CN101280281B - Paclitaxel genome rearrangement bacterial strain HDFS4-26 - Google Patents
Paclitaxel genome rearrangement bacterial strain HDFS4-26 Download PDFInfo
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- CN101280281B CN101280281B CN2008100640483A CN200810064048A CN101280281B CN 101280281 B CN101280281 B CN 101280281B CN 2008100640483 A CN2008100640483 A CN 2008100640483A CN 200810064048 A CN200810064048 A CN 200810064048A CN 101280281 B CN101280281 B CN 101280281B
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Abstract
The invention relates to paclitaxel genome shuffling bacterial strain HDFS4-26 and a seed selection method of high yield paclitaxel bacterial strain, which belongs to the paclitaxel genome shuffling bacterial strain and the seed selection method of the paclitaxel engineering bacterial strain and solves the problem that the paclitaxel output of the bacterial strain which can be used in the paclitaxel microorganism fermentation at present is still relatively low. The paclitaxel genome shuffling bacterial strain HDFS4-26 is nodulisporium sylviforme and belongs to the nodulisporium and is preserved in the China Center for Type Culture Collection, the preservation date is February 28, 2008, the preservation number is CCTCC M 208026. The seed selection method of the high yield paclitaxel bacterial strain adopts firstly, the protoplast preparation; secondly, the inactivation of the protoplast; thirdly, the interfusion of the protoplast; fourthly, the regeneration of the protoplast; fifthly, the genome shuffling and sieving, therefore to obtain the high yield paclitaxel bacterial strain. The paclitaxel output of the bacterial strain HDFS4-26 is 516.37ug/L. The seed selection method of thehigh yield paclitaxel bacterial strain provided by the invention is simple, the effect is good, and the paclitaxel genome shuffling bacterial strain HDFS4-26 and the seed selection method of high yield paclitaxel bacterial strain lay the foundation of industrial production of bacterial strain providing high yield paclitaxel.
Description
Technical field
The present invention relates to a kind of paclitaxel genome rearrangement bacterial strain.
Background technology
Malignant tumour is seriously threatening human beings'health, and cancer is the second largest cause of the death that is only second to cardio-cerebrovascular disorder in the present human diseases.
Taxol is the diterpene alkaloid class medicine (molecular formula as shown in Figure 1) with multiple antitumor curative effect that extracts from Chinese yew genus plants.Because Ramulus et folium taxi cuspidatae (having another name called Japanese yew) is a rare tree species; the speed of growth is slow; and its biological characteristics is had relatively high expectations to ecotope; the natural regeneration ability; so population quantity is very limited; belong to protective plant in imminent danger, resource is very limited, so Chinese scholars is all at the source new drugs of researching and developing taxol energetically.
Microbial fermentation production is the Perfected process that reduces cost, obtains in a large number taxol.Chemist Andrea Stierle of U.S. Meng Tana state university in 1993 and phytopathologist Gary Strobel are separated to a strain endogenetic fungus Taxomycesandreanae in the yewtree in park, country glacier, the Meng Tana northwestward, working once delivering of Stierle etc., 1,000,000 dollars of these researchs of supporting Meng Tana state universities of just bidding of U.S. cell clone company.But the still lower (strain HD of taxol output that can be used for the bacterial strain of taxol microbial fermentation at present
100-23Be the high paclitaxel produced mutant strain that the spore of bacterial strain NCEU-1 obtains behind UV and NTG complex mutation, its China fir alcohol output is 356.80 μ g/L, bacterial strain UV
40-19And UL
50-6Be the protoplastis high paclitaxel produced mutant strain that obtains behind ultraviolet ray and ultraviolet ray and lithium chloride complex mutation respectively of bacterial strain NCEU-1, taxol output is respectively 376.38 μ g/L and 392.63 μ g/L), be difficult to satisfy the demand in market.
Summary of the invention
The objective of the invention is in order to solve the still lower problem of taxol output of the bacterial strain that can be used for the taxol microbial fermentation at present, and the paclitaxel genome rearrangement strain HD FS4-26 that provides.
Paclitaxel genome rearrangement strain HD FS4-26 of the present invention is that tree-shaped more piece spore (Nodulisporiumsylviforme) belongs to more piece spore genus (Nodulisporium), be preserved in Chinese typical culture collection center (CCTCC), the preservation address is a Wuhan University, preservation date is on February 28th, 2008, and preserving number is CCTCC M 208026; The paclitaxel genome rearrangement strain HD FS4-26 bacterium colony cultivation initial stage is a white, and the later stage is dark-grey brown, and the back side is black; Give birth in the paclitaxel genome rearrangement strain HD FS4-26 mycelia part, the part hypergene has every, multiple-limb, and mycelia is that Vandyke brown shoals gradually to top property, and diameter is 2.24～5.86 μ m, does not see sclerotium and produces; Conidiophore Dan Sheng, majority is arborizations, and brown is thin out to top property, long 37～162 μ m; The conidiogenous cell column, single giving birth to or the colyliform arrangement, 6.37～22.63 μ m * 2.51～3.86 μ m, the sympodium formula is produced spore; Conidium Dan Sheng, ellipse or oval, the level and smooth or little wart of tool, no barrier film, 5.07～7.89 μ m * 2.45～3.79 μ m, conidium is head and arranges in the product spore point position of conidiogenous cell.
Taxol superior strain of the present invention seed selection by the following method: one, protoplastis preparation; Two, the deactivation of protoplastis; Three, the fusion of protoplastis; Four, protoplast regeneration; Five, genome rearrangement, screening promptly obtain the taxol superior strain; The bacterial strain that wherein is used for the protoplastis preparation is that taxol produces bacterium HD
100-23, UV
40-19And UL
50-6
Step 1 protoplastis preparation in the selection of taxol superior strain of the present invention: get the activatory taxol and produce bacterium HD
100-23, UV
40-19And UL
50-6Put into the PDA liquid nutrient medium and under 28 ± 0.5 ℃ condition, leave standstill cultivation 3 days, bacteria suspension centrifugal 10min under the 4000g condition collects mycelium, again with the mycelium vibration evenly with vortex mixer, then with homeo-osmosis agent washing 2 times, the ratio adding enzymatic lysis liquid that adds 1mL enzymatic lysis liquid afterwards in every 250mg mycelium, place 30 ℃ of environment temperature to bathe 7h again, filter with 3 layers of aseptic lens paper then, filtrate 4000g is centrifugal 10min under the 4000g condition, the protoplasma precipitation obtains the protoplastis of purifying again for 2 times with homeo-osmosis agent washing, afterwards protoplastis is suspended in the homeo-osmosis agent.
The deactivation of step 2 protoplastis:
With the HD behind the purifying
100-23The protoplastis suspension moves in the sterile test tube, and 50 ℃ of water bath with thermostatic control 5min carry out hot deactivation;
With the UV behind the purifying
40-19The protoplastis suspension to move into diameter be in the aseptic plate of 6cm, place in advance under the thermally-stabilised 30W ultraviolet lamp, plate and ultraviolet lamp vertical range are 30cm, shine 100s and carry out ultraviolet inactivation;
UL behind purifying
50-6The protoplastis suspension in to add final concentration be 0.6%LiCl, place immediately in advance then under the thermally-stabilised 30W ultraviolet lamp, plate and ultraviolet lamp vertical range are 30cm, shine 70s and carry out the compound deactivation of ultraviolet ray+lithium chloride.
The fusion of step 3 protoplastis: from deactivation protoplastis suspension, respectively get the 1mL suspension and mix in twos, mixed solution is centrifugal 10min under the condition of 3000r/min, after removing centrifuged supernatant protoplastis is suspended in the agent of 0.2mL homeo-osmosis, add 1.8mL, temperature again and be 30 ℃, mass concentration and be 35% PEG fusogen, water bath heat preservation 90s under 30 ℃ of conditions, the homeo-osmosis agent that adds 5mL, temperature again and be 4 ℃ stops merging, centrifuge washing is removed fusogen then, is suspended in the homeo-osmosis agent more again through the protoplastis that merges; Wherein be added with the CaCl that concentration is 0.01mol/L in the fusogen
2
The step 4 protoplast regeneration adopts the double-layer plate culture method: with the protoplastis suspension doubling dilution of step 3 through merging, diluent is respectively drawn 0.5mL and 4.5mL PDA regeneration semisolid medium mixing, be poured into then on the PDA regeneration solid medium flat board, put into 28 ℃ environment and cultivated 3～5 days.
The step 5 genome rearrangement: whole regeneration bacterium colonies of collecting the step 4 cultivation are that F1 is for merging bacterium, prepare protoplastis with F1 for the heterozygosis mycelium then, and adopt the compound deactivation method of hot deactivation, ultraviolet inactivation and ultraviolet ray+lithium chloride in the step 2 that the F1 protoplastis is carried out deactivation respectively, three and four merge and regenerate set by step then, promptly obtain F2 for merging bacterium; Repetitive operation is up to obtaining F4 for merging bacterium, be that the nystatin PDA substratum of 135 μ g/mLs carry out resistance screen for merging bacterium with concentration with F4 again, to have drug-fast fusion bacterium then and carry out the shake flask fermentation cultivation, extraction, purifying meta-bolites, analyze, obtain the taxol superior strain.
Paclitaxel genome rearrangement strain HD FS4-26 adopts the selection seed selection of taxol superior strain of the present invention to come out.Adopt with background technology in identical fermentation process and fermentation condition ferment, the taxol output of paclitaxel genome rearrangement strain HD FS4-26 is 516.37 μ g/L, improved 64.41% than bacterial strain NCEU-1 taxol output, improved 31.52%～44.72% than parent strain taxol output.Its taxol output still keeps taxol output to be higher than 500 μ g/L after the paclitaxel genome rearrangement strain HD FS4-26 continuous passage 6 times, and the high taxol output stabilization characteristics of genetics of paclitaxel genome rearrangement strain HD FS4-26 is described.
Seed selection is simple by the following method for taxol superior strain of the present invention, and is effective, lays a good foundation for high paclitaxel produced industrial producing strain is provided.
Paclitaxel genome rearrangement strain HD FS4-26 of the present invention is that tree-shaped more piece spore (Nodulisporiumsylviforme) belongs to more piece spore genus (Nodulisporium), be preserved in Chinese typical culture collection center (CCTCC), the preservation address is a Wuhan University, preservation date is on February 28th, 2008, and preserving number is CCTCC M 208026.
Description of drawings
Fig. 1 is the taxol molecular formula, Fig. 2 is the microscopic examination figure of the conidiophore of strain HD FS4-26, Fig. 3 is conidial microscopic examination figure of strain HD FS4-26, Fig. 4 is the conidial head of the strain HD FS4-26 mode of arranging and the microscopic examination figure of the wart on the conidiophore, Fig. 5 is a taxol standard substance HPLC collection of illustrative plates, Fig. 6 is a strain HD FS4-26 fermented product extract HPLC collection of illustrative plates, and Fig. 7 is a paclitaxel genome rearrangement strain HD FS4-26 fermenting, abstracting and purifying material collection of illustrative plates.
Embodiment
Embodiment one: present embodiment paclitaxel genome rearrangement strain HD FS4-26 is that tree-shaped more piece spore (Nodulisporium sylviforme) belongs to more piece spore genus (Nodulisporium), be preserved in Chinese typical culture collection center (CCTCC), preservation date is on February 28th, 2008, and preserving number is CCTCCM 208026; The paclitaxel genome rearrangement strain HD FS4-26 bacterium colony cultivation initial stage is a white, and the later stage is dark-grey brown, and the back side is black; Give birth in the paclitaxel genome rearrangement strain HD FS4-26 mycelia part, the part hypergene has every, multiple-limb, and mycelia is that Vandyke brown shoals gradually to top property, and diameter is 2.24～5.86 μ m, does not see sclerotium and produces; Conidiophore Dan Sheng, majority is arborizations, and brown is thin out to top property, long 37～162 μ m; The conidiogenous cell column, single giving birth to or the colyliform arrangement, 6.37～22.63 μ m * 2.51～3.86 μ m, the sympodium formula is produced spore; Conidium Dan Sheng, ellipse or oval, the level and smooth or little wart of tool, no barrier film, 5.07～7.89 μ m * 2.45～3.79 μ m, conidium is head and arranges in the product spore point position of conidiogenous cell.
The conidiophore of present embodiment paclitaxel genome rearrangement strain HD FS4-26 as shown in Figure 2, conidium as shown in Figure 3, mode that conidial head is arranged and the wart on the conidiophore are as shown in Figure 4.
Present embodiment paclitaxel genome rearrangement strain HD FS4-26 adopts following method activation: the paclitaxel genome rearrangement strain HD FS4-26 of 4 ℃ of preservations is inoculated on the PDA slant medium, under 28 ℃ condition, activate 2～3 days (repetitive operation once), the strain HD FS4-26 of twice of slant activation is transferred in the triangular flask that 50mL PDA liquid nutrient medium is housed, put into 28 ℃, the shaking table of 120r/min and continued activation culture 2 days.
To insert in the S-7 substratum of improveing (the S-7 substratum of improvement is on the former S-7 medium base of Strobel phenylalanine, tyrosine, linolic acid to be increased to final concentration 1.5～5.0mg/L in 1993 to make) through the ratio of activatory paclitaxel genome rearrangement strain HD FS4-26 suspension in 3% (v/v), at 25 ℃, the condition bottom fermentation of 150r/min, fermentation time is 12 days.
Fermentation back taxol clicks step and extracts: will cultivate the thalline fermented liquid suction filtration of different time, filtrate and solid all reclaim; At twice filtrate is extracted the static 1h layering of separating funnel with isopyknic ethyl acetate; Be dissolved with taxol in the upper organic phase, lower floor's water discards; Grind remaining thalline behind the suction filtration simultaneously, and add ethyl acetate concussion extraction 30min and filter, filtrate is mixed with the organic phase after fermented liquid extracts; Remove solvent ethyl acetate with Rotary Evaporators, treat that surplus solution is 1/10 o'clock of stoste volume, surplus solution is poured in the plate, dry under 50 ℃ of conditions; Wash the taxol of the trace on plate surface more repeatedly with a spot of acetonitrile, fixed molten, 4 ℃ of preservations.
Embodiment two: the difference of present embodiment and embodiment one is: paclitaxel genome rearrangement strain HD FS4-26 cultivated 3 days on 25～28 ℃, PDA substratum, and bacterium colony is open and flat, and colony diameter is 4.1～5.8cm.Other is identical with embodiment one.
Embodiment three: present embodiment taxol superior strain seed selection by the following method: one, protoplastis preparation; Two, the deactivation of protoplastis; Three, the fusion of protoplastis; Four, protoplast regeneration; Five, genome rearrangement, screening promptly obtain the taxol superior strain; The bacterial strain that wherein is used for the protoplastis preparation is that taxol produces bacterium HD
100-23, UV
40-19And UL
50-6
Embodiment four: the difference of present embodiment and embodiment three is: the preparation of step 1 protoplastis: get the activatory taxol and produce bacterium HD
100-23, UV
40-19And UL
50-6Put into the PDA liquid nutrient medium and under 28 ± 0.5 ℃ condition, leave standstill cultivation 3 days, bacteria suspension centrifugal 10min under the 4000g condition collects mycelium, again with the mycelium vibration evenly with vortex mixer, then with homeo-osmosis agent washing 2 times, the ratio adding enzymatic lysis liquid that adds 1mL enzymatic lysis liquid afterwards in every 250mg mycelium, place 30 ℃ of environment temperature to bathe 7h again, filter with 3 layers of aseptic lens paper then, filtrate 4000g is centrifugal 10min under the 4000g condition, the protoplasma precipitation obtains the protoplastis of purifying again for 2 times with homeo-osmosis agent washing, afterwards protoplastis is suspended in the homeo-osmosis agent.Other step and parameter are identical with embodiment three.
Stablizer in the present embodiment is that concentration is the NaCl solution of 0.7mol/L.
Enzymatic lysis liquid in the present embodiment is the prozyme system of cellulase, helicase and N,O-Diacetylmuramidase, the pH value of enzymatic lysis liquid is 5.5～6.0, the concentration of cellulase is 30mg/mL in the enzymatic lysis liquid, and the concentration of helicase is 20mg/mL, and the concentration of N,O-Diacetylmuramidase is 10mg/mL.
Embodiment five: the difference of present embodiment and embodiment four is: protoplastis is suspended in the homeo-osmosis agent, and the concentration of protoplastis is 1.0 * 10
7Cfu/mL.Other step and parameter are identical with embodiment four.
Embodiment six: the difference of present embodiment and embodiment three is: the deactivation of step 2 protoplastis:
With the HD behind the purifying
100-23The protoplastis suspension moves in the sterile test tube, and 50 ℃ of water bath with thermostatic control 5min carry out hot deactivation;
With the UV behind the purifying
40-19The protoplastis suspension to move into diameter be in the aseptic plate of 6cm, place in advance under the thermally-stabilised 30W ultraviolet lamp, plate and ultraviolet lamp vertical range are 30cm, shine 100s and carry out ultraviolet inactivation;
UL behind purifying
50-6The protoplastis suspension in to add final concentration be 0.6%LiCl, place immediately in advance then under the thermally-stabilised 30W ultraviolet lamp, plate and ultraviolet lamp vertical range are 30cm, shine 70s and carry out the compound deactivation of ultraviolet ray+lithium chloride.Other step and parameter are identical with embodiment three.
Embodiment seven: present embodiment with the difference of embodiment three is: the fusion of step 3 protoplastis: respectively get the 1mL suspension and mix in twos from deactivation protoplastis suspension, mixed solution is centrifugal 10min under the condition of 3000r/min, after removing centrifuged supernatant protoplastis is suspended in the agent of 0.2mL homeo-osmosis, add 1.8mL again, temperature is 30 ℃, mass concentration is 35% PEG fusogen, water bath heat preservation 90s under 30 ℃ of conditions, add 5mL again, temperature is that 4 ℃ homeo-osmosis agent stops merging, centrifuge washing is removed fusogen then, is suspended in the homeo-osmosis agent more again through the protoplastis that merges; Wherein be added with the CaCl that concentration is 0.01mol/L in the fusogen
2Other step and parameter are identical with embodiment three.
Embodiment eight: the difference of present embodiment and embodiment three is: the step 4 protoplast regeneration adopts the double-layer plate culture method: with the protoplastis suspension doubling dilution of step 3 through merging, diluent is respectively drawn 0.5mL and 4.5mL PDA regeneration semisolid medium mixing, be poured into then on the PDA regeneration solid medium flat board, put into 28 ℃ environment and cultivated 3～5 days.Other step and parameter are identical with embodiment three.
Embodiment nine: the difference of present embodiment and embodiment three is: the step 5 genome rearrangement: whole regeneration bacterium colonies of collecting the step 4 cultivation are that F1 is for merging bacterium, prepare protoplastis with F1 for the heterozygosis mycelium then, and adopt the compound deactivation method of hot deactivation, ultraviolet inactivation and ultraviolet ray+lithium chloride in the step 2 that the F1 protoplastis is carried out deactivation respectively, three and four merge and regenerate set by step then, promptly obtain F2 for merging bacterium; Repetitive operation is up to obtaining F4 for merging bacterium, be that the nystatin PDA substratum of 135 μ g/mLs carry out resistance screen for merging bacterium with concentration with F4 again, to have drug-fast fusion bacterium then and carry out the shake flask fermentation cultivation, extraction, purifying meta-bolites, analyze, obtain the taxol superior strain.Other step and parameter are identical with embodiment three.
Embodiment ten: the difference of present embodiment and embodiment nine is: adopt thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) and mass spectrum (MS) method to analyze.Other step and parameter are identical with embodiment nine.
Embodiment 11: present embodiment adopts embodiment three method seed selections to obtain paclitaxel genome rearrangement strain HD FS4-26.
The high yield paclitaxel genome rearrangement strain HD FS4-26 that utilizes the present embodiment seed selection to obtain carries out fermentation culture and taxol extracts, purifying, adopt high performance liquid chromatography (HPLC) and mass spectrum (MS) method to analyze then respectively, the analytical results difference is (Fig. 5 is the collection of illustrative plates of taxol standard substance HPLC, and arrow is expressed as the taxol peak among Fig. 5, Fig. 6 and Fig. 7) as shown in Figure 6 and Figure 7.
Claims (1)
1. paclitaxel genome rearrangement strain HD FS4-26, it is characterized in that paclitaxel genome rearrangement strain HD FS4-26 is that tree-shaped more piece spore (Nodulisporium sylviforme) belongs to more piece spore genus (Nodulisporium), be preserved in Chinese typical culture collection center, preservation date is on February 28th, 2008, and preserving number is CCTCC M 208026; The paclitaxel genome rearrangement strain HD FS4-26 bacterium colony cultivation initial stage is a white, and the later stage is dark-grey brown, and the back side is black; Give birth in the paclitaxel genome rearrangement strain HD FS4-26 mycelia part, the part hypergene has every, multiple-limb, and mycelia is that Vandyke brown shoals gradually to top property, and diameter is 2.24～5.86 μ m, does not see sclerotium and produces; Conidiophore Dan Sheng, majority is arborizations, and brown is thin out to top property, long 37～162 μ m; The conidiogenous cell column, single giving birth to or the colyliform arrangement, 6.37～22.63 μ m * 2.51～3.86 μ m, the sympodium formula is produced spore; Conidium Dan Sheng, ellipse or oval, the level and smooth or little wart of tool, no barrier film, 5.07～7.89 μ m * 2.45～3.79 μ m, conidium is head and arranges in the product spore point position of conidiogenous cell.
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| CN103194502B (en) * | 2013-04-24 | 2015-05-27 | 黑龙江大学 | Separating and purifying method of taxol synthesized by biological fermentation of nodulisporium sylviforme entophytic fungi as well as precursors of taxol |
| CN108865899A (en) * | 2018-07-06 | 2018-11-23 | 上海市农业科学院 | A kind of agaricus bisporus bacterial strain and its selection |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1377957A (en) * | 2001-03-28 | 2002-11-06 | 齐齐哈尔大学 | New taxol producing fungus and taxol producing fermentation process |
| CN1511943A (en) * | 2002-12-30 | 2004-07-14 | 黑龙江大学 | Engineering strain and its preparing method, and method for preparing taxol thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1377957A (en) * | 2001-03-28 | 2002-11-06 | 齐齐哈尔大学 | New taxol producing fungus and taxol producing fermentation process |
| CN1511943A (en) * | 2002-12-30 | 2004-07-14 | 黑龙江大学 | Engineering strain and its preparing method, and method for preparing taxol thereof |
Non-Patent Citations (2)
| Title |
|---|
| 赵凯等.产紫杉醇菌株原生质体诱变育种的研究.生物工程学报21 5.2005,21(5),848-851. |
| 赵凯等.产紫杉醇菌株原生质体诱变育种的研究.生物工程学报21 5.2005,21(5),848-851. * |
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