CN105969714A - Xiamenensis mycin high-producing strain and culture medium and application thereof - Google Patents
Xiamenensis mycin high-producing strain and culture medium and application thereof Download PDFInfo
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Abstract
The invention discloses a Xiamenensis mycin high-producing strain and a culture medium and application thereof, belongs to the technical field of biology, and particularly relates to establishment of a high-producing genetic engineering strain (Streptomyces xiamenensis) 318Pls1 CGMCC No.12366 bio-synthesized from Xiamenensis mycin and culture medium optimization. Loci of biosynthetic gene clusters are specifically integrated, additional Xiamenensis mycin biosynthetic gene clusters are guided into a core region of a Streptomyces xiamenensis genome, and the bio-synthetic capacity of the Xiamenensis mycin is enhanced. Through optimized culture medium combination, the yield of the Xiamenensis mycin is greatly increased.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of Xiamen mycin superior strain and culture medium, purposes.
Background technology
The type strain CGMCC No.4.3534 of Streptomyces xiamenensis is isolatable from Fujian province Rhizophora apiculata Blume
Woods environmental samples.The Xiamen mycin that this bacterial strain produces is the benzopyran compounds with aminoacid replacement and isopentene group,
There is suppression pulmonary fibrosis and the biological activity of scar hyperplasia, be new anti-fibrosis medicine material standed for.At the GYM generally used
In (Glucose Yeast-extract Maltose) culture medium, in the streptomycete of wild type Xiamen, the yield of Xiamen mycin is only
15mg/L.For meeting the demand of further pharmacology activity research and drug development, the yield of Xiamen mycin is in urgent need to be improved.
The biosynthesis pathway of Xiamen mycin is yet completely resolved, the biological synthesis gene cluster being cloned into include ximA-E this
5 genes, are each responsible for catalysis and are formed 4-HBA by chorismic acid cracking, form tall building through isopentene group transfer, cyclisation
Portamycin B, and load the process of L-threonine formation end-product Xiamen mycin.Xiamen mycin biosynthesis pathway is the simplest
Single, the main metabolic pathway (TCA, PPP, MEP etc.) that the precursor needed for synthesis generally exists both from microorganism.Mould based on Xiamen
Element biosynthesis pathway is oriented molecular breeding to starting strain, is expected to rapid build superior strain.Medium component and training
Foster condition is to affect thalli growth and the key factor of fermentation product element efficiency, and being optimized correlative factor is to improve chemical combination produce
The traditional method of amount.
Summary of the invention
It is an object of the invention to provide the Xiamen mycin superior strain that a kind of Xiamen mycin biological synthesis gene cluster doubles
And culture medium, purposes.The present invention is imported by site-specific integration at the core space of Xiamen streptomycete wild strain genome
Extra Xiamen mycin biological synthesis gene cluster, doubles the biological synthesis gene cluster in the streptomycete of Xiamen, it is thus achieved that tool
There is engineering strain Xiamen streptomycete (Streptomyces xiamenensis) the 318Pls1 CGMCC of high-yield character
No.12366.Added and orthogonal experiment by training systern, single factor test, it is determined that the optimization training of Xiamen mycin shake flask fermentation
Support base.Bacterial strain and culture medium prescription that the present invention provides have the beneficial effect that Xiamen mycin yield is greatly improved.
It is an object of the invention to be achieved through the following technical solutions:
First aspect, the present invention relates to the engineering strain of a kind of Xiamen streptomycete, and described engineering strain is tall building
Door chain mycete (Streptomyces xiamenensis) 318Pls1 CGMCC No.12366.
Preferably, described engineering strain is to be existed by Xiamen mycin biological synthesis gene cluster by site-specific integration
Wild type Xiamen streptomycete (Streptomyces xiamenensis) CGMCC No.4.3534 carries out doubling and obtaining.
Preferably, described Xiamen mycin biological synthesis gene cluster is positioned on integrative plasmid pLMO09404, is led to by this plasmid
Cross the joint transfer between escherichia coli-streptomycete, Xiamen mycin biological synthesis gene cluster can be realized at wild type Xiamen strepto-
Doubling in bacterium, it is thus achieved that engineering strain (Streptomyces xiamenensis) 318Pls1 of high yield Xiamen mycin
CGMCC No.12366。
It is furthermore preferred that the artificial Xiamen mycin biological synthesis gene cluster imported is positioned at the core space of genome, and protect simultaneously
Stay the primitive organism synthetic gene bunch in genome arm district.
Second aspect, the present invention relates to the culture medium of the engineering strain of aforesaid Xiamen streptomycete, every liter of culture medium
In containing glucose 20g, KNO31g, yeast extract 10g, malt extract 15g.
The third aspect, the present invention relates to the engineering strain of a kind of aforesaid Xiamen streptomycete in producing Xiamen mycin
Purposes.
Preferably, described engineering strain selects above-mentioned culture medium, and fermented cultivation obtains Xiamen mycin.
Preferably, described engineering strain uses shake-flask culture to produce Xiamen mycin；Described shake-flask culture working condition
For:
Seed activation: picking on solid medium SFM (soybean cake powder 20g/L, mannitol 20g/L, agar 20g/L) flat board
Produce single bacterium colony that spore is good, be inoculated in 100mL seed activation culture medium TSB (Tryptone Soya in 500mL triangular flask
Broth 30.1g/L) in, 30 DEG C, 220r/min, cultivates 48h；
Fermentation culture: 500ml triangular flask, liquid amount 100mL, it is inoculated in such as claim 4 institute by 5% inoculum concentration (V/V)
The fermentation medium stated, 30 DEG C, the concussion of 220r/min rotary shaker is cultivated 7 days.
In the present invention, described producing strains-Xiamen streptomycete (Streptomyces xiamenensis) 318Pls1 in
On April 19th, 2016 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address
For Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, the numbered CGMCC of culture presevation
No.12366。
Wild type Xiamen streptomycete (Streptomyces xiamenensis) is preserved in Chinese micro-life in April, 2007
Thing culture presevation administration committee's common micro-organisms center (CGMCC), preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
No. 3, Institute of Microorganism, Academia Sinica, culture presevation numbered CGMCC No.4.3534.
The device have the advantages that for: by the high yield base providing a kind of Xiamen mycin biological synthesis gene cluster to double
Because of engineered strain and preferably culture medium prescription and shake flask culture conditions, the yield of Xiamen mycin obtains and is greatly improved.
Accompanying drawing explanation
By the detailed description non-limiting example made with reference to the following drawings of reading, the further feature of the present invention,
Purpose and advantage will become more apparent upon:
Fig. 1 is the PCR checking that Xiamen mycin biological synthesis gene cluster doubles bacterial strain 318Pls1；Wherein, A is integration site
The PCR of attL identifies, B is that the PCR of xim upstream region of gene recombination sequence on pLMO09404 identifies, C is xim base on pLMO09404
Because the PCR of downstream recombination sequence identifies, M:1Kb DNAmaker；The PCR primer of 1:318::pSET152；The PCR of 2:318Pls1
Product；3:318WT negative control；
Fig. 2 is that gene cluster doubles Xiamen mycin yield in bacterial strain 318Pls1 medium optimization orthogonal experiment；
Fig. 3 is Xiamen mycin HPLC examination criteria curve；
Fig. 4 is Xiamen mycin yield (Fig. 4 A) and the HPLC spectrogram (Fig. 4 B) that gene cluster doubles bacterial strain 318Pls1.
Detailed description of the invention
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.Following example will assist in this area
Technical staff be further appreciated by the present invention, but limit the present invention the most in any form.It should be pointed out that, general to this area
For logical technical staff, without departing from the inventive concept of the premise, it is also possible to make certain adjustments and improvements.These broadly fall into
Protection scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition, or according to
Condition proposed by manufacturer.
Embodiment 1, Xiamen mycin biological synthesis gene cluster double structure and the checking of bacterial strain
■ step one, escherichia coli shift with the joint of streptomycete
■ by single for E.coli ET12567 (pUZ8002) bacterium colony in 10mL LB (adding Chl, Kana) culture medium 37 DEG C shake
Swing overnight incubation；
■ takes 100 μ L overnight culture and is seeded to LB (adding Chl, Kana) culture medium fresh for 10mL, 37 DEG C cultivate 4～
6h, to OD600=0.4；
■ 10mL fresh LB washs above-mentioned thalline 2 times to remove antibiotic, and remaining thalline is suspended in 1mL
In LB culture medium；
■ simultaneously, takes 1mL (10-8) streptomycete spore (No.4.3534) in 5mL TES buffer, 50 DEG C of heat shocks
10min, cooling；Adding 5mL 2 × YT broth, 37 DEG C, 200rpm cultivates 2h；
■ is by the streptomycete spore mixing after 1mL E.coli ET12567 (pUZ8002) cell and sprouting, the most centrifugal,
Discard major part supernatant, residue about 50 μ L liquid suspension is precipitated；
■ is diluted to 10 successively-1～10-3Times, each dilution factor takes 100 μ L and coats SFM solid medium, 30 DEG C of cultivations
16～20h；
■ 1mL contains 25mg/mL nalidixic acid and 30mg/mL A Bo draws the sterilized water of antibiotic to cover flat board, 30 DEG C
Continue to cultivate to single bacterium colony occurs；
The single bacterium colony every plate picking 10 grown on flat board is lined twice cultivation of transferring on SFM (Apr) flat board by ■, enters
Performing PCR is verified, selects the successful recon of conversion.
■ step 2, integron to apramycin resistance carry out PCR checking after purification
■ is first with the sequence of primer Pls-attF/R amplification integration site attL, pcr amplification product size and target bar
Band 1Kb is consistent, it was demonstrated that pSET152 plasmid is successfully integrated in 318 chromosomes (Fig. 1-A).Secondly, primer Pls-is used respectively
M13A-F/R and Pls-M13E-F/R, the recombination sequence of xim gene cluster front and back end in amplification recombiant plasmid pLMO09404, PCR expands
Volume increase thing size is consistent with the set goal band 2.2Kb and 2.3Kb, it was demonstrated that second xim gene cluster success that plasmid carries
It is integrated in 318 chromosomes (Fig. 1-B, 1-C).
■ PCR reaction system:
■ PCR response procedures:
The integrative plasmid pSET152 imported in transfer mutant ET12567/pSET152 is engaged for detecting
The primer of Apramycin resistant gene:
Apr-F:5 '-ATGTCATCAGCGGTGGAGT-3 ' SEQ ID NO:1
Apr-R:5 '-CGGCATCGCATTCTTCGCA-3 ' SEQ ID NO:2
Double bacterial strain 318Pls1 for detecting Xiamen mycin gene cluster (xim) and integrate the bacterial strain 318: of empty plasmid:
The primer of pSET152 upstream recombination site attL:
Pls-att-F:5 '-GTAAAACGACGGCCAGTGCCAAG-3 ' SEQ ID NO:3
Pls-att-R:5 '-ATTTGCCGTTGCCGTTGCGG-3 ' SEQ ID NO:4
Double integrated plasmid pLMO09404 on bacterial strain 318Pls1 genome take for detecting Xiamen mycin gene cluster (xim)
Primer with xim gene cluster:
Pls-M13A-F:5 '-GTAAAACGACGGCCAGTGCCAA-3 ' SEQ ID NO:5
PIs-M13A-R:5 '-GCCTGACGGACGTTACCCAGTA-3 ' SEQ ID NO:6
Pls-M13E-F:5 '-CAGGAAACAGCTATGACATGAT-3 ' SEQ ID NO:7
Pls-M13E-R:5 '-AAGCCGTCAAGGGCGAAAT-3 ' SEQ ID NO:8
■ step 3, electrophoresis detection PCR primer: 120V, after electrophoresis 30min, with fresh Gel Red dye liquor dye 40min,
Ultraviolet detection target stripe, Fig. 1.
■ step 4, target stripe is carried out glue recovery, be connected in pMD-18T carrier, send company to check order, sequence alignment
After filter out the recombinant bacterial strain successfully constructed.
Xiamen mycin gene cluster is doubled to build process LAN bacterial strain (Streptomyces xiamenensis) by the ■ present invention
The purpose of 318Pls1 CGMCC No.12366 is to improve Xiamen mycin yield, to obtain the Xiamen mycin mutant of high yield.This
Outward, owing to Xiamen mycin gene cluster is positioned on a genomic islands in 318 chromosome left arm districts, there is genetic instability, its base
Because of group core space sequence inC31 phage specificities integration site attB, is integrated another by specific position
Xiamen mycin gene cluster is incorporated into core space, too increases hereditary stability while adding gene cluster copy number, is conducive to
The structure of subsequent gene engineered strain (add strong promoter, delete negative regulation etc.) on the basis of this bacterial strain.
Embodiment 2, Xiamen streptomycete fermentation condition optimizing
■ step one, designs experiment of single factor, respectively with regard in sweat, whether adds spring, defoamer in shaking flask, cultivates
Temperature: 30 DEG C, 37 DEG C, shaking speed: 180r/min, 220r/min, 300r/min, the seed activation time: 24h, 36h, 48h,
Inoculum concentration 2%, 5%, 10%, has carried out experiment of single factor；
■ step 2, in observing fermentation 7 days, strain growth situation, mycelium crack conditions, shaking flask state, and record；
■ step 3, after fermenting 7 days, receives bacterium, extracts Xiamen mycin and carry out HPLC detection, and three parallel laboratory test groups take
Meansigma methods；
■ step 4, comprehensive thalli growth and product element result, summing up optimal culture conditions is:
■ a. seed activation: 500mL triangular flask, liquid amount 100mL, picking produces single bacterium colony that spore is good, is inoculated in seed
In activation medium TSB (Tryptone Soya Broth 30.1g/L, pH 7.2), 30 DEG C, 220r/min, cultivates 48h；
■ b. fermentation culture: 500mL triangular flask, liquid amount 100mL, it is inoculated in GYM fermentation training with 5% inoculum concentration (V/V)
Supporting base (glucose 4g/L, yeast extract 4g/L, malt extract 10g/L, pH 7.2), 30 DEG C, 220r/min rotary shaker shakes
Swing cultivation 7 days, shaking flask needs add spring, be not added with defoamer.
Embodiment 3, Xiamen mycin bio-synthesis medium optimize
■ step one, uses L9 (34) table carries out the orthogonal (table 1) of 4 factor 3 levels, train by adjusting GYM
Support the proportioning of 4 kinds of raw materials in base, obtain the formula (table 2) of 9 kinds of culture medium；
■ step 2, uses the cultural method after above-mentioned optimization that 318Pls1 bacterial strain has carried out the optimization of fermentation medium
Screening, with Xiamen mycin yield at end for research index.Each experimental group is in triplicate；
■ step 3, carries out efficient liquid phase chromatographic analysis, in conjunction with extreme difference and method of analysis of variance to the tunning after extracting
Evaluate fermentation condition optimization combination and significance level (Fig. 2)；
■ step 4, preferably goes out Xiamen mycin high-yield culture medium GYM-9 (glucose 20g/L, yeast extract 10g/L, Fructus Hordei Germinatus
Extractum 15g/L, KNO31g/L, pH 7.2), its Xiamen mycin yield is up to 76mg/L.Relatively 318 wild strains, unloaded bacterial strain and
Doubling bacterial strain 318Pls1 yield in GYM culture medium and the GYM-9 that preferably goes out, 3 bacterial strains yield in GYM-9 all improves 2 times
Many (Fig. 4).Additionally, no matter gene cluster doubles bacterial strain 318Pls1 in GYM culture medium or in the GYM-9 culture medium preferably gone out,
Xiamen mycin yield all compares wild strain increases about 2.5 times.
Embodiment 4, the HPLC quantitative analysis method of Xiamen mycin yield
■ step one, the drafting of Xiamen mycin concentration standard curve: Xiamen mycin standard substance are diluted to 7 concentration ladders
Degree, carries out HPLC sample detection, and each concentration correspondence peak area data are as follows:
■ draws standard curve (Fig. 3) formula: y=14831812.72x-39728.96, R2=1, y peak area, x Xiamen
Mycin concentration；
■ step 2, Xiamen mycin product extracting method: by 100mL fermentation liquid equal-volume ethyl acetate soaked overnight,
Repeating to extract 3 times, upper organic phase merging is evaporated, and extractum is dissolved in 6mL methanol；
■ step 3, HPLC detection method: after above-mentioned methanol is dissolved extractum dilution 3 times, carry out according to sample size 10 μ L
High performance liquid chromatography (HPLC) is analyzed, and testing conditions is: C18 reversed-phase column (Agilent Extend-C18,4.6 × 150mm, 5 μ
m)；Flowing is the first alcohol and water (20%-80% gradient elution) being separately added into 0.5% trifluoroacetic acid mutually；Flow velocity 1mL/min；Post
Temperature is 40 DEG C；Ultraviolet detection wavelength 254nm；
■ step 4, Xiamen mycin volume analysis method: use standard Xiamen mycin concentration curve formula, by detect
Peak area, is converted into corresponding Xiamen mycin concentration, is multiplied by extension rate, calculate Xiamen mycin yield.
The specific embodiment of the present invention is described by more than ■.It is to be appreciated that the invention is not limited in
Stating particular implementation, those skilled in the art can make various deformation or amendment within the scope of the claims, and this is not
Affect the flesh and blood of the present invention.
Claims (7)
1. the engineering strain of an Xiamen streptomycete, it is characterised in that described engineering strain is Xiamen streptomycete
(Streptomyces xiamenensis)318Pls1 CGMCC No.12366。
2. the engineering strain of Xiamen as claimed in claim 1 streptomycete, it is characterised in that described engineering strain is
By site-specific integration by Xiamen mycin biological synthesis gene cluster at wild type Xiamen streptomycete (Streptomyces
Xiamenensis) CGMCC No.4.3534 is carried out doubling and obtaining.
3. the engineering strain of Xiamen as claimed in claim 2 streptomycete, it is characterised in that the artificial Xiamen mycin imported
Biological synthesis gene cluster is positioned at the core space of genome, and remains the primitive organism synthetic gene bunch in genome arm district simultaneously.
4. the culture medium of the engineering strain of an Xiamen as claimed in claim 1 streptomycete, it is characterised in that every liter of training
Support in base containing glucose 20g, KNO31g, yeast extract 10g, malt extract 15g.
5. the engineering strain of Xiamen as claimed in claim 1 streptomycete purposes in producing Xiamen mycin.
6. purposes as claimed in claim 5, it is characterised in that described engineering strain is selected as claimed in claim 4
Culture medium, fermented cultivation obtains Xiamen mycin.
7. purposes as claimed in claim 5, it is characterised in that it is mould that described engineering strain uses shake-flask culture to produce Xiamen
Element；Described shake-flask culture working condition is:
Seed activation: picking produces single bacterium colony that spore is good on solid medium SFM flat board, is inoculated in 500mL triangular flask
In 100mL seed activation culture medium TSB, 30 DEG C, 220r/min, cultivates 48h；
Fermentation culture: 500mL triangular flask, liquid amount 100mL, the inoculum concentration of 5% is inoculated in as claimed in claim 4 by volume
Fermentation medium, 30 DEG C, 220r/min rotary shaker concussion cultivate 7 days.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108531429A (en) * | 2018-04-27 | 2018-09-14 | 山东农业大学 | It is a kind of to produce protease, the Xiamen bacillus of anti-botrytis cinerea and its application |
| CN112940999A (en) * | 2019-12-11 | 2021-06-11 | 上海交通大学 | Streptomyces xiamenensis and method for biologically synthesizing benzopyran and 4-hydroquinoline compounds by Streptomyces xiamenensis |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108531429A (en) * | 2018-04-27 | 2018-09-14 | 山东农业大学 | It is a kind of to produce protease, the Xiamen bacillus of anti-botrytis cinerea and its application |
| CN108531429B (en) * | 2018-04-27 | 2021-02-02 | 山东农业大学 | Bacillus mansion capable of producing protease and resisting botrytis cinerea and application thereof |
| CN112940999A (en) * | 2019-12-11 | 2021-06-11 | 上海交通大学 | Streptomyces xiamenensis and method for biologically synthesizing benzopyran and 4-hydroquinoline compounds by Streptomyces xiamenensis |
| CN112940999B (en) * | 2019-12-11 | 2022-08-23 | 上海交通大学 | Streptomyces xiamenensis and method for biologically synthesizing benzopyran and 4-hydroquinoline compounds by Streptomyces xiamenensis |
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