CN110066744A - The application that one pnca gene group resets Producing Strain and its is bestowed by heaven in mycin in production - Google Patents
The application that one pnca gene group resets Producing Strain and its is bestowed by heaven in mycin in production Download PDFInfo
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Abstract
The application resetting Producing Strain the invention discloses a pnca gene group and its being bestowed by heaven in mycin in production, the genome rearrangement Producing Strain areStreptomyces sp.CB03234-GS26, is deposited in China typical culture collection center on July 16th, 2018, and deposit number is CCTCC M 2018485.The present invention passes through based on existing superior strainStreptomyces sp.CB03234-S(deposit number is CCTCC M 2017538) and gentamicin resistance ribosome engineering mutant bacteriaStreptomyces sp.The genome rearrangement of CB03234-G obtains stable novel Enediyne natural products and is bestowed by heaven mycin-A(TNM-A) the fermentation production rate of superior strain CB03234-GS26, TNM-A be more than 38 mg/L.
Description
Technical field
The application resetting Producing Strain the present invention relates to a pnca gene group and its being bestowed by heaven in mycin in production, belongs to biological medicine
Technical field.
Background technique
Enediyne natural products has unique conjugation alkynes-alkene-alkynes molecular structure and superpower bioactivity, is most to open
A kind of antitumor antibiotics of hair prospect, according to its nuclear structure can be divided into including neoearcinostain (neocarzinotatin,
NCS), the nine-atomic ring enediyne including lidamycin (C-1027), kedarcidin and maduropepti etc., and including card
Including miramycin (calicheamicin, CAL), esperamicin, dynemicin (DYN) and uncialamycin (UCM) etc.
Ten-ring enediyne.The bioactivity of Enediyne Antitumor Antibiotics depends on the DNA damage mechanism of its induction, i.e.,
Temporary phenyl ring diradical is formed by electron rearrangement, is formed after carrying out nucleophillic attack to DNA ditch with DNA
Free radical centered on carbochain, and cause under the action of molecular oxygen the fracture of single-stranded or double-stranded DNA.As finding so far
The strongest molecule of cytotoxicity, Enediyne natural products can be used as the bullet of anti-tumour antibody coupling drug (ADC)
Molecule has high patent medicine prospect.In presently found 13 kinds of enediyne molecules, NCS and CAL are had been developed that as clinical medicine
Object, wherein be mainly used for treating liver cancer by the SMANCS (polystyrene maleic acid is conjugated NCS) of Japan's exploitation, and Pfizer is public
Taking charge of the ADC drug Mylotarg (CD33 monoclonal antibody is coupled CAL) developed recently and Besponsa, (CD22 monoclonal antibody is even
Connection CAL) then it is respectively used to treatment acute myelogenous leukemia and the white blood of the adult acute leaching of relapsed or stubborn B cell precursor
Disease.
Mycin-the A (Tiancimycin-A, breviary TNM-A) that is bestowed by heaven is to pass through within 2016 genome digging technology from streptomycete
A kind of novel ten-ring alkene two of discovery is separated in the tunning of (Streptomyces sp.CB03234, abbreviation CB03234)
Alkynes antitumor antibiotics, structure are as follows:
There is no carbon former at the right-angled intersection on right side in structural formula
Son.
Mycin-the A that is bestowed by heaven is similar with UCM and DYN structure, there is the activity of superelevation to Several Kinds of Malignancy cell, is more than current
Nearly thousand times of clinical front-line chemotherapeutic agents mitomycin, and show more rapidly and more complete tumor cell killing potential, will be
The antitumor ideal bullet drug molecule of ADC drug, the achievement have been published in (2016 on international microorganism authoritative journal MBio.；
7(6).pii:e02104-16).Yield of the TNM-A in original strain (Streptomyces sp.CB03234) is extremely low, only
0.3mg/L or so, and reach at present the yield of other Enediyne natural products of preparation of industrialization level 20mg/L with
On, therefore the source of existing TNM-A is far from satisfying the application demand of clinical research and industrialized production.Simultaneously TNM-A because
Its complicated unique molecular structure, can not be obtained by traditional chemical synthesis process, be to make at present by microbial fermentation
The standby most practicable means of TNM-A.
Genome rearrangement is mainly recombinated and is handed over using the segment that protoplast fusion carries out full-length genome range to microorganism
It changes, is taken turns recursion screening fusant excessively constantly to accumulate beneficial mutation, thus realize that the positive of target strain is evolved, with
Traditional breeding way, which is compared, has many clear superiorities.Based on ribosome engineering to the original producing bacterial strain of TNM-A
Streptomyces sp.CB03234 is mutated, and obtains the mutant strain with different antibiotic resistances, TNM- respectively
The yield of A is significantly improved than original strain.On this basis, it is parent with above-mentioned ribosome engineering mutant strain, uses
Genome rearrangement based on protoplast fusion, screening obtain the superior strain that TNM-A yield is further promoted, and will be to realize it
Large-scale production and preparation, the preclinical phases research such as Anticancer Activity Analysis, mechanism of action for pushing TNM-A subsequent, and it is related
Important foundation is established in the exploitation of antitumor ADC new drug.
Summary of the invention
Present invention solves the technical problem that being the yield for improving TNM-A, to meet the application demand of industrialized production.
The present invention is by being bestowed by heaven the original production of mycin-A (TNM-A) to the extremely strong novel Enediyne natural products of activity
Bacterial strain Streptomyces sp.CB03234 carries out the ribosome engineering induction mutation of bacterium based on gentamicin resistance, and screening obtains
The superior strain CB03234-G of TNM-A, and (fermentation production rate reaches 7mg/L to the superior strain CB03234-S based on existing TNM-A
Left and right, for the bacterial strain in the preservation on the 25th of September in 2017 to China typical culture collection center, deposit number is CCTCC M
2017538) it, by carrying out the genome rearrangement based on protoplast fusion to CB03234-G and CB03234-S, finally screens
Obtain TNM-A superior strain CB03234-GS26.
The technical scheme is that providing a pnca gene group resets high yield mutant bacteria, streptomycete is equally also belonged to, it is described
Streptomycete is Streptomyces sp.CB03234-GS26, has been deposited in Chinese Typical Representative culture guarantor on July 16th, 2018
Hiding center, deposit number are CCTCC M 2018485.
It is as follows that the present invention prepares the step of above-mentioned CB03234-GS26 bacterial strain:
1) the gentamicin resistance ribosome engineering mutagenesis of streptomycete CB03234；
2) streptomycete CB03234 mutant strain bioactivity high flux screening and TNM-A superior strain CB03234-G are obtained
?；
3) bacterial strain CB03234-S and CB03234-G is subjected to genome rearrangement and obtains TNM-A superior strain CB03234-
GS26。
The streptomycete CB03234-GS26 can be applicable to the preparation of be bestowed by heaven mycin-A and its derivative, it is described be bestowed by heaven it is mould
The structural formula of element-A are as follows:
Fermentation is carried out using above-mentioned streptomycete and prepares the mycin-A that is bestowed by heaven, and the composition of culture medium used in fermentation process is such as
Under: 40g/L soluble starch, 20g/L cotton seed meal, 0.1g/L CuSO4·5H2O, 0.005g/L NaI and 2g/L CaCO3, training
The pH for supporting base is 7.0, and 10g HP20 macroporous absorbent resin is added in every liter of culture medium.
The present invention obtains stable TNM-A superior strain by ribosome engineering mutagenesis and high-throughput bioactivity screening
The fermentation production rate of CB03234-G, TNM-A reach 3.7mg/L or so, and bacterial strain CB03234-S and CB03234-G is carried out gene
Group resets screening and obtains TNM-A superior strain CB03234-GS26, and yield reaches 38mg/L or so.
Preservation information
Strain name: streptomycete CB03234-GS26；The Latin generic name of strain: Streptomyces sp.；Preservation is compiled
Number: CCTCC No.:M 2018485；Preservation date: on July 16th, 2018；Depositary institution: in China typical culture collection
The heart；Depositary institution address: Wuhan, China, Wuhan University.
Specific embodiment
It is explained further and illustrates the present invention below with reference to embodiment, unless otherwise instructed, be related to eluent, mobile phase
Percentage is percentage by volume, and other percentages are mass percentage.
Embodiment 1: the culture and fermentation of streptomycete CB03234 and the bioactivity detection of TNM-A
Streptomycete CB03234 is inoculated with Zhi Gaoshi 1 (G1) solid medium (G1 solid medium are as follows: 10g/L can
Soluble starch, 0.5g/L MgSO4·7H2O、0.5g/L K2HPO4、1g/L NaCl、1g/L KNO3、0.01g/L FeSO4·
7H2O, 20g/L agar, pH=7.0) on inclined-plane, cultivated 8-15 days or so under 30 DEG C of constant temperatures, it is molten with sterile 20% glycerol
It is spare in -80 DEG C that liquid collects refrigeration after spore obtains spore suspension.To obtain target product TNM-A, by 50 μ L CB03234
Spore suspension is seeded to 50mL pancreas peptone soybean broth (TSB) seed culture medium (TSB seed culture medium are as follows: 17g/L
Tryptone, 3g/L phytone, 2.5g/L K2HPO4, 5g/L NaCl, 2.5g/L glucose, pH=7.3) in, at 30 DEG C
After being cultivated 48 hours under the conditions of 200rpm, 5mL seed is forwarded to containing the 50mL production medium (production medium
Are as follows: 10g/L soluble starch, 5g/L cotton seed meal, 2g/L CaCO3、0.05g/L CuSO4, 0.005g/L NaI, and every liter train
Support and add 10g HP20 macroporous absorbent resin in base) 250mL conical flask in, cultivated 7 days at 30 DEG C and under the conditions of 200rpm.
Supernatant will be taken after the centrifugation of gained fermentation liquid, is that biology is living with micrococcus luteus (Micrococcus luteus) ATCC10240
Property indicator bacteria paper disk method test (20 μ L fermented supernatant fluids/piece) are carried out on standard LB culture medium flat plate, pass through measurement inhibition zone
Size carry out the content of TNM-A in preresearch estimates fermentation liquid.
Embodiment 2: the gentamicin resistance ribosome engineering mutagenesis of streptomycete CB03234
Streptomycete CB03234 is seeded in G1 culture medium slant, a 8-15 days left sides are cultivated under 30 DEG C of constant temperatures
Spore is collected with sterile 20% glycerite in the right side, and gained spore mixed liquor is filtered after oscillation is broken up with sterile sand core funnel, and
Spore count is carried out by the sparse coating of plate, is finally made the spore suspension of homogenization concentration.Compound concentration is simultaneously
Gentamicin (Gen) aqueous solution of 10mg/mL simultaneously carries out filtration sterilization, configures the G1 containing various concentration Gen on this basis
Solid medium tablets；100 μ L and 5mL are taken to contain various concentration Gen after the spore suspension of CB03234 is diluted 50-100 times
G1 soft agar medium (the other ingredients of culture medium are identical as G1, agar 10g/L) tile after mixing to containing correspondence it is dense
It spends in the G1 solid medium tablets of Gen (3 pieces of plates of every kind of Gen concentration parallel culture), observes bacterium after cultivating 4-5 days at 30 DEG C
Survival condition is fallen, the final minimal inhibitory concentration (MIC) for determining Gen is 15mg/L.On the basis of the studies above, 4 kinds are chosen not
Same Gen concentration (15mg/L, 20mg/L, 40mg/L, 60mg/L, 80mg/L) carries out the ribosome engineering of streptomycete CB03234
Mutagenesis (table 2).Every kind of concentration is inoculated with 10 pieces of G1 plates according to above-mentioned inoculate flow process in parallel, cultivates 7-8 under 30 DEG C of constant temperatures
The single colonie of survival is selected after it.
Embodiment 3: streptomycete CB03234 mutant strain bioactivity high flux screening
The single colonie of the CB03234 selected and its related mutation bacterial strain is seeded to two piece of 96 orifice plate G1 solid in parallel respectively
In growth medium.96 orifice plates of inoculation after constant temperature incubation 8-15 days, are selected into the agar block of corresponding single colonie in 30 DEG C, are placed
In on the LB plate of punching, indicated by biological activity test of micrococcus luteus (Micrococcus luteus) ATCC10240
1mL ATCC10240 bacterium solution and 4mL LB soft agar medium are poured over after mixing and are equipped with single colonie fine jade to be screened by bacterium
It on the LB plate of rouge block, is stayed overnight after its solidification in 37 DEG C of constant temperature incubations, using original strain as reference, by measuring corresponding single bacterium
The inhibition zone size of agar block is fallen quickly to screen potential TNM-A superior strain, finally filters out 34 from 59 mutant strains
Strain carries out further fermentation verifying (table 2).
The ribosome engineering mutagenesis and bioactivity high flux screening result of table 2.CB03234 bacterial strain
| Gen screening concentration (mg/L) | 15 | 20 | 40 | 60 | 80 |
| Grow single colonie number | 10 | 20 | 4 | 18 | 7 |
| Inhibition zone is greater than original bacteria strain number | 3 | 9 | 0 | 17 | 5 |
The determination of embodiment 4:TNM-A superior strain CB03234-G
Inhibition zone is greater than to the mutation single colonie of original strain CB03234, is forwarded to the pancreas that 50mL contains corresponding concentration Gen
Tryptone soya broth (TSB) seed culture medium (TSB seed culture medium are as follows: 17g/L tryptone, 3g/L vegetable protein
Peptone, 2.5g/L K2HPO4, 5g/L NaCl, 2.5g/L glucose, pH=7.3) in, cultivate 48 in 30 DEG C and 200rpm under the conditions of
Hour, it samples freezing and is forwarded to G1 culture medium slant；(remainder by 10% inoculum concentration be forwarded to containing
50mL production medium (the production medium are as follows: 10g/L soluble starch, 5g/L cotton seed meal, 2g/L CaCO3、0.1g/L
CuSO4, 0.005g/L NaI, and add 0.5g (1% mass volume ratio, the dosage of resin be quality (g) in all embodiments
Volume (mL) ratio) HP20 macroporous absorbent resin) 250mL conical flask in, cultivated 7-10 days at 30 DEG C and under the conditions of 200rpm.
Macroreticular resin is collected after fermentation, and methanol impregnates and eluent merging is concentrated into 2mL after ultrasound elution, analyzes by HPLC
It is prominent finally to filter out G-60-10 (referred to as: CB03234-G) from 34 plants of potential superior strains for the TNM-A yield for calculating mutant strain
Become bacterium, TNM-A yield reaches 3.7mg/L or so (table 3).
The fermentation yield of the potential TNM-A superior strain in 3. part of table
| Bacterial strain | Yield (mg/L) | Bacterial strain | Yield (mg/L) |
| G-60-3 | 1.4±0.3 | G-80-6 | 2.8±0.3 |
| G-60-7 | 2.6±0.5 | G-80-7 | 3.1±0.4 |
| G-60-10 | 3.7±0.2 | G-80-14 | 2.2±0.3 |
| G-60-14 | 1.9±0.3 | CB03234 | 0.8±0.1 |
Embodiment 5: the genome rearrangement based on CB03234-S and CB03234-G
It is parent with TNM-A superior strain CB03234-S and CB03234-G, it is primary prepares its corresponding high quality respectively
Plastid, main flow are as follows: (1) take 0.2mL filter spore suspension be inoculated into 50mL seed culture medium (add 3g micro glass beads and
0.1% glycine) in, it is cultivated 36-40 hours under the conditions of 30 DEG C of constant-temperature table 200rpm；(2) after mycelium being collected by centrifugation,
15mL P-buffer (the P-buffer buffer are as follows: 103g/L sucrose, 0.25g/L K is added2SO4、2g/L MgCl2·
6H2O, 3.7g/L CaCl2·2H2O, 0.05g/L KH2PO4) inhaled and beaten repeatedly with pipette tips, keep mycelium loose, repeated washing 3 times；
(3) every 0.5g mycelium is suspended with 5mL P-buffer, and be added 100 μ L glusulases and 50 μ L lysozymes (be 10mg/mL,
P-buffer is prepared), slightly digested 3 hours under the conditions of 32 DEG C of constant-temperature table 100rpm after mixing；(4) it is added again
10mL P-buffer is slowly inhaled and is beaten, after filtering out mycelium with sterilising filtration funnel (four layers of lens wiping paper are made), low-speed centrifugal
(1000rpm) collects protoplast, and is cleaned with 10mL P-buffer, is repeated 2 times；(5) 5mL P-buffer is finally used
Again suspend protoplast, is distributed into 500 μ L/ pipe (107-108/mL).By the Protoplast suspension of prepare two parents
It is isometric to mix (each 500 μ L), liquid is discarded supernatant after low-speed centrifugal, and it is equal that 5mL 50%PEG-1000 fusion buffer suspension is added
Even, 37 DEG C of waters bath with thermostatic control stand 10min (soft mixing in every 3 minutes is primary) and are merged；5mL P- is immediately added
Buffer cleaning, and being suspended again the protoplast of fusion with 5mL P-buffer, and be coated on after suitably diluting containing
Regenerated plate (the 103g/L sucrose of 50mg/L streptomysin and 50mg/L gentamicin；10g/L glucose；0.1g/L sour water solution junket
Albumen；5g/L yeast powder；0.25g/LKH2PO4；10.12g/L MgCl2·6H2O；5.73g/L TES；20g/L agar.With
After the packing sterilizing of 100mL volume, independent sterile components: 1mL 5g/L KH are sequentially added2PO4；0.8mL 367.5g/L
CaCl2·2H2O；0.7mL 1M NaOH) on, it is cultivated 7-10 days in 30 DEG C of incubators, picking individual colonies carry out subsequent screening.
The screening and verifying of embodiment 6:TNM-A superior strain CB03234-GS26
Inhibition zone is greater than to the mutation single colonie of original strain CB03234,50mL is forwarded to and contains corresponding concentration streptomysin
With pancreas peptone soybean broth (TSB) seed culture medium (TSB seed culture medium are as follows: 17g/L tryptose of gentamicin
Peptone, 3g/L phytone, 2.5g/L K2HPO4, 5g/L NaCl, 2.5g/L glucose, pH=7.3) in, at 30 DEG C and
It is cultivated 36-48 hours under the conditions of 200rpm.It is forwarded to by 10% inoculum concentration containing the 50mL production medium (productive culture
Base are as follows: 10g/L soluble starch, 5g/L cotton seed meal, 2g/L CaCO3、0.1g/L CuSO4, 0.005g/L NaI, and add
0.5g (1% mass volume ratio, the dosage of resin is quality (g) volume (mL) ratio in all embodiments) HP20 macroporous absorption tree
Rouge) 250mL conical flask in, cultivated 7-10 days at 30 DEG C and under the conditions of 200rpm.Macroreticular resin, first are collected after fermentation
Alcohol impregnates and eluent merging is concentrated into 2mL after ultrasound elution, by the TNM-A yield of HPLC analytical calculation mutant strain, most
CB03234-GS26 is filtered out from 26 plants of fusion mutant bacterias eventually, TNM-A yield reaches 16mg/L or so (table 4), than original
Output increased nearly 2.5 times of TNM-A Producing Strain CB03234-S.
The TNM-A fermentation yield of 4. part GS of table fusion mutant bacteria
Subsequent stability test shows the TNM-A in CB03234-GS26 continuous four generation under the conditions of original production medium
Yield all at 16mg/L or so (table 5), has good genetic stability.
The genetic stability of table 5.CB03234-GS26 is verified
| Spore algebra | The first generation | The second generation | The third generation | Forth generation |
| Yield (mg/L) | 16.1±0.3 | 16.0±0.2 | 16.2±1.8 | 16.7±2.3 |
In addition, CB03234-GS26 is in the Optimal Medium (Optimal Medium are as follows: 40g/L soluble starch, 20g/L
Cotton seed meal, 0.1g/L CuSO4·5H2O, 0.005g/L NaI and 2g/L CaCO3(pH 7.0)) in TNM-A yield it is further
It is promoted to 38.7 ± 0.2mg/L, compared with the yield of original bacteria CB03234 about 0.8mg/L, improves about 48 times.
The Genetic Variation Analysis of embodiment 7:CB03234-GS26
The total DNA for extracting CB03234-GS26 carries out genome and resurveys sequence, and by the reference base of sequencing result and CB03234
Gene difference is found because a group sequence compares.
The Genetic Variation Analysis of table 6.CB03234-GS26
The result shows that 230 single nucleotide polymorphism mutation (SNP) are detected in CB03234-GS26 altogether, wherein non-same
At justice mutation 96, the albumen for causing 75 genes to encode has occurred amino acid sequence and changes (table 6)；Detect that gene encodes at 3
Region missing, opening code-reading frame frameshift mutation at 2, the albumen for causing 4 genes to encode have occurred amino acid sequence and change (table
6), and these mutation are evenly distributed in the full-length genome of CB03234-GS26.In addition, the genome structure of CB03234-GS26
Also obvious variation has occurred, the variation such as missing, inversion and dystopy has occurred in the DNA fragmentation of many places.The above results show genome
Resetting Producing Strain CB03234-GS26, there are significant hereditary differences with original bacteria CB03234.
Claims (3)
1. a pnca gene group resets Producing Strain, which is characterized in that the genome rearrangement Producing Strain is Streptomyces
Sp.CB03234-GS26 is deposited in China typical culture collection center, deposit number CCTCC on July 16th, 2018
M 2018485。
2. the application that genome rearrangement Producing Strain described in claim 1 is bestowed by heaven in mycin-A and its derivative in preparation, described
It is bestowed by heaven the structural formula of mycin are as follows:
3. application as claimed in claim 2, which is characterized in that using genome rearrangement Producing Strain described in claim 1 into
Row fermentation prepares the mycin-A that is bestowed by heaven, and the composition of culture medium used in fermentation process is as follows: 40g/L soluble starch, 20g/L cotton
Seed powder, 0.1g/L CuSO4·5H2O, 0.005g/L NaI and 2g/L CaCO3, the pH of culture medium is 7.0, and in every liter of culture
10g HP20 macroporous absorbent resin is added in base.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974464A (en) * | 2010-10-18 | 2011-02-16 | 中国科学院南海海洋研究所 | Streptomyces and process for preparing antimycin antibiotics by fermentation using same |
| WO2015093839A1 (en) * | 2013-12-18 | 2015-06-25 | 동국제약 주식회사 | New streptomyces filamentosus variant and method for producing daptomycin using same |
| WO2016155568A1 (en) * | 2015-03-27 | 2016-10-06 | 浙江海正药业股份有限公司 | Streptomyces and method for producing milbemycin a3 using same |
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2018
- 2018-11-30 CN CN201811453409.3A patent/CN110066744B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974464A (en) * | 2010-10-18 | 2011-02-16 | 中国科学院南海海洋研究所 | Streptomyces and process for preparing antimycin antibiotics by fermentation using same |
| WO2015093839A1 (en) * | 2013-12-18 | 2015-06-25 | 동국제약 주식회사 | New streptomyces filamentosus variant and method for producing daptomycin using same |
| WO2016155568A1 (en) * | 2015-03-27 | 2016-10-06 | 浙江海正药业股份有限公司 | Streptomyces and method for producing milbemycin a3 using same |
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