pro Val Gly Gly His Ala Val Phe Leu Asp Ala
340 345 350
Arg Arg Phe Cys Pro His Ile Pro Gln Glu Glu Phe Pro Ala Gln Ala
355 360 365
Leu Ala Ala Ala Ile Tyr Val Glu Cys Gly Val Arg Thr Met Glu Arg
370 375 380
Gly Ile Ile Ser Ala Gly Arg Asp Val Lys Thr Gly Glu Asn His Lys
385 390 395 400
Pro Lys Leu Glu Thr Val Arg Val Thr Ile Pro Arg Arg Val Tyr Thr
405 410 415
Tyr Lys His Met Asp Val Val Ala Glu Gly Ile Ile Lys Leu Tyr Lys
420 425 430
His Lys Glu Asp Ile Lys Pro Leu Glu Phe Val Tyr Glu Pro Lys Gln
435 440 445
Leu Arg Phe Phe Thr Ala Arg Phe Gly Ile Lys Lys
450 455 460
Claims (10)
1. a kind of Fusobacterium nucleatum tyrosine phenol lyase mutant, it is characterised in that the mutant is by SEQ ID NO.2
Shown amino acid sequence the 84th and 129 carries out what the double mutation of fallibility PCR were obtained.
2. Fusobacterium nucleatum tyrosine phenol lyase mutant as claimed in claim 1, it is characterised in that double mutation be by
The glutamic acid mutation of the 84th is lysine, while the threonine of the 129th is sported into isoleucine.
3. a kind of encoding gene of Fusobacterium nucleatum tyrosine phenol lyase mutant described in claim 1, it is characterised in that institute
Encoding gene nucleotides sequence is stated to be classified as shown in SEQ ID NO.3.
4. it is a kind of as described in claim 3 encoding gene build recombinant vector.
5. it is a kind of that the recombination engineering bacteria for preparing is converted as recombinant vector described in claim 4.
6. application of the Fusobacterium nucleatum tyrosine phenol lyase mutant described in a kind of claim 1 in levodopa is synthesized.
7. application as claimed in claim 6, it is characterised in that the application is prominent with tyrosine phenol lyase containing Fusobacterium nucleatum
The wet thallus that the fermented culture of recombination engineering bacteria of variant encoding gene is obtained are catalyst, with catechol, pyruvic acid
Sodium and ammonium acetate are substrate, with sodium sulfite and EDTANa2It is auxiliary agent, with phosphopyridoxal pyridoxal phosphate as coenzyme, with pH5.0~9.0
Buffer solution constitute transformation system for reaction medium, conversion reaction is carried out under the conditions of 5~30 DEG C of temperature, after reaction terminates, will
Reaction solution is isolated and purified, and obtains levodopa.
8. application as claimed in claim 7, it is characterised in that in the transformation system, when initial, catechol adds dense eventually
5~50g/L of degree, Sodium Pyruvate adds 5~50g/L of final concentration, ammonium acetate to add 5~100g/L of final concentration, ammonium nitrate to add eventually
0.1~5g/L of concentration, sodium sulfite adds final concentration 0.5~10g/L, EDTANa2Add 0.5~10g/L of final concentration, phosphoric acid
Pyridoxal adds 0.05~5mM of final concentration, and wet thallus consumption is 2~50g/L.
9. application as claimed in claim 7, it is characterised in that during the conversion reaction to catechol, Sodium Pyruvate and
Ammonium acetate carries out feed supplement, and every 0.5~4h feed supplements once, the wherein each 0.1~10g/L of feed supplement of catechol, Sodium Pyruvate is every
Secondary 0.1~10g/L of feed supplement, each 1~20g/L of feed supplement of ammonium acetate.
10. application as claimed in claim 7, it is characterised in that the catalyst is prepared as follows：
(1) inclined-plane culture：The recombination engineering bacteria of the mutant code gene of tyrosine phenol lyase containing Fusobacterium nucleatum is connect
Plant to the slant medium containing 50 μ g/ml kanamycins, 8~16h are cultivated at 37 DEG C, obtain inclined-plane thalline；The inclined-plane culture
Matrix amount final concentration is constituted：Peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, 1.5% agar, solvent is to go
Ionized water, pH 7.0；
(2) seed culture：Inclined-plane thalline is seeded to seed culture medium, 8~10h is cultivated at 37 DEG C, obtain seed liquor；The kind
Sub- culture medium final concentration is constituted：Peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, 50 μ g/ml kanamycins,
Solvent is deionized water, pH 7.0；
(3) fermented and cultured：Seed liquor is seeded to the aseptic 5L equipped with 3L fermentation mediums with the inoculum concentration of volumetric concentration 3%
In mechanical agitation ventilation general-purpose type fermentation tank, sterilized final concentration of 15g/L lactose is directly appended in fermentation tank in 28
Fiber differentiation at DEG C, wet thallus are collected after tank is put after 6~8h of culture；The fermentation medium final concentration is constituted：Peptone
15g/L, dusty yeast 12g/L, NaCl10g/L, glycerine 15g/L, (NH4)2SO45g/L, KH2PO41.36g/L, K2HPO4·3H2O
2.28g/L, MgSO4·7H2O 0.375g/L, solvent is deionized water, pH.
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| CN201611094369.9A CN106754846B (en) | 2016-12-02 | 2016-12-02 | Fusobacterium nucleatum tyrosine phenol lyase mutant, gene, vector, engineering bacterium and application thereof |
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| CN107586797A (en) * | 2017-09-25 | 2018-01-16 | 长兴制药股份有限公司 | The method that one pot of enzyme process prepares levodopa |
| CN108642130A (en) * | 2018-03-29 | 2018-10-12 | 浙江工业大学 | A kind of high-throughput screening method of tyrosine phenol lyase high dynamic strain |
| CN108715827A (en) * | 2018-06-08 | 2018-10-30 | 鲁东大学 | The extracellular expression of tyrosine phenol lyase and its application |
| CN109161568A (en) * | 2018-08-10 | 2019-01-08 | 浙江工业大学 | A method of improving levodopa product quality and yield |
| CN109897845A (en) * | 2019-04-18 | 2019-06-18 | 江南大学 | It is a kind of express thermostable type tyrosine phenol-lyase Escherichia coli and its application |
| 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 |
| CN110373421A (en) * | 2019-06-28 | 2019-10-25 | 浙江工业大学 | A kind of tyrosine phenol lyase gene recombination plasmid and application |
| CN110903998A (en) * | 2019-10-23 | 2020-03-24 | 上海市第十人民医院 | Intestinal tract separated fusobacterium nucleatum Wenzeri strain and application thereof |
| CN111411132A (en) * | 2020-05-14 | 2020-07-14 | 山东惠仕莱生物科技有限公司 | Conversion and extraction method for producing levodopa by enzyme method |
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| CN113621548A (en) * | 2021-08-25 | 2021-11-09 | 上海交通大学 | Method for producing levodopa based on vibrio natriegens |
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| CN107586797A (en) * | 2017-09-25 | 2018-01-16 | 长兴制药股份有限公司 | The method that one pot of enzyme process prepares levodopa |
| CN108642130A (en) * | 2018-03-29 | 2018-10-12 | 浙江工业大学 | A kind of high-throughput screening method of tyrosine phenol lyase high dynamic strain |
| CN108642130B (en) * | 2018-03-29 | 2021-10-15 | 浙江工业大学 | High-throughput screening method for high-activity strain of tyrosine phenol lyase |
| CN108715827A (en) * | 2018-06-08 | 2018-10-30 | 鲁东大学 | The extracellular expression of tyrosine phenol lyase and its application |
| CN109161568A (en) * | 2018-08-10 | 2019-01-08 | 浙江工业大学 | A method of improving levodopa product quality and yield |
| CN111793615A (en) * | 2019-04-07 | 2020-10-20 | 宁波酶赛生物工程有限公司 | Engineered polypeptides and their use in the synthesis of tyrosine or tyrosine derivatives |
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| 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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| CN110331153B (en) * | 2019-06-24 | 2021-04-30 | 浙江工业大学 | Kluyveromyces tyrosol lyase mutant and application thereof |
| CN110373421A (en) * | 2019-06-28 | 2019-10-25 | 浙江工业大学 | A kind of tyrosine phenol lyase gene recombination plasmid and application |
| CN110903998B (en) * | 2019-10-23 | 2021-09-21 | 上海市第十人民医院 | Intestinal tract separated fusobacterium nucleatum Wenzeri strain and application thereof |
| CN110903998A (en) * | 2019-10-23 | 2020-03-24 | 上海市第十人民医院 | Intestinal tract separated fusobacterium nucleatum Wenzeri strain and application thereof |
| CN111411132A (en) * | 2020-05-14 | 2020-07-14 | 山东惠仕莱生物科技有限公司 | Conversion and extraction method for producing levodopa by enzyme method |
| CN112063610A (en) * | 2020-09-23 | 2020-12-11 | 浙江工业大学 | Tyrosine phenol lyase mutant, engineering bacterium and application |
| CN112063610B (en) * | 2020-09-23 | 2022-02-11 | 浙江工业大学 | Tyrosine phenol lyase mutant, engineering bacterium and application |
| CN114250237A (en) * | 2020-09-23 | 2022-03-29 | 浙江工业大学 | Tyrosine phenol lyase mutant, engineering bacteria and application of tyrosine phenol lyase mutant in catalytic synthesis of levodopa |
| CN114250237B (en) * | 2020-09-23 | 2024-05-03 | 浙江工业大学 | Tyrosine phenol lyase mutant, engineering bacterium and application thereof in catalytic synthesis of levodopa |
| CN113621548A (en) * | 2021-08-25 | 2021-11-09 | 上海交通大学 | Method for producing levodopa based on vibrio natriegens |
| CN113621548B (en) * | 2021-08-25 | 2023-11-24 | 上海交通大学 | Method for producing levodopa based on vibrio natrii |
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