CN108841852A - A kind of high yield 5-ALA produces construction method and the application of bacterial strain - Google Patents
A kind of high yield 5-ALA produces construction method and the application of bacterial strain Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
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- C12N15/09—Recombinant DNA-technology
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- C12N15/90—Stable introduction of foreign DNA into chromosome
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
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- C12Y203/00—Acyltransferases (2.3)
- C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
- C12Y203/01037—5-Aminolevulinate synthase (2.3.1.37)
Abstract
The invention discloses a kind of construction method of high yield 5-ALA production bacterial strain and applications, the present invention is host with saccharomyces cerevisiae by4741, by cloning high activity saccharomyces cerevisiae 5-ALA synzyme hem1 gene, hem1 gene cloning is entered into expression vector Prs426.Promoter selects constitutive promoter TDH3P.After this contains the plasmid linearization of hem1 gene, saccharomyces cerevisiae is transferred to by electrotransformation mode.Obtained transformant is engineering strain needed for the method for the present invention.And bacterial strain to be used for the production of 5-ALA and the addition of animal feed.The beneficial effects of the invention are as follows：5-ALA synthase gene is successfully imported into eukaryon bacterium saccharomyces cerevisiae for the first time, is not had been reported that in research before.The high yield 5-ALA recombinant Saccharomyces cerevisiae of building, the yield for obtaining 5-ALA reach 2.0 g/L and compare with other researchs, and the yield of 5-ALA is relatively high.The high yield 5-ALA recombinant Saccharomyces cerevisiae of building, can be applied in fermentative feedstuff of microbe industry, have the characteristics that pollution-free, environmentally protective.
Description
Technical field
The present invention relates to a kind of construction method of high yield 5-ALA production bacterial strain and applications, belong to genetic engineering
And technical field of microbial fermentation.
Background technique
5-ALA (5-aminolevulinic acid, 5-ALA) is a kind of oxygen-containing and nitrogen nytron
Object is that one kind is widely present in as the required precursor substance of the tetrapyrroles class such as ferroheme, chlorophyll and vitamin B12 compound
Nonprotein amino acid in the living organisms living cells such as bacterium, fungi, animal and plant is that animals and plants vital movement is required
, the active physiological activator of metabolism, can be synthesized, can also be synthesized with artificial chemistry by biological approach, made that poison is not secondary
With degradable noresidue.It is widely used in agricultural, medicine and chemical field, is the high added value biology base of great Development volue
Chemicals.5-ALA can be prepared by chemical synthesis and biological fermentation process, and the biological fermentation process of clean and effective gradually becomes
The emphasis of research and development, and industrial application has been obtained.
The production bacterial strain of ALA mainly passes through mutation breeding at present or genetic engineering transformation obtains protokaryon bacterium, such as large intestine
Bacillus, corynebacterium glutamicum etc. are at home and abroad rarely reported by the research that S. cervisiae is transformed in genetic engineering.And root
According to No. 2045 bulletins of the Ministry of Agriculture of China in 2013《Catalogue of feed additive varieties》Middle regulation is in feed and feed addictive
Available 35 kinds of microbial strains types, both bacterium of Escherichia coli, corynebacterium glutamicum are not arranging.
Summary of the invention
In order to overcome drawbacks described above, the present invention provides a kind of construction methods of high yield 5-ALA production bacterial strain
And application.Purport of the invention is to weaken the activity of amber acid enzyme by recombinantly expressing external source 5-ALA synzyme, change
The metabolic pathway of cell itself, so that constructing new engineered strain carrys out green production 5-ALA；On the other hand, new strains are led to
The fermentative feedstuff of microbe for crossing solid fermentation production, is added in animal feed and feeds weanling pig, and it is young to significantly improve wean
The content of indices in the growth performance and blood of pig.
To achieve the goals above, the technical scheme is that：In a first aspect, the present invention provides a kind of high yield 5-
Amino-laevulic acid produces the construction method of bacterial strain, the synthesis of succinyl-coenzyme A in reduction 5-ALA production bacterial strain
The activity of pathway key enzyme.
Preferably, the route of synthesis key enzyme of the succinyl-coenzyme A is amber acid enzyme.
Preferably, the reduction refers to that the activity by amber acid enzyme is reduced to the 40-60% of original activity.
It is further preferred that the reduction refers to that the activity by amber acid enzyme is reduced to the 48-50% of original activity,
Optimal value is 48.17%.
Preferably, the 5-ALA production bacterial strain is saccharomyces cerevisiae.
Preferably, 5-aminolevulinate synthetase hem1 gene cloning in saccharomyces cerevisiae is entered into expression vector
Prs426；Promoter selects constitutive promoter TDH3P；After this contains the plasmid linearization of hem1 gene, by electrotransformation side
Formula is transferred to saccharomyces cerevisiae；Obtained transformant is high yield 5-ALA saccharomyces cerevisiae engineered yeast strain.
In second aspect, the present invention provides a kind of high yield 5-ALAs to produce bacterial strain, the 5- glycyl
The activity of the route of synthesis key enzyme of succinyl-coenzyme A is weakened in propionic acid production bacterial strain.
Preferably, the route of synthesis key enzyme of the succinyl-coenzyme A is amber acid enzyme.
In the third aspect, the present invention provides a kind of methods for producing 5-ALA, from above-mentioned 5- amino second
5-ALA is obtained in the fermentation culture system of acyl propionic acid production bacterial strain.
In fourth aspect, the present invention provides a kind of preparation methods of fermentative feedstuff of microbe, by above-mentioned 5- amino second
Acyl propionic acid is produced bacterial strain and is accessed in solid medium with 1% inoculum concentration, dries pulverizing after fermentation.
At the 5th aspect, the present invention provides a kind of microorganisms of the preparation method of fermentative feedstuff of microbe utilized preparation
Fermented feed.
At the 6th aspect, the present invention provides a kind of application of above-mentioned fermentative feedstuff of microbe in feed.
The present invention is host with saccharomyces cerevisiae by4741, by cloning high activity saccharomyces cerevisiae 5-ALA synzyme hem1 base
Cause, the present invention implement the 5-ALA synthase gene for being derived from saccharomyces cerevisiae utilized, and enzyme gene without being limited thereto is also possible to
The 5-ALA synthase gene in other sources.Hem1 gene cloning is entered into expression vector Prs426.Promoter selection composing type opens
Mover TDH3P.After this contains the plasmid linearization of hem1 gene, saccharomyces cerevisiae by4741 is transferred to by electrotransformation mode.It obtains
Transformant be engineering strain needed for the method for the present invention.
There are two subunits for amber acid enzyme, are generated respectively by gene Lsc1 and Lsc2, the two genes is selected to weaken
It is expressed, reduces succinic acid synthesis flux, and to provide more, succinyl-coenzyme A is synthesized for 5-ALA.
It constructs and converts plasmid prs426-gRNA1 and Lsc1 recombinant fragment, help incision in Cas9 albumen after expression gRNA1
Lsc1 gene on genome is cut, then carries out double exchange reorganization at point of contact using Lsc1 recombinant fragment, replace promoter and is repaired
Multiple genes group, and verified in Sc-leu-ura plate screening, it is coated on 5-FOA plate after mentioning gene order-checking, by what is grown
Transformant is verified using bacterium colony PCR, the bacterial strain that screening Lsc1 gene promoter is replaced successfully.In kind screening obtains again
The bacterial strain that Lsc2 gene promoter is replaced successfully.
Finally utilize CRISPR-Cas9 system combination hem1 gene to the site chromosome YBR128C.
The plasmid is integrated into cell chromosome DNA by way of homologous recombination in this way, so that exogenous DNA synthesizes
Enzyme can obtain stable expression in recombinant cell.
Solid fermentation is carried out using the high yield 5-ALA recombinant Saccharomyces cerevisiae built, produces fermentative feedstuff of microbe.The hair
Ferment feed addition feeds weanling pig into animal feed, can significantly improve containing for indices in its growth performance and blood
Amount.
Saccharomyces cerevisiae contains higher thick protein, amino as one of strain most common in fermentative feedstuff of microbe
Acid and multivitamin, simultaneously containing digestive ferment needed for many animals（Alpha-amylase, protease, cellulase, hemicellulose
Enzyme etc.）, play an important role to the digestion and absorption of nutriment.Yeast cells can also directly and enteron aisle in pathogen knot
It closes, neutralizes the toxin in stomach and intestine.Saccharomycete has strong yeast fragrance simultaneously, increases appetite to domestic animal, enhancing is digested and assimilated
Etc. there is certain effect.As fermentative feedstuff of microbe, it is added in complete diet pellet, there is the growth performance for improving animal, enhancing
Animal immunizing power and other effects.So the high yield 5-ALA recombinant Saccharomyces cerevisiae bacterium obtained by genetic engineering is raised in microbial fermentation
There is vast potential for future development in material.
The beneficial effects of the invention are as follows：5-ALA synthase gene is successfully imported into the wine brewing of eukaryon bacterium for the first time by the present invention
In yeast, do not had been reported that in research before.
The high yield 5-ALA recombinant Saccharomyces cerevisiae that the present invention constructs obtains 5- by weakening the activity of amber acid enzyme
The yield of ALA reaches 2.0 g/L and compares with other researchs, and the yield of 5-ALA is relatively high.
The high yield 5-ALA recombinant Saccharomyces cerevisiae that the present invention constructs, can be applied in fermentative feedstuff of microbe industry, has
The features such as pollution-free, environmentally protective.
Detailed description of the invention
Fig. 1 is the canonical plotting of 5-ALA of the present invention.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with attached drawing, it is clear that described implementation
Example is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ordinary skill
Personnel's every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
The following examples are intended to illustrate the invention, but is not limited to protection scope of the present invention.In the following example not
The experimental method of actual conditions is indicated, usually according to normal condition such as Sambrook et al., molecular cloning：Laboratory manual
(New York:Cold Spring Harbor LaboratoryPress, 1989) condition described in, or according to manufactory
Condition proposed by quotient.
Unless otherwise defined, all technical and scientific terms used herein and one skilled in the art of the present invention
Normally understood meaning is identical.Implement or examine although can utilize to described herein similar or of equal value any method and material
The present invention, but preferably method described herein and material.
The present invention can be furtherd elucidate by following embodiments.
Experimental material brief description.
Taq archaeal dna polymerase, restriction enzyme, plasmid extraction kit, DNA extraction kit and the recycling examination of DNA glue
Agent box is purchased from Takara company；Saccharomyces cerevisiae type strain by4741, expression plasmid Prs426 are purchased from Beijing Hua Yue ocean biology
Science and Technology Ltd..
Yeast extract and peptone are purchased from Britain's Oxoid Products；Glycine, IPTG, 5-ALA and to dimethylamino
Benzaldehyde etc. is purchased from the Sigma company in the U.S.；Agar powder and antibiotic are purchased from Beijing Suo Laibao Science and Technology Ltd.；Glucose,
It is close europeanized that glacial acetic acid, perchloric acid, trichloroacetic acid, acetylacetone,2,4-pentanedione, chloroform and other common chemical reagent are purchased from Tianjin section
Reagent Co., Ltd is learned, is that domestic analysis is pure.
YPD culture medium：1 % yeast extract（Yeast Extract）, 2 % peptones（Peptone）, 2 % glucose
（glucose）If solid medium processed, 2 % agar powders are added.
Fermentation medium：20 g/L of glucose, 7.5 g/L of glycine, yeast extract 7.5 g/L, (NH4)2SO4
(10 g/L), KH2PO4(1 g/L), K2HPO4(1 g/L), MgSO4∙7H2O (0.25 g/L), CaCl2(10 mg/L),
FeSO4∙7H2O (0.01 mg/L), MnSO4∙H2O (0.1 mg/L), ZnSO4∙7H2O (1 mg/L), CuSO4∙5H2O (0.2
Mg/L), NiCl2∙6H2O (20 μ g/L), biotin (0.4 mg/L) (pH 7.0).
Embodiment 1
Step 1: the building of expression 5-Aminolevulinate synthase recombination yeast
1. extracting saccharomyces cerevisiae by4741 DNA
The saccharomyces cerevisiae by4741 of activation is inoculated into YPD fluid nutrient medium, 30 °C, 150 rpm shaken cultivation, 24 h.
Constitutive promoter TDH3P PCR amplification primer sequence：
TDH3P-U:CGCGGATCCAGTGAATTTACTTTAAATCTTGC（BamHI）
TDH3P-L:CCTCGCAAAAATGGAGCGTTGCATGGAATCTGTGTATATTACTGC
According to the saccharomyces cerevisiae in GenBank（Saccharomyces cerevisiae）5-Aminolevulinate synthase hem1
Gene（No. GenBank: M26329）, design two PCR primers：
hem1-U：ATGCAACGCTCCATTTTTGCGAGG
hem1-L：CGCGAATTCTAGCCTATATATACTCATACG（EcoRI）
PCR amplification parameter is：94 °C 5 min；94 °C of 30 sec, 55 °C of 30 sec, 72 °C of 2 min are recycled 30 times；
72 °C 10 min.PCR after reaction, takes 1 μ L reaction solution to carry out agarose gel electrophoresis, detects PCR amplification effect, warp
PCR product after crossing agarose gel electrophoresis is recycled with DNA QIAquick Gel Extraction Kit.
2. the acquisition of recombinant plasmid
The promoter fragment of acquisition is connected with hem1 genetic fragment by fusion DNA vaccine, segment and plasmid prs426 after connection
Double digestion is carried out with BamHI and EcoRI respectively, the plasmid prs426-TDH3P- of Escherichia coli screening building is imported after connection
Hem1, screening verification obtain recombinant plasmid.
3. the preparation of Saccharomyces cerevisiae competent cell
Picking saccharomyces cerevisiae by4741 is in 300 mL YPD culture mediums, and 30 °C, 250 rpm are cultivated to OD600Reach 1.3-
1.5,5000 rpm are centrifuged 5min, and bacterial sediment uses 100 mL, 20 mL ice baths pre-cooling sterile water and 20 mL, 1 mol/L respectively
Ice bath pre-cooling sorbierite respectively wash once, every time clean after be centrifuged and collect thallus, finally the ice bath with 200 μ L 1mol/L is pre-
Cold sorbierite suspends.
4. the electrotransformation of saccharomyces cerevisiae by4741 cell
The saccharomyces cerevisiae by4741 competent cell prepared is taken out, be placed on makes its thawing on ice, and plasmid to be transformed is added
DNA is uniformly mixed it with pipettor pressure-vaccum competent cell；By the recombinant expression plasmid prs426-hem1 Avr of building
The linearisation of II restriction enzyme, phenol chloroform drawer, ethanol precipitation recycling linear DNA are simultaneously dissolved in 10 μ L sterile waters, simultaneously
Empty plasmid using identical linearization for enzyme restriction and is recycled as control.The linearisation DNA of acquisition and saccharomyces cerevisiae by4741 is felt
By state mixing with cells, electrotransformation, condition is：1500 V of voltage, 50 μ F of capacitor, 4 K Ω of resistance.1 is added into electrotransformation cup
Electrotransformation product is moved into respectively in a sterile centrifugation tube after the sorbierite of mL ice bath pre-cooling, 30 °C stand overnight.Take 300-
500 μ L are coated in the YPD culture medium containing ampicillin, and 30 °C of cultures to single colonie occur, and are cultivated and are verified the positive
Bacterial strain obtains the recombination yeast that can express hem1 gene.
Step 2: the activity of reduction amber acid enzyme
There are two subunits for amber acid enzyme, are generated respectively by gene Lsc1 and Lsc2, select the two genes to weaken its table
It reaches, reduce succinic acid synthesis flux, succinyl-coenzyme A is synthesized for 5-ALA to provide more.Utilize CRISPR-Cas9 system
System substitutes the former promoter of gene weak promoter cyc1 promoter.
1. constructing plasmid prs426-gRNA1 and prs426-gRNA2
The selection of gRNA sequence
The site Lsc1：W chain：CCCAACCAGCGGCTGGATAA
C chain：TTATCCAGCCGCTGGTTGGG
The site Lsc2：W chain：GGAAGCTAACAGATAGTTCT
C chain：AGAACTATCTGTTAGCTTCC
Double-strand Lsc1 and Lsc2 gRNA segment is obtained after Oligo annealing annealing, constructs gRNA expression plasmid respectively
Prs426-gRNA1 and prs426-gRNA2.Escherichia coli are converted, bacterium colony verifying is chosen, plasmid are extracted, after universal primer M13 amplification
Sequencing identification expression plasmid.
2. the building of Lsc1 recombinant fragment
Homology arm 1, CYC1T, CYC1P and 2 four sub-pieces of homology arm are respectively obtained by PCR amplification first, using fusion DNA vaccine
Connect into Lsc1 recombinant fragment.
The building of 2.Lsc2 recombinant fragment
Homology arm 3, CYC1T, CYC1P and 4 four sub-pieces of homology arm are respectively obtained by PCR amplification, are connected using fusion DNA vaccine
At Lsc2 recombinant fragment.
4. the conversion of yeast
The plasmid prs415-case9 provided using other laboratories obtains Cas9 protein expression strain, and flat using Sc-leu
Plate screening verification.
According to the method for transformation of step 1 obtain plasmid prs426-gRNA1 and Lsc1 recombinant fragment, express gRNA1 after
Cas9 albumen helps Lsc1 gene on lower cutting genome, then carries out double crossing over weight at point of contact using Lsc1 recombinant fragment
Group, replacement promoter and revision points group, and verified in Sc-leu-ura plate screening, 5- is coated on after mentioning gene order-checking
On FOA plate, the transformant grown is verified using bacterium colony PCR, screens and obtain the bacterium that Lsc1 gene promoter is replaced successfully
Strain.
Conversion plasmid prs426-gRNA2 and segment Lsc2 is obtained according to the method for transformation of step 1, is repeated the above steps,
Screen and obtain the bacterial strain that Lsc2 gene promoter is replaced successfully.
Step 3: integrant expression
Utilize CRISPR-Cas9 system combination hem1 gene to the site chromosome YBR128C.
The selection of 1.gRNA sequence
The site YBR128C gRNA sequence design：
W chain：TCACAGGACCTCATGACCGA
C chain:TCGGTCATGAGGTCCTGTGA
3. constructing plasmid prs426-gRNA3
Building of the method with prs426-gRNA1 and prs426-gRNA2.
The building of the site 4.YBR128C recombinant fragment
Three above sub-piece is obtained by PCR amplification, wherein the homologous arm region of upstream and downstream is that gRNA sequence upstream and downstream is each
500bp；Three sub-pieces connect into the site YBR128C recombinant fragment by fusion DNA vaccine.
4. recombinant bacterial strain constructs
Method finally obtains with the method for transformation of 4. yeast in step 2 and incorporates the saccharomyces cerevisiae weight of hem1 gene on chromosome
Group bacterial strain.
Step 4: the active detection of recombination yeast
1. the detection of recombination yeast physiological activity
The recombination yeast screened is inoculated into YPD fluid nutrient medium, after 30 °C of 150 rpm culture 12-20 h, then presses 1
Ferment 48 h in % ratio access 5-ALA fermentation medium.Fermentation liquid is diluted to the measurement range of spectrophotometer with distilled water
（Between 0.3-0.8）, with light absorption value of the bacterium solution at 600 nm after ultraviolet specrophotometer measurement dilution, be 4.8 ±
0.23。
2. recombination yeast 5-Aminolevulinate synthase（5-ALAS）The measurement of enzyme activity
The acquisition of 2.1 crude enzyme liquids
In YPD fluid nutrient medium, at 30 °C, 150 rpm are cultivated to logarithmic growth phase recombination yeast.Shift 50 mL bacterium solutions extremely
In 100 mL centrifuge tubes of pre-cooling, 9000 rpm, 4 °C of 5 min of centrifugation simultaneously collect thallus.By the thallus being collected into 20 mL
50 mM contain the Tris-HCl buffer solution of 0.1 mM EDTA and 2 mM dithiothreitol (DTT)s（7.5,4 °C of pH pre-
It is cold）It washs secondary, is suspended in the same buffer of 2 mL.With ultrasonic disruption thalline, using following condition：130 W, 20
KHz；Pulse On 5 s；Pulse Off 5 s；Always broken 30 min of time.Under the conditions of 4 °C, 9000 rpm centrifugation 30
Min removes impurity, and obtained supernatant is crude enzyme liquid, and 4 °C of placement is spare.
The measurement of 2.2 5-ALAS enzyme activity
123.7 μ L reaction solutions are added in 376.3 μ L of supernatant after taking clasmatosis（Such as table 1）, mix.In 37 °C of water-bath
10 min of middle reaction, are then added the 10 % trichloroacetic acids of 150 μ L.Centrifugation, takes 300 μ L supernatants, according to the side for surveying 5-ALA
The content of method test 5-ALA.
By weakening the generation of succinic acid, before reduction and after reduction, the enzyme activity of recombination yeast is respectively 9.5 ±
0.23 and 19.49 ± 0.19 U/mg protein illustrate the generation for weakening succinic acid, 5-ALA can be improved
The enzyme activity of synthase.
The detection of 2.3 5-ALA yield
（1）Required solution
Modified Ehrlich's reagent（Matching while using）：The glacial acetic acid of 1 mL, the p- dimethyl of 0.2 g are taken respectively
Benzaldehyde, the perchloric acid of 70 % of 1 mL, ice acetic acid constant volume to 10 mL.
Sodium acetate buffer（pH 4.6）：It measures the glacial acetic acid of 5.7 mL, weigh 8.2 g anhydrous sodium acetates, add water
It is settled to 100 mL.
（2）The preparation of 5-ALA standard curve
Weigh the 5-ALA hydrochloride of 0.128 g（5-ALA-HCl）, 100 mL are settled to get the 5- of 1 g/L
ALA.Then gradient dilution measures its absorbance at 554 nm according to the above method.With the concentration of 5-ALA（mg/L）For cross
Coordinate draws the standard curve of 5-ALA using OD554 value as ordinate（Fig. 1）.
（3）The measurement of the yield of 5-ALA
12000 rpm of fermentation liquid is centrifuged 10 min and removes solid impurity, supernatant is diluted according to a certain percentage.Take dilution
250 μ L of liquid is separately added into the sodium acetate buffer of 125 μ L and the acetylacetone,2,4-pentanedione of 62.5 μ L, 100 °C of 15 min of reaction.It is cold
But to room temperature, Modified Ehrlich's reagent reagent is added and reacts 20 min, then measure reaction solution in 554 nm
Under absorbance, the concentration of 5-ALA is calculated according to standard curve as shown in Figure 1, the yield for obtaining 5-ALA reaches 2.0 g/
L。
Embodiment 2
The preparation of fermentative feedstuff of microbe
Solid culture based component：20 % of wheat bran, 20 % of fermented bean dregs, 24 % of corn protein powder, 20 % of corn flour, 0.8 % of urea,
K2HPO4 0.8 %.Material-water ratio 1: 0.8.300 g/500 mL wide-mouth bottle of loading amount, pH are natural.Cultured recombination yeast is sent out
Zymotic fluid is accessed in solid medium with 1 % inoculum concentration, is dried to 10-12 % of moisture for 50-60 °C after 30 °C of 24 h of fermentation,
Being crushed to granularity is 2.0 mm to get the product of microorganism fermented forage.
The fermentation liquid cultivated with original Wine brewing yeast strain by4741, guarantee contain with yeast in recombinant Saccharomyces cerevisiae fermentation liquid
It measures identical, accesses in solid medium in the same scale, with the fermentation of identical condition, dry and crush, as subsequent feeding
The positive control of test is named as " PC fermentative feedstuff of microbe ", and the product prepared with high yield 5-ALA bacterial strain is named as " EX
Fermentative feedstuff of microbe ".
3-5 g of fermented feed product after weighing drying adds 100 mL distilled water, shakes in 250 mL triangular flask
After even, 12000 rpm are centrifuged 10 min and remove solid impurity after 30 min of shaken at room temperature, and supernatant is dilute according to a certain percentage
It releases.250 μ L of dilution is taken, the sodium acetate buffer of 125 μ L and the acetylacetone,2,4-pentanedione of 62.5 μ L, 100 °C of reactions are separately added into
15 min.It is cooled to room temperature, Modified Ehrlich's reagent reagent is added and reacts 20 min, then measures reaction solution
Absorbance at 554 nm calculates the concentration of 5-ALA according to standard curve, and the yield for obtaining 5-ALA reaches 50 mg/kg.
Embodiment 3
Influence of the 5-ALA fermented feed to Growth Performance of Weaning Piglets and physiochemical indice
Choose the administering transgenic that 80 weanling pigs carry out 35 days by a definite date, original body mass（BW）For 7.05 ± 0.46 kg, experiment
Start, this 100 pigs are divided into 5 processing groups at random.Each processing group has 4 repetitions.Basal diet ingredient is shown in Table 2.This 5
Processing group is respectively NC group（Basal diet does not add the negative control of any fermentative feedstuff of microbe）, PC group（Basal diet+2
The positive control of % PC fermentative feedstuff of microbe）, experimental group（+ 2 % EX fermentative feedstuff of microbe of basal diet）.
Feeding process is divided into three phases, first stage（0-7 day）, second stage（8-21 days）, the phase III
（22-35 days）, the average daily gain of each processing group weanling pig in each of which stage is measured respectively（ADG）, average day feeding
Amount（ADF1）And meat feed ratio（G:F）（Table 3）
The results show that experiment three phases and all stage：The growth performance of PC group weanling pig（ADG, ADF1 and G:F）Want excellent
In NC group, but difference is not significant, illustrates using the fermentative feedstuff of microbe of original Wine brewing yeast strain production to weanling pig
Growth performance does not have much influence；And the growth performance of experimental group weanling pig is obviously better than NC group and PC group, illustrates 5-
The success of ALA synzyme is expressed in saccharomyces cerevisiae, and the fermentative feedstuff of microbe of recombinant Saccharomyces cerevisiae bacterial strain production can be significant
Improve the growth performance of weanling pig.
For hematological indices, after feeding experiment, by the blood sample collection of weanling pig to non-heparinized and
K3In EDTA, serum and whole blood are obtained with vacuum tube respectively.Subsequent 4 °C of blood sample, 2000 × g is put into 4 after being centrifuged 30 min
It is saved backup in °C refrigerator.
RBC（Red blood cell）,WBC（White blood cell）Use Automatic Blood Cell Analyzer（Sweden McDonnell Douglas Buddhist nun gram CA620）Measurement, Hb
（Hemoglobin, hemoglobin）And HCT（Hematocrit, hematocrit）Using automatic clinical chemistry analyzer（Hitachi
7180）Measurement；Use flow cytometer（Guave 6HT）Measure Lymphocyte（Lymphocyte）Quantity（Table 4）.NC group and PC
Each indicator difference is little in group blood, and each index content is compared compared with NC group with PC group in experimental group blood, is had and is significantly mentioned
Height, illustrate in basal feed add the fermented feed product containing 5-ALA ingredient can significantly improve Hb in animal blood,
The content of HCT, RBC, WBC and Lymphocyte.
Of the invention substantially having been done to core of the invention by generality explanation and specific embodiment is more detailed
Elaboration, but on the basis of the present invention, those skilled in the art can modify or improve to it, even
It improves, and this improvement and raising are obvious.Therefore, these done without departing from theon the basis of the spirit of the present invention are repaired
Change or improve, should belong to the scope of protection of present invention.
Sequence table
SEQUENCE LISTING
<110>Henan city letter is apt into Bioisystech Co., Ltd
<120>A kind of high yield 5-ALA produces construction method and the application of bacterial strain
<130> 2018
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 501
<212> DNA
<213>It is unknown
<400> 1
agtgaattta ctttaaatct tgcatttaaa taaattttct ttttatagct ttatgactta 60
gtttcaattt atatactatt ttaatgacat tttcgattca ttgattgaaa gctttgtgtt 120
ttttcttgat gcgctattgc attgttcttg tctttttcgc cacatgtaat atctgtagta 180
gatacctgat acattgtgga tgctgagtga aattttagtt aataatggag gcgctcttaa 240
taattttggg gatattggct ttttttttta aagtttacaa atgaattttt tccgccagga 300
taacgattct gaagttactc ttagcgttcc tatcggtaca gccatcaaat catgcctata 360
aatcatgcct atatttgcgt gcagtcagta tcatctacat gaaaaaaact cccgcaattt 420
cttatagaat acgttgaaaa ttaaatgtac gcgccaagat aagataacat atatctagat 480
gcagtaatat acacagattc c 501
<210> 2
<211> 501
<212> DNA
<213>It is unknown
<400> 2
aatttttttc aacgtcttgg caaattcttc agcagcttta atacgtccta gtctcctctt 60
ctccaaaaga tgtagcggga ctttcttccc gggaggtgct tttgcgggat ctagcttcgg 120
atatttctta taacgtttac tagtctgcac agcaggtggt gccctcttat ctctagagct 180
ccctaattca ttttcatttt cgttggcatt atcattatca ttttcgtcct cattgtcaac 240
atcattgtcg tcttcgtctt catcttcgtc ttccccttct tcatcttcat cttcttcgtc 300
tgcaacgtct cttgggtcaa cagaaagacc tgttacactt ctgggtgtct cagatccaac 360
tacgttatcg acactatcgt ctgactcatt ctccgcattt tcttgatccg cctggctgct 420
taaaggtacg ctattttctt cttccagttc atcctcatcg tcatcatcct ctaacggaat 480
atcaatgggt tcctcataaa t 501
<210> 3
<211> 301
<212> DNA
<213>It is unknown
<400> 3
aacaggcccc ttttcctttg tcgatatcat gtaattagtt atgtcacgct tacattcacg 60
ccctcctccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt 120
ccctatttat tttttttaat agttatgtta gtattaagaa cgttatttat atttcaaatt 180
tttctttttt ttctgtacaa acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt 240
gagaaggttt tgggacgctc gaaggcttta atttgcaagc ttcgcagttt acactctcat 300
c 301
<210> 4
<211> 289
<212> DNA
<213>It is unknown
<400> 4
atttggcgag cgttggttgg tggatcaagc ccacgcgtag gcaatcctcg agcagatccg 60
ccaggcgtgt atatatagcg tggatggcca ggcaacttta gtgctgacac atacaggcat 120
atatatatgt gtgcgacgac acatgatcat atggcatgca tgtgctctgt atgtatataa 180
aactcttgtt ttcttctttt ctctaaatat tctttcctta tacattagga cctttgcagc 240
ataaattact atacttctat agacacgcaa acacaaatac acacactaa 289
<210> 5
<211> 501
<212> DNA
<213>It is unknown
<400> 5
atgttaagat ctaccgtttc aaaagcttct ctcaaaattt gtcgtcactt tcacagagaa 60
tctattcctt acgataagac gatcaaaaat ttgctgcttc ccaaggacac caaggtgatc 120
tttcaggggt tcacaggtaa acaaggtact tttcatgcca gcatctctca agaatatggt 180
acaaatgttg tgggtggtac gaacccaaaa aaggcgggtc aaacacattt aggccaacct 240
gtctttgcct ctgtgaagga cgcgattaag gagactggag ccactgccag tgccatcttt 300
gttcctccac cgatcgcagc tgctgccatt aaagaatcta ttgaagctga aattccgtta 360
gctgtatgta tcactgaagg tattcctcaa catgacatgt tatacattgc agaaatgttg 420
cagacacaag ataaaacaag attagttggg cccaactgcc ctggtatcat taacccagca 480
acaaaggtaa gaattggtat c 501
<210> 6
<211> 501
<212> DNA
<213>It is unknown
<400> 6
gctgatactg ctgtgggata tgttgctggt gtaaatatcc atcattcgga gagtttggca 60
cagagcgtgg agtgtttacg cgaacattca tgttcgagaa gatagatgaa acatcactat 120
ttgactgtat ggagccaata gagctctgga aagaggtgaa tgattgcgat ggaactggag 180
tgtatgtaga aattggcata ggagaagttg caggctggaa agatgcagcg gaggcatgaa 240
tgggggctgg ttgggccatg attggtatca ccattgggtg caccgcattg ggagcgtgta 300
taatgttacc tccatttact gcctgaacat ttatggccat atttgctggt aggacttgcg 360
gtattggttg gtgtatcatc acgcccgatg caatggtagg aattccgctt actgtcaaaa 420
actcctgttt ctgtacgcta gctttcttca atctcctttg agattgccct gtatgcgttc 480
tcatgtgcct tttcagttca t 501
<210> 7
<211> 501
<212> DNA
<213>It is unknown
<400> 7
gcatcattca gacaggaaaa gatatattcc tggagggact tatcacaaga agatcctgat 60
gaagttaagg caaagaagta tgatttgaat tttgttaagt tgaagggtaa cattggatgt 120
ttagtcaatg gtgctggttt ggctatggct actatggatg tcatcaaatt aaatggaggc 180
gatcctgcga actttttgga ttgtggtggt ggtgccaccc ctgagaccat caaacaaggt 240
ttcgaattga tcttatccaa taagaacgta gatgcaattt tcgtcaatat tttcggcggt 300
atcgtaagat gtgactatgt tgccctgggg ctggtagaag ccgccagaga actagaagtt 360
agggtcccca ttgtggcacg tttgcaaggt accaaagtag aagaaggccg cgacattatc 420
aataagtcag gcgtgaagat ttattcgttt gatgaattag atcctgctgc taaaaaggtc 480
gttgaattga cccaaaatta a 501
<210> 8
<211> 444
<212> DNA
<213>It is unknown
<400> 8
ccactaatat ctttgaaaaa cttccgtatt ttacctcttg ctataacaaa cgattccatc 60
aacatcatgt ggaagtatat tagcttcttc tcagacattc ttatgattaa acttccctac 120
acaaataaaa tctgtgaaca acccatgttt gagttttccg atagcataca gacagtagta 180
caaaggttga tcaagcttat cataaatatt ttacagatat gtagacattt gaaactcgta 240
ccttcaacac ccatggatat cccatggcta ctggaccagt acgatgtgga tgggctgttc 300
tataatatgg tgaaacggaa caagatgaag tgtaggtccg tctcgctata ttggactttt 360
gggatgttgt actcgatggt tttggataac atgaataatc cacaaagagg acatcctgca 420
aggcggaccg caccacctcc aaca 444
<210> 9
<211> 500
<212> DNA
<213>It is unknown
<400> 9
tacgtggtag gctagagtgc gcactttttt tcctgccgtg tgtataaagt tgcatgtagt 60
cagtcataaa caatagcagc cagtgtaata agcattcggg taagaaccaa ctcagttttt 120
tagttgtttt tttttttttt tttttcaccc cgttgaaaga aaaggataag agataaagga 180
ctaaatgatt ttaatacaga tagacaacca ttgttgacag gattgcgatt gtaagagtag 240
acagtacatc aagcgaaaat aaatattgca ggaatggttt tgtctgataa ggagttgttt 300
gccataaata agaaagccgt cgaacaaggt ttcaatgtga agcctagatt gaactataat 360
acggtcagtg gtgtgaacgg tccattagtc attttggaaa aggtcaagtt cccacgttac 420
aacgaaattg ttaatttgac attgccagat ggaaccgtga gacaaggtca agttttggaa 480
attagaggag atagagccat 500
Sequence table
<110>Henan city letter is apt into Bioisystech Co., Ltd
<120>A kind of high yield 5-ALA produces construction method and the application of bacterial strain
<130> 2018
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 501
<212> DNA
<213>Unknown ()
<400> 1
agtgaattta ctttaaatct tgcatttaaa taaattttct ttttatagct ttatgactta 60
gtttcaattt atatactatt ttaatgacat tttcgattca ttgattgaaa gctttgtgtt 120
ttttcttgat gcgctattgc attgttcttg tctttttcgc cacatgtaat atctgtagta 180
gatacctgat acattgtgga tgctgagtga aattttagtt aataatggag gcgctcttaa 240
taattttggg gatattggct ttttttttta aagtttacaa atgaattttt tccgccagga 300
taacgattct gaagttactc ttagcgttcc tatcggtaca gccatcaaat catgcctata 360
aatcatgcct atatttgcgt gcagtcagta tcatctacat gaaaaaaact cccgcaattt 420
cttatagaat acgttgaaaa ttaaatgtac gcgccaagat aagataacat atatctagat 480
gcagtaatat acacagattc c 501
<210> 2
<211> 501
<212> DNA
<213>Unknown ()
<400> 2
aatttttttc aacgtcttgg caaattcttc agcagcttta atacgtccta gtctcctctt 60
ctccaaaaga tgtagcggga ctttcttccc gggaggtgct tttgcgggat ctagcttcgg 120
atatttctta taacgtttac tagtctgcac agcaggtggt gccctcttat ctctagagct 180
ccctaattca ttttcatttt cgttggcatt atcattatca ttttcgtcct cattgtcaac 240
atcattgtcg tcttcgtctt catcttcgtc ttccccttct tcatcttcat cttcttcgtc 300
tgcaacgtct cttgggtcaa cagaaagacc tgttacactt ctgggtgtct cagatccaac 360
tacgttatcg acactatcgt ctgactcatt ctccgcattt tcttgatccg cctggctgct 420
taaaggtacg ctattttctt cttccagttc atcctcatcg tcatcatcct ctaacggaat 480
atcaatgggt tcctcataaa t 501
<210> 3
<211> 301
<212> DNA
<213>Unknown ()
<400> 3
aacaggcccc ttttcctttg tcgatatcat gtaattagtt atgtcacgct tacattcacg 60
ccctcctccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt 120
ccctatttat tttttttaat agttatgtta gtattaagaa cgttatttat atttcaaatt 180
tttctttttt ttctgtacaa acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt 240
gagaaggttt tgggacgctc gaaggcttta atttgcaagc ttcgcagttt acactctcat 300
c 301
<210> 4
<211> 289
<212> DNA
<213>Unknown ()
<400> 4
atttggcgag cgttggttgg tggatcaagc ccacgcgtag gcaatcctcg agcagatccg 60
ccaggcgtgt atatatagcg tggatggcca ggcaacttta gtgctgacac atacaggcat 120
atatatatgt gtgcgacgac acatgatcat atggcatgca tgtgctctgt atgtatataa 180
aactcttgtt ttcttctttt ctctaaatat tctttcctta tacattagga cctttgcagc 240
ataaattact atacttctat agacacgcaa acacaaatac acacactaa 289
<210> 5
<211> 501
<212> DNA
<213>Unknown ()
<400> 5
atgttaagat ctaccgtttc aaaagcttct ctcaaaattt gtcgtcactt tcacagagaa 60
tctattcctt acgataagac gatcaaaaat ttgctgcttc ccaaggacac caaggtgatc 120
tttcaggggt tcacaggtaa acaaggtact tttcatgcca gcatctctca agaatatggt 180
acaaatgttg tgggtggtac gaacccaaaa aaggcgggtc aaacacattt aggccaacct 240
gtctttgcct ctgtgaagga cgcgattaag gagactggag ccactgccag tgccatcttt 300
gttcctccac cgatcgcagc tgctgccatt aaagaatcta ttgaagctga aattccgtta 360
gctgtatgta tcactgaagg tattcctcaa catgacatgt tatacattgc agaaatgttg 420
cagacacaag ataaaacaag attagttggg cccaactgcc ctggtatcat taacccagca 480
acaaaggtaa gaattggtat c 501
<210> 6
<211> 501
<212> DNA
<213>Unknown ()
<400> 6
gctgatactg ctgtgggata tgttgctggt gtaaatatcc atcattcgga gagtttggca 60
cagagcgtgg agtgtttacg cgaacattca tgttcgagaa gatagatgaa acatcactat 120
ttgactgtat ggagccaata gagctctgga aagaggtgaa tgattgcgat ggaactggag 180
tgtatgtaga aattggcata ggagaagttg caggctggaa agatgcagcg gaggcatgaa 240
tgggggctgg ttgggccatg attggtatca ccattgggtg caccgcattg ggagcgtgta 300
taatgttacc tccatttact gcctgaacat ttatggccat atttgctggt aggacttgcg 360
gtattggttg gtgtatcatc acgcccgatg caatggtagg aattccgctt actgtcaaaa 420
actcctgttt ctgtacgcta gctttcttca atctcctttg agattgccct gtatgcgttc 480
tcatgtgcct tttcagttca t 501
<210> 7
<211> 501
<212> DNA
<213>Unknown ()
<400> 7
gcatcattca gacaggaaaa gatatattcc tggagggact tatcacaaga agatcctgat 60
gaagttaagg caaagaagta tgatttgaat tttgttaagt tgaagggtaa cattggatgt 120
ttagtcaatg gtgctggttt ggctatggct actatggatg tcatcaaatt aaatggaggc 180
gatcctgcga actttttgga ttgtggtggt ggtgccaccc ctgagaccat caaacaaggt 240
ttcgaattga tcttatccaa taagaacgta gatgcaattt tcgtcaatat tttcggcggt 300
atcgtaagat gtgactatgt tgccctgggg ctggtagaag ccgccagaga actagaagtt 360
agggtcccca ttgtggcacg tttgcaaggt accaaagtag aagaaggccg cgacattatc 420
aataagtcag gcgtgaagat ttattcgttt gatgaattag atcctgctgc taaaaaggtc 480
gttgaattga cccaaaatta a 501
<210> 8
<211> 444
<212> DNA
<213>Unknown ()
<400> 8
ccactaatat ctttgaaaaa cttccgtatt ttacctcttg ctataacaaa cgattccatc 60
aacatcatgt ggaagtatat tagcttcttc tcagacattc ttatgattaa acttccctac 120
acaaataaaa tctgtgaaca acccatgttt gagttttccg atagcataca gacagtagta 180
caaaggttga tcaagcttat cataaatatt ttacagatat gtagacattt gaaactcgta 240
ccttcaacac ccatggatat cccatggcta ctggaccagt acgatgtgga tgggctgttc 300
tataatatgg tgaaacggaa caagatgaag tgtaggtccg tctcgctata ttggactttt 360
gggatgttgt actcgatggt tttggataac atgaataatc cacaaagagg acatcctgca 420
aggcggaccg caccacctcc aaca 444
<210> 9
<211> 500
<212> DNA
<213>Unknown ()
<400> 9
tacgtggtag gctagagtgc gcactttttt tcctgccgtg tgtataaagt tgcatgtagt 60
cagtcataaa caatagcagc cagtgtaata agcattcggg taagaaccaa ctcagttttt 120
tagttgtttt tttttttttt tttttcaccc cgttgaaaga aaaggataag agataaagga 180
ctaaatgatt ttaatacaga tagacaacca ttgttgacag gattgcgatt gtaagagtag 240
acagtacatc aagcgaaaat aaatattgca ggaatggttt tgtctgataa ggagttgttt 300
gccataaata agaaagccgt cgaacaaggt ttcaatgtga agcctagatt gaactataat 360
acggtcagtg gtgtgaacgg tccattagtc attttggaaa aggtcaagtt cccacgttac 420
aacgaaattg ttaatttgac attgccagat ggaaccgtga gacaaggtca agttttggaa 480
attagaggag atagagccat 500
Claims (10)
1. a kind of construction method of high yield 5-ALA production bacterial strain, it is characterised in that：Weaken 5-ALA
Produce the activity of the route of synthesis key enzyme of succinyl-coenzyme A in bacterial strain；The route of synthesis key enzyme of the succinyl-coenzyme A is
Amber acid enzyme.
2. the construction method of high yield 5-ALA production bacterial strain according to claim 1, it is characterised in that：It is described
Reduction refers to that the activity by amber acid enzyme is reduced to the 40-60% of original activity.
3. the construction method of high yield 5-ALA production bacterial strain according to claim 2, it is characterised in that：It is described
Reduction refers to that the activity by amber acid enzyme is reduced to the 48-50% of original activity, optimal value 48.17%.
4. the construction method of high yield 5-ALA production bacterial strain according to claim 2 or 3, it is characterised in that：
The 5-ALA production bacterial strain is saccharomyces cerevisiae.
5. the construction method of high yield 5-ALA production bacterial strain according to claim 4, it is characterised in that：It will make
5-aminolevulinate synthetase hem1 gene cloning enters expression vector Prs426 in brewer yeast；Promoter selection composing type opens
Mover TDH3P；After this contains the plasmid linearization of hem1 gene, saccharomyces cerevisiae is transferred to by electrotransformation mode；Obtained conversion
Son is the saccharomyces cerevisiae engineered yeast strain of high yield 5-ALA.
6. a kind of high yield 5-ALA produces bacterial strain, it is characterised in that：In the 5-ALA production bacterial strain
The activity of the route of synthesis key enzyme of succinyl-coenzyme A is weakened；The route of synthesis key enzyme of the succinyl-coenzyme A is amber
Amber acid enzyme.
7. a kind of method for producing 5-ALA, it is characterised in that：From 5-ALA as claimed in claim 6
It produces in the fermentation culture system of bacterial strain and obtains 5-ALA.
8. a kind of preparation method of fermentative feedstuff of microbe, it is characterised in that：By 5-ALA as claimed in claim 6
It produces bacterial strain to access in solid medium with 1% inoculum concentration, dries pulverizing after fermentation.
9. fermentative feedstuff of microbe prepared by a kind of preparation method of fermentative feedstuff of microbe according to any one of claims 8.
10. a kind of application of fermentative feedstuff of microbe as claimed in claim 9 in feed.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113475634A (en) * | 2021-06-28 | 2021-10-08 | 河南邑鸿善成生物技术有限公司 | Pig feed containing 5-aminolevulinic acid, preparation method and application of pig feed to piglets |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981203A (en) * | 2013-02-07 | 2014-08-13 | 中国科学院天津工业生物技术研究所 | 5-amino levulinic acid (ALA) high-yield strain and preparation method and application thereof |
| CN104195190A (en) * | 2014-09-04 | 2014-12-10 | 山东大学 | Method for producing 5-aminolevulinic acid by carrying out anaerobic fermentation by utilizing recombinant escherichia coli |
| CN106967627A (en) * | 2017-04-11 | 2017-07-21 | 济南大学 | A kind of fermenting and producing ALA recombinant bacterial strain and preparation method thereof |
-
2018
- 2018-05-31 CN CN201810545863.5A patent/CN108841852A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103981203A (en) * | 2013-02-07 | 2014-08-13 | 中国科学院天津工业生物技术研究所 | 5-amino levulinic acid (ALA) high-yield strain and preparation method and application thereof |
| CN104195190A (en) * | 2014-09-04 | 2014-12-10 | 山东大学 | Method for producing 5-aminolevulinic acid by carrying out anaerobic fermentation by utilizing recombinant escherichia coli |
| CN106967627A (en) * | 2017-04-11 | 2017-07-21 | 济南大学 | A kind of fermenting and producing ALA recombinant bacterial strain and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| B PRZYBYLA-ZAWISLAK，ET AL: "Genes of succinyl-CoA ligase from Saccharomyces cerevisiae", 《EUR J BIOCHEM》 * |
| GUO-QIANG CHEN，ET AL: "Microbial Synthesis of 5-Aminolevulinic Acid and Its Coproduction with Polyhydroxybutyrate", 《ACS SYNTH BIOL》 * |
| 张熙等: "酿酒酵母异源合成己二酸", 《食品与发酵工业》 * |
| 沈佳佳等: "蛋白质赖氨酸琥珀酰化修饰研究进展", 《医学研究生学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113475634A (en) * | 2021-06-28 | 2021-10-08 | 河南邑鸿善成生物技术有限公司 | Pig feed containing 5-aminolevulinic acid, preparation method and application of pig feed to piglets |
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Application publication date: 20181120 |