CN109609560A - Biosynthesis 3,4- dihydroxyphenyl acetic acid method - Google Patents
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Abstract
Biosynthesis 3,4- dihydroxyphenyl acetic acid method be related to biotechnology and metabolic engineering field.The invention discloses biosynthesis 3, the approach of 4-DHPA, host and its methods.The metabolic pathway of 3,4-DHPA production has been imported in the host for producing 3,4-DHPA.It more precisely, be that high efficient expression 4- hydroxyl phenylacetic acid hydroxylase (HpaBC) carrys out bioconversion 4- hydroxyl phenylacetic acid is in host 3,4-DHPA.It in encoded channel genes host, will as a result obtain can use 4- hydroxyl phenylacetic acid, glucose, glycerol etc. produces the host of 3,4-DHPA.The invention also discloses a kind of strategies of gene regulation simultaneously, so that 3,4-DHPA production is highly efficient.
Description
Technical field
The present invention relates to biotechnologys and metabolic engineering field, more precisely, the present invention relates to heterologous organisms synthesis 3,
The method of the metabolic pathway and gene regulation of 4- dihydroxyphenyl acetic acid (3,4-DHPA), selection derive from bacterium, fungi or albumen
The engineered exogenous enzymes of matter select bacterium original or being transformed, and fungi, plant cell or zooblast are thin as host
Born of the same parents, and by RED recombination edit carry out gene regulation, finally improve 3,4- dihydroxyphenyl acetic acid yield and
Carbon source yield.
Background technique
Phenolic acid because its is anti-oxidant, antibacterial and other biological activities and be widely used in food, cosmetics and pharmaceuticals industry.3,4-
Dihydroxyphenyl acetic acid is naturally occurring phenolic acid, shows powerful free radical scavenging activity.It can reduce lipid peroxy
Change, and natural can be used potentially as to replace butylated hydroxytoluene (BHT) and butylated hydroxyanisol
(BHA) with stable edible oil.3,4-DHPA also shows several activity beneficial to health.It is quercetin glycoside in diet
One of with phenolic acid the most abundant in the primary bioactivity catabolite of flavonoids and fruits and vegetables, and to cell
Apoptosis, mitochondria dysfunction and oxidative stress etc. have protective effect, can induce cystine transhipment enzyme, Glutathione Transport
The expression of enzyme, heme oxygenase, cytochromes enzyme etc..In prostate cancer and colon cancer cell, 3,4-DHPA are also showed
Strong antiproliferative activity out.In addition, proinflammatory thin in 3, the 4-DHPA peripheral blood mononuclear cells that lipopolysaccharides can also be inhibited to stimulate
The expression of p-selectin in the secretion of intracellular cytokine and resting platelets.
Although 3, the 4-DHPA significant health benefits having, economical and environmentally friendly 3,4-DHPA are also lacked so far
Production method.Therefore, it is necessary to effective synthetic strategy be established, to provide 3, the 4-DHPA compound of sufficient amount for visiting
Rope and apply its multiple functions.3,4-DHPA is extracted by low-purity, poor efficiency, high-cost limitation from natural origin.For
Chemical synthesis, regioselectivity neighbour's hydroxylating on phenyl ring are challenging always, and chemical synthesis also will be in face of height
Pollution, low yield, Gao Chengben, the problems such as time-consuming.Previous studies produce 3 using the electrochemical conversion of phenylacetic acid (PAA),
4-DHPA, but the mixture of dihydroxy PAA is formd, make product be difficult to separate.Therefore, it is closed using green and efficient biology
Just become the best selection of production 3,4-DHPA at method.
Present invention is primarily aimed at efficiently synthesizing for the biological method for realizing 3,4-DHPA.Screened catalytic efficiency compared with
High 4-Hydroxyphenylacetate 3-monooxygenase HpaBC realizes the bioconversion from 4- hydroxyl phenylacetic acid to 3,4-DHPA.Equally
The new metabolic pathway of engineer realizes the de novo formation of 3,4-DHPA, and in addition the invention also discloses a kind of knockout places
Pure grape is respectively adopted in the gene regulation strategy of the gene PYKA and PYKF of encoding pyruvate acid kinase and one kind in key-gene group
Sugar, pure glycerin, glucose and glycerol are mixed into carbon source to realize the dynamic regulation strategy of yield and growth coordination.Experimental result table
Bright, under optimization of fermentation conditions, the bioconversion of 4- hydroxyl phenylacetic acid to 3,4-DHPA can reach 100% conversion ratio in 48h；
And the maximum carbon source receipts of the maximum production of 1856 ± 67 mg/L and 0.1741% can be reached by de novo formation, 3,4-DHPA
Rate.
Summary of the invention
The object of the present invention is to provide high yield 3, the host of 4-DHPA, by bacterium original or being transformed, fungi,
The approach for efficiently producing 3,4-DHPA is imported in plant cell or zooblast, selection derives from bacterium, fungi or albumen
The engineered 4-Hydroxyphenylacetate 3-monooxygenase HpaBC of matter realizes 3,4- by the high efficient expression of HpaBC in host
Biosynthesis of the DHPA from 4- hydroxyl phenylacetic acid.
A kind of biosynthesis 3,4- dihydroxyphenyl acetic acid method, it is characterised in that: under being 6-8 at 30-37 DEG C and pH, 4-
Hydroxyl phenylacetic acid 3- monooxygenase HpaBC is catalyzed 3 to 60 hours, and catalysis 4- hydroxyl phenylacetic acid is generated 3,4- dihydroxy benzenes second
Acid.
Screening derives from bacterium, fungi or the coding 4-Hydroxyphenylacetate 3-monooxygenase by protein engineering transformation
Gene HpaBC.3, the host of 4- dihydroxyphenyl acetic acid production ways includes bacterium, fungi, plant cell or zooblast,
Including the bacterium being transformed, fungi, plant cell or zooblast.
Gene PYKA, PYKF or PYKA of encoding pyruvate acid kinase in host genome and PYKF are knocked out, raising 3,
4- dihydroxyphenyl acetic acid yield.
The present invention also provides the methods that microorganism produces 3,4-DHPA: first method is external addition substrate 4- hydroxy benzenes
Acetic acid, to bacterium original or being transformed, fungi imports 4-Hydroxyphenylacetate 3-monooxygenase in plant cell or zooblast
HpaBC, and be building up on the carrier of middle copy and expressed.Then, this is produced into the host of 3,4-DHPA in LB Tube propagation base
In be incubated overnight, later transfer 1%-4% volume bacterium solution into the M9 culture medium for the 4- hydroxyl phenylacetic acid for being added to 3g/L,
37 DEG C of culture 48h.It is sampled after addition inducer every 12h and surveys OD, and measure the concentration of 3,4-DHPA with high performance liquid chromatography.The
Two kinds of methods are de novo formations, and to bacterium original or being transformed, it is de- to import prephenic acid in plant cell or zooblast for fungi
Hydrogen enzyme TyrA, phosphoenolpyruvate synthase PpsA, transketolase TktA, 3- deoxidation-D-arabinose-heptonic acid -7- phosphoric acid close
At enzyme AroG, aminopherase AT, keto acid decarboxylase ARO10, the mono- oxygenation of phenylacetaldehyde dehydrogenase FeaB, 4- hydroxyl phenylacetic acid 3-
Enzyme HpaBC, expressed in high copy number plasmid respectively expression HpaBC, TyrA in ARO10, FeaB and middle copy plasmid, PpsA,
TktA,AroG.Then at 37 DEG C, by this strain inoculated to glucose (glucose) or glycerol (glycerol) for carbon source
M9 culture medium in ferment, and sampled from fermentation liquid, using high performance liquid chromatography to 12h, for 24 hours, the hair of 36h, 48h
The concentration of zymotic fluid product is analyzed.
The present invention also provides microbe high-yield 3, the method for 4-DHPA specifically has following several: first is to optimize table
Up to module, HpaBC is optimized for middle copy from high copy expression and is expressed, makes upstream and downstream enzyme amount expression balance.Second is the introduction of
Gene regulation strategy, gene PYKA, the PYKF for having knocked out encoding pyruvate acid kinase reduce the generation of by-product, increase product
Formation.The dynamic regulation strategy that third is the introduction of yield and growth is coordinated selects to realize place with glucose for single carbon source
Main fast-growth selects the efficient production for realizing 3,4-DHPA for single carbon source with glycerol, and selection is with glucose and glycerol
The dynamic regulation of mixed carbon source (2:1) realization yield and growth.Finally, in vitro in addition experiment, in 48h, the 4- hydroxyl of 3g/L
Base phenylacetic acid is converted into 3,4-DHPA by 100%；In de novo formation, from the mixed carbon source of glycerol and glucose, 3,4-
DHPA can reach the maximum production of 1856 ± 67mg/L and 0.1714% carbon source yield；From glycerol, 3,4-DHPA
The yield of 1750 ± 73mg/L and 0.1741% maximum carbon source yield can be reached.
As described above, the present invention relates to effective production method of 3,4-DHPA and high yield host, the method and hosts
Be characterized by that screening activity is high and the enzyme of high efficient expression, construct biosynthesis 3, the artificial approach of 4-DHPA designs base
Because of the method for the dynamic regulation strategy that regulating strategy, yield and growth are coordinated, and optimize the culture medium in fermentation process and training
The condition of supporting, to realize efficiently synthesizing for 3,4-DHPA.
Detailed description of the invention
Fig. 1 de novo formation and addition experiment produce the path of 3,4-DHPA；
The bacterium that Fig. 2 imports the original of 4-Hydroxyphenylacetate 3-monooxygenase (HpaBC) gene or was transformed, fungi are planted
The fermentation results that object cell or zooblast bioconversion 4-HPA are 3,4-DHPA；
Fig. 3 imports aminopherase (AT), keto acid decarboxylase (ARO10), phenylacetaldehyde dehydrogenase (FeaB), 4- hydroxy benzenes
Acetic acid 3- monooxygenase (HpaBC), shikimic acid pathway TyrAfbr, PpsA, TktA and AroGfbrEnzyme gene original was transformed
Bacterium, fungi, plant cell or zooblast, from glucose, the glycerol head that sets out synthesizes the fermentation results of 3,4-DHPA；
Fig. 4 knocks out gene PYKA, PYKF or PYKA of encoding pyruvate acid kinase in host genome and PYKF respectively,
It effectively prevents after PEP is largely converted into pyruvic acid, from glucose, the glycerol head that sets out synthesizes the fermentation results of 3,4-DHPA；
Specific embodiment
Specific embodiment 1
3,4-DHPA is produced by adding 4- hydroxyl phenylacetic acid in vitro
Pass through the homologous modeling point of sequence alignment (https: //blast.ncbi.nlm.nih.gov) and Modeller software
Analysis, screening derive from the 4-Hydroxyphenylacetate 3-monooxygenase HpaBC of bacterium, fungi or protein engineering transformation.Wherein HpaBC
Transformation include F101A, S112G, A128N, E144T, P152Y.It is obtained from plasmid or microbial genome by round pcr
The genetic fragment for obtaining 4-Hydroxyphenylacetate 3-monooxygenase (HpaBC), then, using restriction endonuclease to genetic fragment
Digestion is carried out with plasmid vector, the segment after digestion is subjected to glue recycling or column and is recycled, is then added 4- hydroxyl phenylacetic acid 3- is mono-
Oxygenase (HpaBC) target gene is inserted on plasmid pCS27 (middle copy), acquisition pCS-HpaBC recombinant plasmid, and utilization PCR,
Digestion carries out plasmid verifying.
Using electric robin preparation Escherichia coli BW25113 (knocked out in original Escherichia coli lacZWJ16, hsdR514,
AraBADAH33, rhaBADLD78) competent cell, and draw 100 μ L had been built up in the EP pipe of 1.5 mL with 4 μ L
Plasmid pCS-HpaBC be sufficiently mixed, be placed on ice 10min pre-cooling.Then sufficient bacterium solution will be mixed and electricity is added in plasmid
It hits in cup, and is rotated into plasmid electricity into competent cell using electroporation.After the completion of electricity turns, 700 μ L LB culture mediums are added, mix
It closes uniformly, and mixture is transferred in 1.5mL centrifuge tube, the recovery 30min at 37 DEG C.It takes 100 μ L bacterium solutions to be coated onto later to contain
On the plate for having kanamycins antibiotic, 37 DEG C are incubated overnight.Bioconversion 4- hydroxyl phenylacetic acid is built into 3,4-DHPA's
Bacterial strain BW (pCS-HpaBC).
Three parallel single colonies of picking on the plate of bacterial strain BW (pCS-HpaBC) are connected to the mould with that is blocked of 4mL
In the liquid LB of plain resistance, 12h is cultivated at 37 DEG C, and the bacterium solution of 1mL in LB (2% volume ratio) is inoculated into having for 50mL later
(medium component includes 7g/L Na to the M9 culture medium of kalamycin resistance2HPO4, 3g/L KH2PO4, 0.5g/L NaCl, 1g/L
NH4Cl, 2.5g/L glucose, 5g/L yeast powder in 2g/L 3- N-morpholinyl (MOPS), and directly add final concentration of
Inducer IPTG is added after then cultivating 3 hours, 3 hours in 37 DEG C, the shaking table of 200r/min in the 4- hydroxyl phenylacetic acid of 3g/L
It is induced, concentration 1mM.It is sampled when 12 hours, 24 hours, 36 hours and 48 hours respectively later, use is ultraviolet
Its growing state of spectrophotometric determination, and the concentration of 3,4-DHPA and the consumption feelings of substrate are measured with high performance liquid chromatography
Condition.It is tested by addition, be that 4- hydroxyl phenylacetic acid will be completely converted in 48h, 3g/L is 3,4-DHPA, conversion ratio 100%.
(Fig. 2)
Specific embodiment 2
The single carbon that conforms to the principle of simplicity source de novo formation 3,4-DHPA
Pass through the enzyme reported in sequence alignment (https: //blast.ncbi.nlm.nih.gov) analysis and document
Activity comparison, screening derive from the prephenate dehydrogenase TyrA of bacterium, fungi or protein engineering transformation, phosphoenolpyruvate
Synthase PpsA, transketolase TktA, 3- deoxidation-D-arabinose-heptonic acid -7- phosphate synthase AroG, aminopherase AT, ketone
Acid decarboxylase ARO10, phenylacetaldehyde dehydrogenase FeaB, 4-Hydroxyphenylacetate 3-monooxygenase HpaBC can be by glucose and glycerol point
It is not converted into the red moss ketone (E4P) of 4- phosphoric acid and phosphoenolpyruvate (PEP), is then branch by shikimic acid pathway catalysis
Acid (chorismate), Single-chip microcomputer, para hydroxybenzene acetaldehyde (4-HPAA), p-hydroxyphenylaceticacid (4-HPA), then
It is converted into 3,4-DHPA.Wherein the transformation of HpaBC includes F101A, S112G, A128N, E144T, P152Y, and TyrA is transform as
TyrAfbr, AroG transform AroG asfbr.Then aminopherase (AT) is obtained with PCR, keto acid decarboxylase (ARO10), phenylacetaldehyde
Dehydrogenase FeaB, 4-Hydroxyphenylacetate 3-monooxygenase (HpaBC) and the TyrA for enhancing shikimic acid pathwayfbr, PpsA, TktA and
AroGfbrThe genetic fragment of enzyme, later by target gene fragment be inserted into plasmid pZE12-luc (height copy) and pCS27 (in copy
Shellfish) on, obtain plasmid pZE-ARO10-FeaB-HpaBC, pZE-ARO10-FeaB, pCS-TPTA and pCS-TPTA-HpaBC.
It takes 2 μ L of the plasmid pZE-ARO10-FeaB-HpaBC built to mix with 2 μ L of pCS-TPTA, takes pZE-ARO10-
2 μ L of FeaB is mixed with 2 μ L of pCS-TPTA-HpaBC, and electricity is gone in the 100 μ L Escherichia coli for having been made into competence respectively,
After the completion of electricity turns, 700 μ L LB culture mediums is added, and mixture is transferred in 1.5mL centrifuge tube, recovers at 37 DEG C
30min.100 μ L bacterium solutions are taken to be coated onto containing ammonia benzyl mycin later, on the plate of kanamycins antibiotic, 37 DEG C are incubated overnight.Shape
At de novo formation 3, the bacterial strain BW (pZE-ARO10-FeaB-HpaBC, pCS-TPTA) of 4-DHPA, BW (pZE-ARO10-FeaB,
pCS-TPTA-HpaBC)。
At coli strain BW (pZE-ARO10-FeaB-HpaBC, pCS-TPTA), BW (pZE-ARO10-FeaB,
PCS-TPTA-HpaBC three parallel single colonies of picking on plate), be connected to 4mL has ammonia benzyl mycin, and kanamycins is anti-
Property liquid LB in, 37 DEG C of culture 12h, later respectively by the bacterium solution of 1mL (volume ratio 2%) be inoculated into 50mL with ammonia benzyl
1mM is added after then cultivating 3 hours in 37 DEG C, the shaking table of 200r/min in mycin, the M9 culture medium of kalamycin resistance
IPTG is induced.It is sampled every 12h, and measures the concentration of 3,4-DHPA with high performance liquid chromatography.Finally, discovery module
The yield ratio BW (pZE-ARO10- of bacterial strain BW (pZE-ARO10-FeaB, pCS-TPTA-HpaBC) 3,4-DHPA of optimization
FeaB-HpaBC, pCS-TPTA) high 600mg/L, up to 916 ± 48mg/L.(Fig. 3)
Specific embodiment 3
3,4-DHPA yield is improved by gene regulation strategy
Among the approach for producing 3,4-DHPA, intermediate phosphate enol pyruvic acid (PEP) will largely be converted into acetone
Sour (pyr), and enter tricarboxylic acid cycle (TCA cycle).Therefore on the basis of host strain BW25113, by host genome
Gene PYKA, PYKF or PYKA and PYKF of middle encoding pyruvate acid kinase are knocked out, and prevent PEP from being largely converted into acetone for effective
Sour (pyr) provides a large amount of precursors for the biosynthesis of 3,4-DHPA, decreases the generation of by-product.Pass through RED gene
Editing technique obtains bacterial strain Δ PYKA BW, Δ PykK BW and Δ PYKA PYKF BW.Next, respectively by plasmid pZE-
Electricity rotates into three kinds of engineered strains ARO10-FeaB and pCS-TPTA-HpaBC together, obtains bacterial strain Δ PYKA BW (pZE-
ARO10-FeaB, pCS-TPTA-HpaBC), Δ PykK BW (pZE-ARO10-FeaB, pCS-TPTA-HpaBC) and Δ PYKA
PYKF BW (pZE-ARO10-FeaB、pCS-TPTA-HpaBC)。
Three parallel single colonies that above-mentioned three kinds of bacterial strains are picked them separately on plate, be connected to 4mL has ammonia benzyl mycin,
In the liquid LB of kalamycin resistance, and it is transferred in M9 culture medium and ferments respectively after 37 DEG C of culture 12h.Exist respectively
12h, for 24 hours, 36h, 48h be sampled, and with high performance liquid chromatography measure 3,4-DHPA concentration.Finally, bacterial strain Δ PYKA
PYKF BW (pZE-ARO10-FeaB, pCS-TPTA-HpaBC) has reached the maximum output (Fig. 4) of 1856 ± 67mg/L.
Claims (4)
1. a kind of method of biosynthesis 3,4- dihydroxyphenyl acetic acid, it is characterised in that: under being 6-8 at 30-37 DEG C and pH, 4- hydroxyl
Base phenylacetic acid 3- monooxygenase HpaBC is catalyzed 3 to 60 hours, and catalysis 4- hydroxyl phenylacetic acid is generated 3,4- dihydroxyphenyl acetic acid.
2. according to the method described in claim 1, it is characterized by: screening is from bacterium, fungi or by protein engineering
The gene HpaBC of the coding 4-Hydroxyphenylacetate 3-monooxygenase of transformation.
3. the method as described in claim 1-2 any one, it is characterised in that: 3, the places of 4- dihydroxyphenyl acetic acid production ways
Main includes bacterium, fungi, plant cell or zooblast, also includes the bacterium being transformed, fungi, plant cell or animal are thin
Born of the same parents.
4. according to the method described in claim 1, it is characterized by: by the gene of encoding pyruvate acid kinase in host genome
PYKA, PYKF or PYKA and PYKF are knocked out, and improve 3,4- dihydroxyphenyl acetic acid yield.
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