CN101440357A - Gene engineering strain of accumulating validoxylamine A and construction method thereof - Google Patents

Abstract

The invention discloses a genetic engineering strain for accumulating validoxylamine A in the technical field of biological medicine and a consturction method thereof. The engineering strain is to introduce genetic mutation on a chromosome, so as to cause a glycosyl transferase gene valG in a biosynthesis way of jinggangmycin to produce the mutation. The construction method for the strain comprises the following concrete steps: designing and constructing a homologous recombinant plasmid vector pJTU609 used for mutating the glycosyl transferase gene valG; constructing the plasmid vector pJTU609 into a receptor, namely sreptomyces 5008 cells for homologous recombination through conjugal transfer; and finally screening an LL-1 mutant strain through the screening of mutant strains and the replica screening of verifying the resistance of thiostrepton first and the difference of fragment size of a PCR product then. The yield of the genetic engineering strain for accumulating a jinggangmycin precursor-the validoxylamine A is 5 times of that of a wild strain. The validoxylamine A with high purity can be produced on a large scale through the engineering strain.

Description

The engineering strain of accumulating validoxylamine A and construction process thereof

Technical field

The present invention relates to the engineering strain and the construction process thereof in a kind of biological medicine technology field, specifically is a kind of engineering strain and construction process thereof of accumulating validoxylamine A.

Background technology

Microbiotic is produced by microorganism, plant and animal etc., and microbiotic plays a role by one or more physiological metabolism processes of disturbing target cell.Jingganmycin (jinggangmycin) is a kind of agricultural antibiotic that is produced by streptomyces hygroscopicus well ridge mutation 5008 (Streptomyces hygroscopicus var.jinggangensis 5008), and it has consistent chemical structure with the validamycin (validamycin) that is produced by streptomyces hygroscopicus lemon mutation (Streptomyces hygroscopicus var.limoneus).Rice sheath blight disease is the regular incidence frequently-occurring disease of paddy rice, it is the major obstacle of China's paddy rice high and stable yields, jingganmycin then is mainly used in the control of rice sheath blight disease (Rhizoctonia solani), it has efficiently, economy, toxicity are low, with characteristics such as Environmental compatibility is good, be first agricultural antibiotic of China's independent development and scale operation, become one of biological pesticide of output maximum at present.The main effective constituent of jingganmycin is jinggangmycin A, and it is synthetic by glucosyl transferase catalysis by the mould ylidene amines A in well ridge (validoxylamine A) and β-D-glucose.The mould ylidene amines A in well ridge is a kind of weakly alkaline polyol, and main group has a secondary amino group (NH-), two methylol (CH ₂OH) and six hydroxyls, molecular formula is C ₁₄H ₂₅NO ₈The chemical structure of mould ylidene amines A in well ridge and trehalose is quite similar, and trehalase is had had strong inhibitory effects.Trehalase extensively is present in insect and the invertebrates, is playing an important role aspect the transhipment of glucose and the energy storage.Studies show that the mould ylidene amines A of Jiang Jinggang injects in the insect body, can block the hydrolysis of trehalose, suppresses the transhipment of glucose in the insect body, causes insect to lose flight performance.The mould ylidene amines A in well ridge can be used for controlling some harmful insects and fungi growth, and therefore, the mould ylidene amines A in well ridge can be developed to and be biotic pesticide.Glycosyltransferase gene valG is responsible for mould ylidene amines A in well ridge and the synthetic jinggangmycin A of β-D-glucose, if after this transgenation, the mould ylidene amines A in precursor substance well ridge of jinggangmycin A component is accumulated in a large number, thereby obtain a well ridge mould ylidene amines A high productive mutant, the mould ylidene amines A in development and utilization well ridge is become a reality.

Summary of the invention

The objective of the invention is to overcome deficiency of the prior art, a kind of engineering strain and construction process thereof of accumulating validoxylamine A is provided, make the mould ylidene amines A in development and utilization well ridge become a reality.

The present invention is achieved by the following technical solutions.

The present invention relates to a kind of engineering strain of accumulating validoxylamine A, this project bacterial strain is to produce on bacterium streptomyces hygroscopicus 5008 (the Streptomyces hygroscopicus var.jinggangensis5008) karyomit(e) at jingganmycin to introduce transgenation, make valG gene inside that replacement mutation take place, thereby cause the glycosyltransferase gene valG in the jingganmycin biosynthetic pathway to lose catalytic activity, the mould ylidene amines A in precursor substance well ridge of jinggangmycin A component is accumulated in a large number, thereby obtain the engineering strain LL-1 of an accumulating validoxylamine A.

Described replacement mutation is specially: 806 bases of glycosyltransferase gene valG are replaced with the apramycin resistant gene.

The donor of described apramycin resistant gene is plasmid pSET152.

806 bases of described valG gene are specially:

tcgaggactt?cgaggtcgtg?gtgtccgacg?atggctcgac?agacaccacc?cgcgacgtag 60

tgcggtccta?cgaggaccgg?ctgcgcatca?agtacgtctt?ccaggaagac?cttggatacc 120

gagtcgccag?cgcgcgcaac?ggtggggcac?gcctggcctc?cgcgccactc?ctggccttcc 180

tcgacactgg?tgtcctggcg?gggccgcagt?acgtccagtc?ggtccttgcc?gcccatgccg 240

ggccggcgcc?cgccaaggtg?gtcctcggct?gctgctacgg?ctacgatccg?cggaatcccc 300

atcccgaact?ccactccctt?gtcgaggagt?tccccccgga?ggaggccgtg?agaagggtcg 360

gggacgcccc?ctggttccag?gacatgcggc?ttccggagtt?cactgcggtc?gacttcgacc 420

taagccgtat?gcacatgccc?tggctgtggt?tctggacgct?caacgtgtcg?ctgccggcag 480

ccgacttctg?gcgggtcggc?gggttcgacg?aggacttcac?cggctggggc?ggggaggaca 540

tcgaactcgg?ctaccggctg?cacgcacacg?gcatccccat?gacggtctca?cgtgagagct 600

gggggatcga?agcgccgcac?gagcgcactc?atgaggcgaa?cgtctcctcc?ctcatgctca 660

attgcgaccg?gttcgtccgc?aagcacccct?ccctgctccc?cgagctgttc?tgggcggtga 720

ccaacagggg?catcttcggc?tccgtcgaga?ctgagcggct?tcgcttcgag?gaatgggcaa 780

gccaggcccg?cgggcaacag?gtcctg 806

Described apramycin resistant gene sequence is specially:

tcatgagctc?agccaatcga?ctggcgagcg?gcatcgcatt?cttcgcatcc?cgccctctgg 60

cggatgcagg?aagatcaacg?gatctcggcc?cagttgaccc?agggctgtcg?ccacaatgtc 120

gcgggagcgg?atcaaccgag?caaaggcatg?accgactgga?ccttccttct?gaaggctctt 180

ctccttgagc?cacctgtccg?ccaaggcaaa?gcgctcacag?cagtggtcat?tctcgagata 240

atcgacgcgt?accaacttgc?catcctgaag?aatggtgcag?tgtctcggca?ccccataggg 300

aacctttgcc?atcaactcgg?caagatgcag?cgtcgtgttg?gcatcgtgtc?ccacgccgag 360

gagaagtacc?tgcccatcga?gttcatggac?acgggcgacc?gggcttgcag?gcgagtgagg 420

tggcaggggc?aatggatcag?agatgatctg?ctctgcctgt?ggccccgctg?ccgcaaaggc 480

aaatggatgg?gcgctgcgct?ttacatttgg?caggcgccag?aatgtgtcag?agacaactcc 540

aaggtccggt?gtaacgggcg?acgtggcagg?atcgaacggc?tcgtcgtcca?gacctgacca 600

cgagggcatg?acgagcgtcc?ctcccggacc?cagcgcagca?cgcagggcct?cgatcagtcc 660

aagtggccca?tcttcgaggg?gccggacgct?acggaaggag?ctgtggacca?gcagcacacc 720

gccgggggta?accccaaggt?tgagaagctg?accgatgagc?tcggcttttc?gccattcgta 780

ttgcac 786

The invention still further relates to a kind of construction process of engineering strain of accumulating validoxylamine A, this method is specially:

Step 1, design and make up are used for homologous recombination plasmid vector that glycosyltransferase gene valG is suddenlyd change;

Step 2 imports to the plasmid vector conjugal transfer of step 1 gained in receptor chain mould 5008 cells and to carry out homologous recombination;

Step 3 is chosen the LL-1 mutant strain earlier by screening mutant strains and checking thiostrepton resistance replica screening, and then by the difference finishing screen of PCR product clip size.

The present invention undergos mutation glycosyltransferase gene valG, thereby obtains corresponding mutant strain by the method for replacement mutation on karyomit(e).The present invention obtains 1 LL-1 engineering bacteria, and the glycosyltransferase gene valG of this bacterial strain undergos mutation, but this project bacterial strain accumulating validoxylamine A.

The present invention has following useful effect: the present invention can accumulate the mould ylidene amines A in jingganmycin precursor substance well ridge, and its output is 5 times of this component output in the wild strain, and the mould ylidene amines A in development and utilization well ridge is become a reality.Can the mould ylidene amines A in the highly purified well of scale operation ridge by this project bacterium, have significant using value.

Bacterial strain streptomyces hygroscopicus 5008 related among the present invention is at document " Bai Linquan, Li Lei, XuHui, Minagawa K, Yu Yi, Zhang Yirong, Zhou Xiufen, Floss H G., Mahmud T, and Deng Zixin.2006.Functional analysis of the validamycin biosyntheticgene cluster and engineered production of validoxylamine A.Chem Biol 13:387-397 " in open.

Intestinal bacteria related among the present invention are at document " Paget, M.S., L.Chamberlin, A.Atrih, S.J.Foster, and M.J.Buttner.1999.Evidence that theextracytoplasmic function sigma factor sigmaE is required for normal cellwall structure in Streptomyces coelicolor A3 (2) .J.Bacteriol.181:204-211 " in open.

The engineering strain of accumulating validoxylamine A of the present invention, its engineering bacteria LL-1 has submitted China Committee for Culture Collection of Microorganisms common micro-organisms center to, and its deposit number is CGMCC NO:2567, and storage life is 30 years of rising 2008.07.02 day.

Description of drawings

Fig. 1 is the secondary relationship synoptic diagram between mutant strain LL-1 and the wild type strain 5008;

Wherein: with 806 bases of glycosyltransferase gene valG in the wild type strain 5008 apramycin resistant gene replacement mutation, obtain mutant strain LL-1 by plasmid pJTU609.

Fig. 2 is the efficient liquid phase chromatographic analysis figure of mutant strain LL-1 and wild type strain 5008 tunnings;

Wherein: I represents the mould ylidene amines A in well ridge, and II represents jinggangmycin A.

Embodiment

Following example will the invention will be further described in conjunction with the accompanying drawings.Present embodiment has provided detailed embodiment and process being to implement under the prerequisite with the technical solution of the present invention, but protection scope of the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.

Embodiment

Step 1, the structure of plasmid

With positive Coase plasmid 2239-AA-G07,2239-AA-G08 carries out double digestion with restriction enzyme EcoRI and HindIII in 37 ℃ of water-baths, obtain 1.0-kb and two fragments of 2.4-kb of being used to exchange respectively, two fragments are carried out three fragments with carrier intestinal bacteria-streptomycete shuttle plasmid pHZ1358 in 37 ℃ of water-baths be connected, with two fragment clonings on the EcoRI of the site of plasmid pHZ1358, at last A Bo is drawn resistant gene to be cloned into two HindIII sites among the fragment, wherein to draw resistant gene be to draw resistant gene to obtain by the A Bo on the HindIII digested plasmid pSET152 to A Bo, and wherein digested plasmid pSET152 purchases the company in Invitrogen.Finally obtain being used for plasmid pJTU609 with karyomit(e) generation homologous recombination by above-mentioned steps.Under 37 ℃ of water bath condition, utilize restriction enzyme BglII enzyme to cut checking plasmid pJTU609, obtain 4.1-kb and 11.0-kb two bar segment, conform to, prove that plasmid construction is correct with expection.

Step 2 imports the wild-type host to the plasmid that is used for karyomit(e) generation homologous recombination

Plasmid pJTU609 must could import in receptor chain mould 5008 cells by conjugal transfer under the assistance of helper plasmid pUZ8002.Cultivate earlier the intestinal bacteria of carrying pUZ8002 and plasmid to be transferred under 50 μ g/mL penbritins, 25 μ g/mL kantlex and 25 μ g/mL paraxin existence conditions, collect thalline after 12 hours, it is standby to wash thalline 3 times with fresh LB substratum.Streptomycete spore as acceptor needs to handle through heat shock and pre-the sprouting.The streptomycete spore is suspended in the 5mLTES damping fluid, and the correlation parameter of TES damping fluid is: 0.05mol/L, the pH value is 8.0.Heat shock 5min in 50 ℃ of water-baths, add the pre-germination medium of equal-volume 2 * spore after being cooled to 25 ℃, the component of this substratum is respectively: massfraction is 1% Difco yeast powder, and massfraction is 1% Difco casamino acids, the CaCl of 0.01mol/L ₂Then under 37 ℃, 220rpm cultivates 2h in shaking table, centrifugal collection spore and evenly being suspended in again in the 5mLLB substratum, by volume for the ratio of 1:4 be coated on the culture plate after intestinal bacteria mix, carry out bacterium parents conjugal transfer, the component and the massfraction of culture plate are respectively: agarose 2%, N.F,USP MANNITOL 2%, soybean cake powder 2%, the pH value is 7.2～7.5.It is dull and stereotyped to use the 1mL sterilized water that contains nalidixic acid and thiostrepton to cover after 16 hours, and the ultimate density of nalidixic acid is 50ng/mL after covering, and thiostrepton is 25ng/mL, can see transconjugant after cultivating 3 days under 30 ℃.

Step 3, screening mutant strains and checking

Select single conjugal transfer from wrapper plate and be inoculated into further affirmation resistance on the flat board with thiostrepton resistance, be transferred to the cultivation that relaxes on the flat board of added with antibiotic not again, and then by resistant panel and non-anti-dull and stereotyped bacterial strain of xeroxing experiment screening, with of the alternative strain of these bacterial strains as mutant strain to some thiostrepton sensitivities.Prepare against the total DNA of roguing as pcr template; Primer P1 and P2 are used for the LL-1 screening, wherein,

The sequence of P1 is: 5 '-AGAGCCGATCTGGTGGTGAG-3 ',

The sequence of P2 is: 5 '-GGTGATGATTAGCCCTTCTCG-3 '.

2150-bp stems from the PCR product of mutant strain LL-1, and 1150-bp stems from the PCR product of wild-type, and the difference in size of two PCR products has been verified the exactness of mutant strain LL-1.

PCR reaction system: 0.1 μ g template DNA, each 50pmol of primer, 4 μ L DMSO, 2 μ L Mg ²⁺, 5 μ L dNTP, 5 μ L damping fluids and 1 unit K OD archaeal dna polymerase add pure water to 50 μ L.

PCR reaction cycle condition is: 95 ℃ 5 minutes; 95 ℃ 30 seconds, 55 ℃ of 30 seconds and 72 ℃ 30 seconds, 30 circulations; Be at last 72 ℃ 5 minutes.

Step 4, the fermentation culture of mutant strain

The cultivation liquid ingredient is: the 4g yeast extract paste, and the 4g malt extract, 10g glucose, 1L water, the pH value was 7.2～7.4,37 ℃ of fermentation culture 6 days.

Step 5 utilizes high performance liquid chromatography that tunning is detected

Use the Water alliance 2690 of Waters company to carry out efficient liquid phase chromatographic analysis, adopt Waters 996 PDA diode array detectors and Waters Millenium ³²Workstation.The high performance liquid phase working conditions is: the concrete parameter of pillar is Agilent ZORBAX-SB-C18,5 μ m, 4.6 * 250mm; Flow velocity 1.0mL/min; Moving phase: volume fraction is that 98% 0.005M phosphoric acid buffer and volume fraction are the methyl alcohol of 2% HPLC level; Detect wavelength: 210nm; Column temperature: 25 ℃.

Fig. 2 is the efficient liquid phase chromatographic analysis result of mutant strain LL-1 and wild type strain 5008 tunnings, and detected result indicates, the accumulation volume of peak I among the mutant strain LL-1 (the mould ylidene amines A in well ridge) is 5 times of peak I accumulation volume in the wild-type 5008.

Sequence table

＜110〉Shanghai Communications University

＜120〉806 of the valG gene bases

<170>PatentIn?Version?3.5

<210>1

<211>806

<212>DNA

＜213〉streptomyces hygroscopicus well ridge mutation 5008 (Streptomyces hygroscopicus var.jinggangensis 5008)

<400>1

＜110〉Shanghai Communications University

＜120〉apramycin resistant gene

<170>PatentIn?Version?3.5

<210>1

<211>786

<212>DNA

＜213〉streptomyces hygroscopicus well ridge mutation 5008 (Streptomyces hygroscopicus var.jinggangensis 5008)

<400>2

Claims (6)

1, a kind of engineering strain of accumulating validoxylamine A, it is characterized in that, on jingganmycin generation bacterium streptomyces hygroscopicus 5008 karyomit(e)s, introduce transgenation, thereby cause the glycosyltransferase gene valG in the jingganmycin biosynthetic pathway to undergo mutation.

2, the engineering strain of accumulating validoxylamine A according to claim 1 is characterized in that, described transgenation is: 806 bases and the apramycin resistant gene of glycosyltransferase gene valG are replaced.

3, the engineering strain of accumulating validoxylamine A according to claim 2 is characterized in that, 806 bases of described valG gene are specially:

tcgaggactt?cgaggtcgtg?gtgtccgacg?atggctcgac?agacaccacc?cgcgacgtag?60

tgcggtccta?cgaggaccgg?ctgcgcatca?agtacgtctt?ccaggaagac?cttggatacc?120

gagtcgccag?cgcgcgcaac?ggtggggcac?gcctggcctc?cgcgccactc?ctggccttcc?180

tcgacactgg?tgtcctggcg?gggccgcagt?acgtccagtc?ggtccttgcc?gcccatgccg?240

ggccggcgcc?cgccaaggtg?gtcctcggct?gctgctacgg?ctacgatccg?cggaatcccc?300

atcccgaact?ccactccctt?gtcgaggagt?tccccccgga?ggaggccgtg?agaagggtcg?360

gggacgcccc?ctggttccag?gacatgcggc?ttccggagtt?cactgcggtc?gacttcgacc?420

taagccgtat?gcacatgccc?tggctgtggt?tctggacgct?caacgtgtcg?ctgccggcag?480

ccgacttctg?gcgggtcggc?gggttcgacg?aggacttcac?cggctggggc?ggggaggaca?540

tcgaactcgg?ctaccggctg?cacgcacacg?gcatccccat?gacggtctca?cgtgagagct?600

gggggatcga?agcgccgcac?gagcgcactc?atgaggcgaa?cgtctcctcc?ctcatgctca?660

attgcgaccg?gttcgtccgc?aagcacccct?ccctgctccc?cgagctgttc?tgggcggtga?720

ccaacagggg?catcttcggc?tccgtcgaga?ctgagcggct?tcgcttcgag?gaatgggcaa?780

gccaggcccg?cgggcaacag?gtcctg 806

4, the engineering strain of accumulating validoxylamine A according to claim 2 is characterized in that, the donor of described apramycin resistant gene is plasmid pSET152.

5, the engineering strain of accumulating validoxylamine A according to claim 2 is characterized in that, described apramycin resistant gene sequence is specially:

tcatgagctc?agccaatcga?ctggcgagcg?gcatcgcatt?cttcgcatcc?cgccctctgg?60

cggatgcagg?aagatcaacg?gatctcggcc?cagttgaccc?agggctgtcg?ccacaatgtc?120

gcgggagcgg?atcaaccgag?caaaggcatg?accgactgga?ccttccttct?gaaggctctt?180

ctccttgagc?cacctgtccg?ccaaggcaaa?gcgctcacag?cagtggtcat?tctcgagata?240

atcgacgcgt?accaacttgc?catcctgaag?aatggtgcag?tgtctcggca?ccccataggg?300

aacctttgcc?atcaactcgg?caagatgcag?cgtcgtgttg?gcatcgtgtc?ccacgccgag?360

gagaagtacc?tgcccatcga?gttcatggac?acgggcgacc?gggcttgcag?gcgagtgagg?420

tggcaggggc?aatggatcag?agatgatctg?ctctgcctgt?ggccccgctg?ccgcaaaggc?480

aaatggatgg?gcgctgcgct?ttacatttgg?caggcgccag?aatgtgtcag?agacaactcc?540

aaggtccggt?gtaacgggcg?acgtggcagg?atcgaacggc?tcgtcgtcca?gacctgacca?600

cgagggcatg?acgagcgtcc?ctcccggacc?cagcgcagca?cgcagggcct?cgatcagtcc?660

aagtggccca?tcttcgaggg?gccggacgct?acggaaggag?ctgtggacca?gcagcacacc?720

gccgggggta?accccaaggt?tgagaagctg?accgatgagc?tcggcttttc?gccattcgta?780

ttgcac 786

6, a kind of construction process of engineering strain of accumulating validoxylamine A is characterized in that, construction process is specially:

Step 1, design and make up are used for homologous recombination plasmid vector that glycosyltransferase gene valG is suddenlyd change;

Step 2 imports to the plasmid vector conjugal transfer of step 1 gained in receptor chain mould 5008 cells and to carry out homologous recombination;

Step 3 is chosen the LL-1 mutant strain earlier by screening mutant strains and checking thiostrepton resistance replica screening, and then by the difference finishing screen of PCR product clip size.

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