CN104388400B - Enzyme and gene of cordyceps sinensis hirsutella sinensis synthesized acetic acid and application thereof - Google Patents
Enzyme and gene of cordyceps sinensis hirsutella sinensis synthesized acetic acid and application thereof Download PDFInfo
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- CN104388400B CN104388400B CN201410653007.3A CN201410653007A CN104388400B CN 104388400 B CN104388400 B CN 104388400B CN 201410653007 A CN201410653007 A CN 201410653007A CN 104388400 B CN104388400 B CN 104388400B
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Abstract
The invention relates to an enzyme and gene of cordyceps sinensis hirsutella sinensis synthesized acetic acid and application thereof, and relates to an enzyme of an acetyl-acyl vector synthesized and metabolized from acetyl coenzyme A by means of participation of cordyceps sinensis hirsutella sinensis from 'Corbrin', as well as gene coding the enzyme and application thereof. The amino acid sequence of the acyl-coenyzme A synthetase is a protein as shown in the SEQ ID No.3. According to the enzyme, the metabolism means of the acyl-coenyzme A synthetase synthesized acetyl-acyl vector protein is studied in details in principle, and the enzyme of the acetyl-acyl vector synthesized and metabolized from acetyl coenzyme A by means of participation of cordyceps sinensis hirsutella sinensis from 'Corbrin' and the coding gene of the enzyme are provided. The cloned DNA of the nucleotide sequence can be transferred into an engineering bacterium by a method of transducing, converting and conjugal transferring, high expression of the host acyl-coenyzme A synthetase is given by regulating expression of the acetyl-acyl vector protein biologically synthesized gene, and an effective means is provided to enlarging the yield of acetic acid. The enzyme has important application prospects.
Description
(1) technical field
The present invention relates to one kind is set out conjunction from the participation S-acetyl-coenzyme-A that " hundred make " produces bacterium Cordyceps China pilose spore
Into the fat-acyl CoA synthase (Acyl-coenzyme A synthetase) of metabolism acetyl group-acyl carrier protein, compile
Code this enzyme gene and its application, acetyl group-acyl carrier protein can again through hydrolysis generate acetic acid.
(2) background technology
Cordyceps (Cordyceps sinensis (Berk.) Sacc.) are that Cordyceps funguss colonize in Lepidoptera
(Lepidoptera) Stroma and larva corpse on Hepialidae insecticide (Hepialus armoricanus Oberthur) larva
Complex on body (including Stroma and polypide).Cordyceps are traditional funguses herb resources that a class is treasured, and are produced with metabolism
The characteristics of thing and diverse biological activities, in biomedicine field huge application and development prospect are shown.Cordyceps are with it
Various medicinal efficacies extensively, substantially receive much concern, and worldwide enjoy high praise.The traditional Chinese medical science thinks that Cordyceps enter lung kidney
Two Jing, can tonifying the lung it is cloudy, and can kidney-replenishing, cure mainly kidney deficiency, impotence and seminal emission, soreness of waist and knee joint, eak after being ill, chronic cough is weak, phthisical cough
Expectorant blood, spontaneous sweating etc., be it is unique it is a kind of can be while balancing, adjusting the Chinese medicine of negative and positive.Modern pharmacology is it has proven convenient that the worm summer in winter
Grass has immunomodulating, antibacterial, antitumor, antioxidation, defying age, reducing blood sugar and blood lipid, gonadotropic Effect etc. extensive biological
Activity.
Cordyceps funguss are a kind of ascomycetess, have Conidial Stage (phorozoon) and ascospore in its life cycle
Stage (epigamouss).And used in the actual productions such as artificial culture, liquid fermentation be the imperfect stage Cordyceps funguss, because
And the identification of Anamorph of Cordyceps Sinensis is extremely important.Chinese scholars Cordyceps Resources investigate, phorozoon confirmation, activity into
Separate analysis and the mechanism of action, development and application aspect are done a lot of work.Cordyceps China pilose spore has proved to be the winter
The worm summer grass phorozoon existence form, with natural cordyceps identical active component and drug effect.
Natural cses have strict parasiticss and special ecological environment, therefore its yield is very low, and price is high.It is wild
Cordyceps are due to by the restriction of the factors such as growing environment, scarcity of resources.It is wild due to making little progress on artificial culture in recent years
The research of Cordyceps succedaneum focuses mostly on liquid fermentation.Using liquid submerged fermentation culture Cordyceps mycelium, extract
Thing or fermentation liquid, are a kind of effective ways for solving Cordyceps medicine source.Chinese caterpillar fungus fermentation produces Cordycepses succedaneum, both can effectively protect
Shield Cordycepses this precious resources, and the not strict restriction of climate, geographical environment and Cordycepses parasitic conditions, are suitable for industrialization big
Large-scale production.Its composition of the succedaneum produced such as mycelium and drug effect are also similar to natural cses, thus cause always both at home and abroad
Power is in the fermentation culture of Cordyceps mycelium.The mycelia that aweto cultured by artificial fermentation China pilose spore is obtained, Jing toxicitys, medicine
Reason, plant research, it was demonstrated that basically identical with natural cses chemical composition, pharmacological action, can replace natural cses to produce Cordycepses system
Product, to make up the shortage of natural resourcess, have bright by the amount of the optimization to fermentation condition, mycelial biomass and metabolite
It is aobvious to improve.
In recent years, developing rapidly with natural product chemistry and modern chromatographic techniques, in researching and developing to worm grass product
Progressively deeper functional metabolic Study on product is turned to by Cordycepses raw material or directly utilizing for crude extract.Both at home and abroad to worm
Careless metabolite has done substantial amounts of research, and metabolite mainly includes a few big class compounds such as nucleoside, polysaccharide, polypeptide, sterol, its
The representative functional metabolic product such as middle purines nucleoside, Cordyceps polysaccharide, Mannitol is in biosynthesiss, pharmacological action etc.
The research of aspect wins initial success.
Unsaturated fatty acid refers to the fatty acid containing one or more double bonds in molecule, the more saturated fatty acid of its fusing point
It is low.Unsaturated fatty acid be constitute body fat a kind of fatty acid, fatty acid needed by human, unsaturated fatty acid according to pair
The difference of key number, is divided into two kinds of monounsaturated fatty acid and polyunsaturated fatty acid.Polyunsaturated fatty acid
There are more effects, it can drop for (Polyunsaturated Fatty Acids, PUFAs) relative saturation fatty acid
Low blood cholesterol and triglyceride, adjust cardiac function, reduce blood viscosity, improve blood microcirculation, improve brain cell
Activity, memory reinforcing and ability of thinking strengthen function of human defensive system etc., and in addition it can also exclude many in human body
Remaining " rubbish ", that is, due to taking the photograph the superabundant fats that the excessive satisfied fatty acid of people is formed, so as to reach the purpose of fat-reducing.
Therefore, its potential medical medical value receives the extensive concern in the world, causes food, the medicine even industry such as cosmetics
Great attention.
Yung-Sheng in 1999 draw cloned Mortierella alpina (Mortiere Uaalpina) △ 6 and △ 12 it is fatty
Dehydrogenase gene is simultaneously expressed in saccharomyces cerevisiae.2004, Dyer et al. proceeded to the desaturases of △ 3 in tung oil tree
Yeast, successfully obtains the linolenic yeast of product.Maali-Amiri in 2007 et al. is by algae green grass or young crops bacterium
(Cyanobacterium) the desaturases of △ 12 proceed to Rhizoma Solani tuber osi, successfully be detected Rhizoma Solani tuber osi fatty acid composition and there occurs substantially
Change.2008, Hao et al. by roll up branch Mucor the desaturases of △ 6 turn in people's transgene tobacco, have successfully been obtained high yield γ-
Linolenic bacterial strain.Additionally, the related gene of also many fatty acid desaturases is cloned and Transformation Application.Due to major part
Desaturase is embrane-associated protein, and it isolates and purifies very difficult, and the desaturase that isolated and purified and identified cans be counted on one's fingers,
And most of research is carried out around delta 8 desaturase genes and its expression regulation.
At present, the unsaturated fatty acid applied produces bacterium based on bacillus subtilises, and as important anabolism
The Cordyceps funguss of unsaturated fatty acid, also only rest in metabolite component analyses and the research of effect, to Cordyceps
The research of related gene and albumen is also rarely found in bacterium unsaturated fatty acid metabolic pathway of synthesizing.
(3) content of the invention
Present invention aim at being directed to the not enough and technical issues that need to address with present on, is produced to " hundred make " the bacterium winter
The enzyme and its encoding gene of worm summer grass China pilose spore anabolism acetic acid is furtherd investigate, there is provided " hundred make " produces the bacterium winter
Worm summer grass China pilose spore participate in S-acetyl-coenzyme-A set out the enzyme of anabolism acetyl group-acyl carrier protein, encoding gene and its
Using.
The technical solution used in the present invention is：
A kind of S-acetyl-coenzyme-A that participates in sets out the fat-acyl CoA synthase of anabolism acetyl group-acyl carrier protein,
There is more than 90% homology with sequence shown in SEQ ID No.1 or SEQ ID No.3.Due to the particularity of aminoacid sequence,
Any fragment or its variant containing the peptide protein of aminoacid sequence shown in SEQ ID NO.1 or SEQ ID No.3, as it is guarded
Property variant, bioactive fragment or derivant, as long as the fragment of the peptide protein or peptide protein variant are same with aforementioned amino acid sequences
Source property belongs to the row of the scope of the present invention more than 90%.The specific change may include amino in aminoacid sequence
Acid disappearance, insertion or replace；Wherein, for the conservative of variant sexually revises, the aminoacid replaced has and original acid phase
As structure or chemical property, such as replace isoleucine with leucine, variant also can change with non-conservation, such as use tryptophan
Replace glycine.
Preferably, the fat-acyl CoA synthase aminoacid sequence is as shown in SEQ ID No.1 or SEQ ID No.3
(unsA is designated as respectively1, unsA2Albumen)；The enzyme can be catalyzed S-acetyl-coenzyme-A and acyl group-carrier protein prepare corresponding acetyl group-
Acyl carrier protein, acetyl group-acyl carrier protein generates acetic acid through the effect of hydrolytic enzyme again.
The present invention states fat-acyl CoA synthase and produces bacterium Cordyceps China pilose spore from " hundred make ".
Correspondence acetyl group-acyl carrier protein, acetyl group-acyl carrier protein water are obtained by S-acetyl-coenzyme-A anabolism
Solution obtains the path (R in HS-R is acyl carrier protein Acyl-carrier protein) as follows of acetic acid：
The invention further relates to described fat-Acyl-coenzyme A synthase prepares the application in acetic acid in living things catalysis, specifically
, the application is：Acetyl group-acyl carrier egg is prepared with fat of the present invention-Acyl-coenzyme A synthase catalysis S-acetyl-coenzyme-A
In vain, again Jing hydrolysis obtains acetic acid to acetyl group-acyl carrier protein.
The invention further relates to the encoding gene of above-mentioned fat-Acyl-coenzyme A synthase, i.e. fat-Acyl-coenzyme A synthase bases
Cause, specifically, the encoding gene can be and SEQ ID NO：Polynucleotide shown in 2 or SEQ ID No.4 have more than 90% together
The gene order of source property.Due to the particularity of nucleotide sequence, any SEQ ID NO：Many nucleoside shown in 2 or SEQ ID No.4
The variant of acid, as long as it has more than 90% homology with the polynucleotide, belongs to the row of the scope of the present invention.It is described many
The variant of nucleotide refers to a kind of polynucleotide sequence changed with one or more nucleotide.The variant of this polynucleotide can
So that raw displacement variant or the variant of non-life, including substitution variants, Deletion variants and insert variation.Such as ability
Known to domain, allelic variant is the alternative forms of a polynucleotide, it be probably a polynucleotide replacement, disappearance or
Insertion, but will not be from the function of the peptide protein for substantially changing its coding.
Preferably, nucleotide sequence (is designated as respectively unsA as shown in SEQ ID No.2 or SEQ ID No.41, unsA2Base
Cause；unsA1Gene code unsA1Albumen, unsA2Gene code unsA2Albumen).
Described gene can be used to building can living things catalysis S-acetyl-coenzyme-A prepare the base of acetyl group-acyl carrier protein
Because of engineering bacteria, to expand the yield of acetic acid or derivatives thereof.
The present invention is characterized by there is provided SEQ ID NO：Aminoacid sequence and SEQ ID NO shown in 1 or 3：2 or 4
Shown nucleotide sequence, in the case of the known aminoacid sequence and nucleotide sequence, the aminoacid sequence and nucleotide
The acquisition of sequence, and the acquisition of relevant carriers, host cell, are to those skilled in the art obvious.
The beneficial effects are mainly as follows：The present invention is carried from principle to S-acetyl-coenzyme-A synthesis of acetyl base-acyl group
Protein metabolism approach is studied in detail, there is provided " hundred make " production bacterium Cordyceps China pilose spore participates in acetylcoenzyme
A sets out the enzyme and its encoding gene of anabolism acetyl group-acyl carrier protein, nucleotide sequence provided by the present invention gram
Grand DNA can be used to be proceeded in engineering bacteria by the method for transduction, conversion, Conjugative tiansfer, by adjusting acetyl group-acyl carrier
The expression of protein biology synthetic gene, give host's fat-acyl CoA synthase high expressivity, be expand acetic acid or its spread out
Biological yield provides effective way, with major application prospect.
(4) illustrate
Fig. 1 is the denaturing formaldehyde gel electrophoretogram that " hundred make " produces bacterium Cordyceps China pilose spore total serum IgE；
Fig. 2 is Fatty acid biosynthesis metabolism approach annotated map；
Fig. 3 is fatty acid metabolism approach annotated map；
Fig. 4 is unsaturated fatty acid metabolic pathway of synthesizing annotated map；
Fig. 5 is fat-Acyl-coenzyme A synthase gene pcr amplification product gel electrophoresis figures；
Fig. 6 is cloning vehicle pMD18-T Vector and expression vector pET-28a physical maps；
Fig. 7 is restructuring cloned plasmids pMD18-T/uns A physical maps；
Fig. 8 is recombinant expression plasmid pET-28a/uns A building process schematic diagrams；
Fig. 9 is recombinant expression plasmid pET-28a/uns A physical maps；
Figure 10 is the SDS-PAGE figures of fat-Acyl-coenzyme A synthase proteins.
(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：
Embodiment 1：" hundred make " produces the culture of bacterium Cordyceps China pilose spore
Bacterium source：Gathering natural cordyceps from Qinghai first, and bring it back into Hangzhou carries out separation screening, obtains
L0106 bacterial strains, and the Jing strain identification bacterial strain is China pilose spore (Hirsutella Sinensis), the culture presevation is in
State's Type Tissue Collection, deposit number is CCTCC No：M 2011278, the patent previously applied
Disclose in CN102373190A.
The strain is inoculated in into inclined-plane, (this is the liquid formulations before solidification to culture medium prescription, good by following proportions
Bevel again afterwards) be：Glucose 2.0% (w/v contains 1g, similarly hereinafter in 1% expression 100mL culture medium), Semen Maydis powder
1.0%th, murphy juice 0.5%, dextrin 0.5%, yeast powder 0.5%, wheat bran 1.0%, dried silkworm chrysalis meal 2.0%, peptone 1.0%, sulfur
Sour magnesium 0.05%, potassium dihydrogen phosphate 0.05%, agar powder 1.0%, balance of water；Cultivate 25 days at 12～16 DEG C；Then by bacterium
Plant and be inoculated in fermentation medium, culture medium prescription is glucose 1.0%, molasses 1.0%, dried silkworm chrysalis meal 0.5%, soybean cake powder
1.0%th, yeast extract 0.5%, magnesium sulfate 0.01%, potassium dihydrogen phosphate 0.02%, balance of water；It is placed on shaking table, temperature 12～
16 DEG C are cultivated 25 days, are cultivated after terminating aseptically, carry out solid-liquid separation, and solid is placed in into sterilized equipment, standby.
Embodiment 2：" hundred make " produces the extraction of bacterium Cordyceps China pilose spore total serum IgE
Total serum IgE is extracted with TRIzol reagents, step is specially：
1) liquid nitrogen grinding：Take the new fresh thallis of 1g to be put in mortar, add liquid nitrogen to be fully ground to powder repeatedly, be dispensed into
In the 1.5mL centrifuge tubes of pre-cooling, 1mL TRIzol reagents are added, mixed, 5min is stood on ice, make nucleic acid-protein compound complete
Separate.
2) RNA is separated：0.2mL chloroforms are added, firmly concussion mixes 15s, 2～3min, 4 DEG C, 12000rpm are stood on ice
Centrifugation 15min, layering takes upper strata aqueous phase, about 600 μ L.
3) RNA precipitate：500 μ L isopropanols are added, 10min, 4 DEG C, 12000rpm centrifugation 10min are stood on ice, abandoned
Clearly.
4) RNA washings：1mL 75% (v/v) ethanol is added, precipitation is hanged, 10min, 4 DEG C, 7500rpm are stood on ice
Centrifugation 15min；Repeat washing step above, then wash one time.
5) RNA is dissolved：Centrifuge tube to be placed in open wide on ice and is dried 5～10min, plus appropriate DEPC water dissolutioies.
Embodiment 3：The RNA sample sequencing of " hundred make " production bacterium Cordyceps China pilose spore
After extracting sample total serum IgE, with the enrichment with magnetic bead mRNA with Oligo (dT).Add fragmentation
MRNA is broken into short-movie section (200～700bp) by buffer, with mRNA as template, with hexabasic base random primer (random
Hexamers first cDNA chain) is synthesized, then synthesis Article 2 cDNA chain, then through QiaQuick PCR kits purification simultaneously
Plus end reparation is done after EB buffer solution elutions, is added polyA and is connected sequence measuring joints, then carry out piece with agarose gel electrophoresiies
Duan great little is selected, and finally enters performing PCR amplification, and the sequencing library built up is sequenced with Illumina GA IIx.What sequencing was obtained
Raw image data Jing base calling are converted into sequence data, i.e. raw data or raw reads.Remove primitive sequencer
It is standby with subsequent analysis containing only the reads of adaptor sequences in reads.
Embodiment 4：The short reading sequence assemblings of " hundred make " production bacterium Cordyceps China pilose spore RNA
Using short reads composite softwares SOAPdenovo (Li, Zhu et al.De novo assembly of human
genomes with massively parallel short read sequencing[J].Genome Res,2010,20:
265-272.) do transcript profile and from the beginning assemble.Reads with certain length overlap is linked to be longer by SOAPdenovo first
The Contig fragments without N.Then reads is compared into back Contig, is determined from same by paired-end reads
The distance between different Contig of transcript and these Contig, SOAPdenovo connect together these Contig, in
Between unknown nucleotide sequence represented with N, thus obtain Scaffold.Scaffold is mended further with paired-end reads
Hole is processed, and finally obtains, Unigene sequence that two ends can not again extend minimum containing N.Finally, by Unigene sequences and albumen number
Blastx is according to storehouse nr, Swiss-Prot, KEGG and COG compare (evalue<0.00001) the best albumen of comparison result, is taken
Determine the sequence direction of Unigene.If the comparison result between different storehouses is contradictory, by nr, Swiss-Prot, KEGG and
The priority of COG determines the sequence direction of Unigene, with Unigene software ESTScan of four storehouses of the above all to being less than
(Iseli,Jongeneel et al.ESTScan:a program for detecting,evaluating,and
reconstructing potential coding regions in EST sequences[J].In Proceedings of
9th InternationalConference on Intelligent Systems for Molecular
Biology.AAAIPress, Menlo Park, CA, pp.1999,138-148.) predict its coding region and determine the side of sequence
To.For the sequence that the Unigene that can determine that sequence direction provides its direction from 5' to 3', for sequence direction cannot be determined
Unigene provides the sequence that composite software is obtained.
Embodiment 5：" hundred make " produces bacterium Cordyceps China pilose spore Unigene functional annotations
Functional annotation information provides protein function annotation, Pathway annotations, COG functional annotations and the Gene of Unigene
Ontology (GO) functional annotation.First, by blastx by Unigene sequence alignments to albumen database nr, Swiss-
Prot, KEGG and COG (evalue<0.00001) albumen with giving Unigene with highest serial similarity, is obtained, so as to
Obtain the protein function annotation information of the Unigene.The Pathway of Unigene can further be obtained according to KEGG annotation informations
Annotation.Unigene and COG data bases are compared, the possible functions of prediction Unigene simultaneously do function classified statistic to it.
According to nr annotation informations, using Blast2GO softwares (Conesa, Gotz et al.Blast2GO:a universal tool
for annotation,visualization and analysis in functional genomics research[J]
.Bioinformatics,2005,21(18):3674-3676.) obtain the GO annotation informations of Unigene.Obtain each
After the GO annotations of Unigene, with WEGO softwares (Ye, Fang et al.WEGO:a web tool for plotting GO
annotations[J].Nucleic Acids Research,2006,34:293-297.) GO functions are done to all Unigene
Classified statistic, from the gene function distribution characteristicss for macroscopically recognizing the species.
Embodiment 6：" hundred make " produces bacterium Cordyceps China pilose spore acetate metabolism path analysis
Fig. 2 is the Fatty acid biosynthesis metabolism (map00061) in KEGG metabolic pathways annotation, and Fig. 3 is KEGG metabolic pathways note
Fatty acid metabolism (map00071) in releasing, Fig. 4 is the unsaturated fatty acid anabolism in KEGG metabolic pathways annotation
(map01040) enzyme for, having annotated is " hundred make " production bacterium Cordyceps China pilose spore acetate metabolism approach phase having detected that
Close enzyme, it can be seen that detect from S-acetyl-coenzyme-A synthesize correspondence acetyl-acyl carrier protein fat-
2 Unigene of Acyl-coenzyme A synthase.By the ORF Finder software on-line checkings in NCBI, this gene is have found
Open reading frame (SEQ ID No.2, SEQ ID No.4) has simultaneously obtained corresponding protein sequence (SEQ ID No.1, SEQ
ID No.3)。
Embodiment 7：" hundred make " produces bacterium Cordyceps China pilose spore fat-Acyl-coenzyme A synthase gene design of primers
The each gene open proofreading dna sequential design obtained according to prediction with GENE RUNNER primer-design softwares
Primer, for cloning fat-Acyl-coenzyme A synthase genes that " hundred make " produces bacterium China pilose spore anabolism acetic acid, primer
By Shanghai, Sheng Gong biological engineering company limited synthesizes, and primer sequence is listed below：
unsA1Gene：The ACGGAATTCATGGCCATGACTTTCGAGACAG of forward primer 5 ' 3 '
The AGCAAGCTTTCACAGGAATGACCGGAAAGG of reverse primer 5 ' 3 '
unsA1Mrna length is 777bp.
unsA2Gene：The ATAGGATCCATGTACGGCACGGGAACCGG of forward primer 5 ' 3 '
The ATAAAGCTTCATCGTCTGGTCAAGGCCTGTG of reverse primer 5 ' 3 '
unsA2Mrna length is 1254bp.
Embodiment 8：" hundred make " produces the preparation of bacterium Cordyceps China pilose spore the first chains of cDNA
The method for first providing according to embodiment 1 is turned out after sutella sinensis fermented mycelium, is carried according still further to embodiment 2
For method the extraction of total serum IgE is carried out to China pilose spore, obtain carrying out " hundred make " production bacterium Cordyceps by following after total serum IgE
The synthesis of the chains of China pilose spore cDNA first, tests for follow-up each gene cloning.
Using PrimeScript 1st Strand cDNA Synthesis Kit test kits (TaKaRa) from Total
The reverse transcription synthesis chains of cDNA first in RNA, experimental procedure is as follows：
1) following mixed liquor is prepared in Microtube pipes.
2) degeneration, annealing operation are conducive to the degeneration of template ribonucleic acid and the specificity annealing of reverse transcription primer and template, can
Reverse transcription reaction efficiency is improved, so carrying out degeneration, annealing reaction in PCR instrument, condition setting is as follows：
65 DEG C, 5min
3) the centrifugation several seconds makes the mixed liquor of template ribonucleic acid/primer etc. be gathered in Microtube bottom of the tube after annealing terminates.
4) following inverse transcription reaction liquid is prepared in above-mentioned Microtube pipes.
5) reverse transcription reaction is carried out by following condition in PCR instrument.
42 DEG C of 15～30min
70℃ 15min
Ordinary circumstance, has a PolyA structure in the end of eukaryote mRNA 3 ', and the quantity of A bases is ten to hundreds of
It is individual, Oligo (dT) primer can be utilized using this structure, in the presence of reverse transcription, with mRNA as templated synthesis
The chains of cDNA first, the present invention adopts sequence (the PrimeScript 1st Strand in the dT regions developed alone by TaKaRa
There is provided in cDNA Synthesis Kit) it is primer, if the mRNA integrity for obtaining is preferable, then can by process of reverse-transcription
The chains of cDNA first of all pheron encoding genes in obtain species.
Embodiment 9：" hundred make " produces bacterium Cordyceps China pilose spore anabolism acetic acid functional gene fat-acyl group-
The clone of CoA synthase unsA genes, expression and the detection of protein vigor
1st, fat-Acyl-coenzyme A synthase unsA1And unsA2The PCR amplifications of gene
The chains of cDNA first obtained with embodiment 8 as template, with embodiment 7 synthesize unsA1Gene primer：5’
The ACG GAA TTC ATG GCC ATG ACT TTC GAG AC AGC AAG CTT TCACAG GAA TGA CCG of AG3 ' and 5 '
GAA AGG3 ', unsA2Gene primer：5 ' the ATA GGA TCC ATG TAC GGCACG GGA ACC ATA AAG of GG3 ' and 5 '
CTT CAT CGT CTG GTC AAG GCC TGT G 3 ' carry out Pfu archaeal dna polymerase pcr amplification reactions, and condition setting is such as
Under：
Pfu pcr amplification reaction systems：
Pfu DNA Ploymerase PCR amplification conditions：
2nd, fat-Acyl-coenzyme A synthase unsA1And unsA2Gene PCR product detected through gel electrophoresis
Specifically detection method is：
1) it is uniformly dissolved it prepare 0.9% agarose gel microwave-oven-heating；
2) 15mL gels are taken, when gel is cooled to 50 DEG C or so, 1 μ L dyeing liquor Gold view is added, after mix homogeneously
Pour on treatments of Electrophoretic Slab Gels, remove and insert after bubble point sample comb；
3) it is careful to take out point sample comb after gel sets, offset plate is put in electrophoresis tank (loading wells one end is near electrophoresis tank
Negative pole), TAE electrophoretic buffers are added in electrophoresis tank；
4) 5 μ L samples are taken and is subsequently adding 6 × Loading Buffer 1.5 μ L and ddH2With on liquid-transfering gun after the μ L of O 4 mixing
Sample, applied sample amount is 10 μ L；
5) power line between electrophoresis tank and electrophresis apparatuses is connected, just extremely red, negative pole is black；
6) power-on, starts electrophoresis, and ceiling voltage is less than 5V/cm；
7) electrophoresis can be terminated when sample ran the 2/3 of offset plate；
8) after cutting off the electricity supply, gel is taken out to be put in gel imaging instrument and is observed, is taken pictures.
Transcript profile sequencing prediction fat-Acyl-coenzyme A synthase unsA1The size of gene is 777bp, unsA2Gene it is big
It is little for 1254bp, agarose gel electrophoresiies result shows that Successful amplification has gone out fat-Acyl-coenzyme A synthase unsA1Gene,
Size is about 770bp, unsA2Gene, size is about 1250bp.Fig. 5 is that " hundred make " produces bacterium China pilose spore anabolism second
Acid function gene PCR product gel electrophoresis figure.
3rd, fat-Acyl-coenzyme A synthase unsA1And unsA2Base A that adds of gene PCR product is processed and purification
Due to Pfu archaeal dna polymerase PCR primers end be flush end, so also need to carry out after glue reclaim plus the process of base A,
Just can be used for carrier T connection after purification.Glue reclaim product adds base A system as follows：
72 DEG C plus A bases 20min, are finally purified with AxyPrep PCR cleaning agents boxes in PCR instrument.
4th, fat-Acyl-coenzyme A synthase unsA1And unsA2The connection of gene and cloning vehicle
Cloning vehicle pMD18-T Vector are purchased from TaKaRa companies (TaKaRa code D101A), its physics
Collection of illustrative plates is shown in Fig. 6, by fat-Acyl-coenzyme A synthase unsA1, unsA2Gene is connected structure restructuring matter with cloning vehicle
Grain pMD18-T/unsA1, pMD18-T/unsA2, physical map is shown in Fig. 7, and linked system and condition of contact are as follows.
Linked system：
Condition of contact：16 DEG C, 16h；Inactivation：65 DEG C, 15min.
5th, fat-Acyl-coenzyme A synthase recombiant plasmid pMD18-T/unsA1And pMD18-T/unsA2Conversion
By recombiant plasmid pMD18-T/unsA1And pMD18-T/unsA2Structure in E. coli JM109 is proceeded to respectively
Build the recombinant bacterium E.coli JM109/pMD18-T/unsA of carrying fat-Acyl-coenzyme A synthase unsA genes1And E.coli
JM109/pMD18-T/unsA2, concretely comprise the following steps：1) 10 μ L reaction systems are gone in competent cell E.coli JM109,
Ice bath 30min；2) thermal shock：42 DEG C, 90s；3) ice bath：2-3min；4) 800 μ L liquid LB of addition, 37 DEG C, 250rpm, 1h；5) apply
Cloth flat board (resistance containing Amp)；6) 37 DEG C of incubator overnight incubations.
6th, fat-Acyl-coenzyme A synthase E.coli JM109/pMD18-T/unsA1With E.coli JM109/pMD18-
T/unsA2The screening of positive restructuring bacterium
Bacterium colony PCR can extract genomic DNA, and directly enter performing PCR as template with exposed DNA after thalline pyrolysis and expand
Increase, whether the method is easy to operate, quick, can be the positive bacterium colony containing purposeful plasmid with Rapid identification bacterium colony, in conversion mirror
It is relatively conventional in fixed.In experiment, will be inoculated into corresponding single bacterium colony in fluid medium carries out bacterium colony PCR, to verify whether to turn
Enter genes of interest.First, added in the 1.5mL centrifuge tubes containing 50 μ L sterilized water with toothpick picking single bacterium colony, boiling water bath 30min,
It is then centrifuged for using supernatant as template, enters performing PCR amplification, PCR program settings is that Taq enzyme expands general procedure.Finally adopt
0.9% agarose gel electrophoresiies detection bacterium colony PCR primer.
7th, fat-Acyl-coenzyme A synthase recombiant plasmid pMD18-T/unsA1And pMD18-T/unsA2Sequencing
The positive restructuring bacteria liquid LB culture medium culturings that bacterium colony PCR is detected overnight after, take 4mL bacterium solutions extract plasmid,
Method presses the operating instruction of AxyPrep plasmid DNA small volume of reagent box offer.Sequencing is complete by Shanghai Sani bio tech ltd
Into.Jing sequence verifications, sequence SEQ ID No.2 and SEQ ID No.4 have recombinated respectively to pMD18-T/unsA1And pMD18-T/
unsA2In.
8th, fat-Acyl-coenzyme A synthase recombinant expression plasmid pET-28a/unsA1And pET-28a/unsA2Structure
Test the principle in expression in escherichia coli according to exogenous gene, and expression vector pET-28a and fat-acyl
Base-CoA synthase unsA genes restriction enzyme site compares situation, it is determined that unsA1With the double enzyme sites of I/Hind of EcoR III, unsA2
With the double enzyme sites of I/Hind of BamH III, and to the E. coli JM109/pMD18-T/unsA that recombinates1And E.coli
JM109/pMD18-T/unsA2Carry out liquid LB test tube shaker cultures, recombiant plasmid to extract.
The recombiant plasmid pMD18-T/unsA of fat-Acyl-coenzyme A synthase unsA genes1And pMD18-T/unsA2And table
Processed in 37 DEG C of difference enzyme action with I/Hind of EcoR III and the restricted enzyme of I/Hind of BamH III respectively up to carrier pET-28a
6h, enzyme action system is as follows：
The double digestion systems of EcoR I (BamH I)/Hind III：
Enzyme action terminate after 65 DEG C of inactivation 15min, then reclaimed with Axygen DNA gel QIAquick Gel Extraction Kits respectively, pure
Change.
Fat-Acyl-coenzyme A synthase unsA genes and expression vector pET-28a Jing double digestions, connected with T4 again after purification
16 DEG C of connections of enzyme overnight, build recombinant expression plasmid pET-28a/unsA, and its building process is shown in Fig. 8, builds the restructuring table for obtaining
Up to plasmid pET-28a/unsA1, pET-28a/unsA2Collection of illustrative plates is shown in Fig. 9.Linked system composition is as follows：
Linked system：
9th, the conversion and the screening of positive monoclonal of fat-Acyl-coenzyme A synthase recombinant expression plasmids
By in the heat-shock transformed BL21 Host Strains to E.coli of the expression plasmid for building, being then applied to containing card, that is mould
On the LB agar plates of plain (Kan) resistance, 37 DEG C of overnight incubations.The random choose single bacterium colony from flat board, with each functional gene
Primer enters performing PCR amplification, selects positive colony.
10th, the abduction delivering of fat-Acyl-coenzyme A synthase recombinant bacteriums
The monoclonal for being accredited as the positive is inoculated in the LB fluid mediums that 5mL contains Kan resistances, 37 DEG C, 250r/
Min overnight incubations.1mL cultures are taken, transferred 37 DEG C, 250r/ in the LB fluid mediums that 50mL contains Kan resistances
Min is cultivated to cell concentration OD600 and is about 0.6～0.8 or so.Certain density IPTG inductions training is separately added into in culture
Foster 8h.Collects thalline is for electrophoretic analysiss and Enzyme activity assay.
11st, fat-Acyl-coenzyme A synthase recombinant bacterium expression products SDS-PAGE analyses
To proceed to the E.coli BL21 bacterium of empty carrier and not add the recombinant bacterium of derivant IPTG as control.It is accredited as
Positive recombinant bacterium takes 0.5mL Induced cultures Jing after IPTG inducing culture 8h, and thalline is collected by centrifugation, and is resuspended in 50 μ L distillations
In water, 50 μ L sample-loading buffers are added, 10min is boiled after mixing, carry out SDS-PAGE electrophoretic analysiss, " A " swimming lane in Figure 10
As recombinant bacterium E.coli BL21/pET-28a/unsA1The fat of expression-Acyl-coenzyme A synthase unsA1(Jing sequence verifications its
Aminoacid sequence is as shown in SEQ ID No.1) SDS-PAGE figures, " B " swimming lane is recombinant bacterium E.coli BL21/pET-
28a/unsA2The fat of expression-Acyl-coenzyme A synthase unsA2(its aminoacid sequence of Jing sequence verifications such as SEQ ID No.3 institutes
Show) SDS-PAGE figure.
12nd, the protein active detection of fat-Acyl-coenzyme A synthase recombinant bacteriums
(1) fat-Acyl-coenzyme A synthase unsA1Protein active detection
It is prepared by enzyme liquid：Weigh the recombinant bacterium E.coli BL21/pET-28a/unsA of collection10.5g phosphate buffers
(50mM, pH8.0) 15mL suspends, ultrasonication (power 350W, broken 2s, altogether interval 2s, ultrasonication 5min).
Fat-Acyl-coenzyme A synthase unsA1Transformation system：E.coli BL21/pET- are added in 50mL conversion bottles
28a/unsA1Each 10mL, 0.1g S-acetyl-coenzyme-A of ultrasonication thalline, 0.1g acyl groups-carrier protein, 30 DEG C, 150r/min turn
Change, after conversion terminates, centrifuging and taking supernatant is standby with subsequent detection.
Detection method：GC conditions：30m × 0.32mm × 0.25mm fused-silica capillary columns；At the beginning of post initial temperature post
190 DEG C of temperature, is incubated 1min, is warming up to 230 DEG C with 6 DEG C/min, then constant temperature；250 DEG C of room temperature of gasification；Carrier gas is high-purity He
(99.999%)；62.6KPa is pressed before post；Flow rate of carrier gas 1.4mL/min；The μ L of sample size 1；Split ratio 60:1.Mass Spectrometry Conditions：Ion
Source is EI sources；230 DEG C of ion source temperature；150 DEG C of quadrupole rod temperature；Electron energy 70eV；260 DEG C of interface temperature；Solvent delay
2min；Mass range 10-550u.
Through above-mentioned chromatographic condition detection and calculating, draw to draw a conclusion：Fat-Acyl-coenzyme A synthase recombinant bacterium institute
The fat of expression-Acyl-coenzyme A synthase unsA1Maximum specific enzyme activity (Specific Activity) be 4mol/min/mg, bottom
Thing conversion ratio is 82.53%.
(2) fat-Acyl-coenzyme A synthase unsA2Protein active detection
It is prepared by enzyme liquid：Weigh the recombinant bacterium E.coli BL21/pET-28a/unsA of collection20.5g phosphate buffers
(50mM, pH8.0) 15mL suspends, ultrasonication (power 350W, broken 2s, altogether interval 2s, ultrasonication 5min).
Fat-Acyl-coenzyme unsA2Synthase unsA transformation systems：E.coli BL21/ are added in 50mL conversion bottles
pET-28a/unsA2Each 10mL, 0.1g S-acetyl-coenzyme-A of ultrasonication thalline, 0.1g acyl groups-carrier protein, 30 DEG C, 150r/min
Conversion, after conversion terminates, centrifuging and taking supernatant is standby with subsequent detection.
Detection method：GC conditions：30m × 0.32mm × 0.25mm fused-silica capillary columns；At the beginning of post initial temperature post
190 DEG C of temperature, is incubated 1min, is warming up to 230 DEG C with 6 DEG C/min, then constant temperature；250 DEG C of room temperature of gasification；Carrier gas is high-purity He
(99.999%)；62.6KPa is pressed before post；Flow rate of carrier gas 1.4mL/min；The μ L of sample size 1；Split ratio 60:1.Mass Spectrometry Conditions：Ion
Source is EI sources；230 DEG C of ion source temperature；150 DEG C of quadrupole rod temperature；Electron energy 70eV；260 DEG C of interface temperature；Solvent delay
2min；Mass range 10-550u.
Through above-mentioned chromatographic condition detection and calculating, draw to draw a conclusion：Fat-Acyl-coenzyme A synthase recombinant bacterium institute
The maximum specific enzyme activity (Specific Activity) of the fat of expression-Acyl-coenzyme A synthase unsA2 is 4.5mol/min/mg,
Substrate conversion efficiency is 84.73%.
Claims (5)
1. it is a kind of participate in S-acetyl-coenzyme-A anabolism acetyl group-acyl carrier protein fat-acyl CoA synthase, its feature
It is the aminoacid sequence of the fat-acyl CoA synthase as shown in SEQ ID No.3.
2. fat-acyl CoA synthase as claimed in claim 1 prepares the application in acetic acid in living things catalysis.
3. the gene of fat-acyl CoA synthase described in claim 1 is encoded.
4. gene as claimed in claim 3, it is characterised in that the nucleotide sequence of the gene is as shown in SEQ ID No.4.
5. gene as claimed in claim 3 build can living things catalysis S-acetyl-coenzyme-A prepare acetyl group-acyl carrier protein
Genetic engineering bacterium in application.
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| CN101182525A (en) * | 2007-11-08 | 2008-05-21 | 南昌大学 | Citrinin biological synthesis gene cluster |
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| CN101182525A (en) * | 2007-11-08 | 2008-05-21 | 南昌大学 | Citrinin biological synthesis gene cluster |
Non-Patent Citations (3)
| Title |
|---|
| Acetate Activation in Methanosaeta thermophila：Characterization of the Key Enzymes Pyrophosphatase and Acetyl-CoA Synthetase;Stefanie Berger,et al;《Archaea》;20120815;第2012卷;1－10 * |
| Purification and characterization of the acetyl-CoA synthetase from Mycobacterium tuberculosis;Ru Li，et al;《Acta Biochim Biophys Sin》;20110906;第43卷;891–899 * |
| Yeast acyl-CoA synthetases at the crossroads of fatty acid metabolism and regulation;Paul N. Black, Concetta C. DiRusso;《Biochimica et Biophysica Acta》;20060516;286－298 * |
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