CN101709297A - Mutagenesis screening method of arachidonic acid producing strain mortierella alpina - Google Patents
Mutagenesis screening method of arachidonic acid producing strain mortierella alpina Download PDFInfo
- Publication number
- CN101709297A CN101709297A CN200910234375A CN200910234375A CN101709297A CN 101709297 A CN101709297 A CN 101709297A CN 200910234375 A CN200910234375 A CN 200910234375A CN 200910234375 A CN200910234375 A CN 200910234375A CN 101709297 A CN101709297 A CN 101709297A
- Authority
- CN
- China
- Prior art keywords
- arachidonic acid
- seed
- mortierella alpina
- mutagenesis
- screening method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 title claims abstract description 86
- 229940114079 arachidonic acid Drugs 0.000 title claims abstract description 43
- 235000021342 arachidonic acid Nutrition 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 35
- 238000002703 mutagenesis Methods 0.000 title claims abstract description 34
- 231100000350 mutagenesis Toxicity 0.000 title claims abstract description 34
- 238000012216 screening Methods 0.000 title claims abstract description 28
- 241000907999 Mortierella alpina Species 0.000 title claims abstract description 25
- 241000894006 Bacteria Species 0.000 claims abstract description 42
- 238000000855 fermentation Methods 0.000 claims abstract description 17
- 230000004151 fermentation Effects 0.000 claims abstract description 17
- 239000007788 liquid Substances 0.000 claims abstract description 10
- JDNTWHVOXJZDSN-UHFFFAOYSA-N iodoacetic acid Chemical compound OC(=O)CI JDNTWHVOXJZDSN-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 8
- 230000037361 pathway Effects 0.000 claims abstract description 8
- 230000012010 growth Effects 0.000 claims abstract description 6
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 claims description 20
- 230000001580 bacterial effect Effects 0.000 claims description 17
- 239000000725 suspension Substances 0.000 claims description 15
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 230000009514 concussion Effects 0.000 claims description 10
- 239000002028 Biomass Substances 0.000 claims description 9
- 239000004519 grease Substances 0.000 claims description 9
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- 230000004913 activation Effects 0.000 claims description 8
- 229940041514 candida albicans extract Drugs 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- 239000002504 physiological saline solution Substances 0.000 claims description 8
- 239000012138 yeast extract Substances 0.000 claims description 8
- 229910019142 PO4 Inorganic materials 0.000 claims description 6
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 6
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 6
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 6
- 235000003170 nutritional factors Nutrition 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 6
- 239000010452 phosphate Substances 0.000 claims description 6
- 229910052700 potassium Inorganic materials 0.000 claims description 6
- 239000011591 potassium Substances 0.000 claims description 6
- 229940001516 sodium nitrate Drugs 0.000 claims description 6
- 235000010344 sodium nitrate Nutrition 0.000 claims description 6
- 239000004317 sodium nitrate Substances 0.000 claims description 6
- 241001192924 Parna Species 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 4
- 239000003112 inhibitor Substances 0.000 claims description 4
- 239000002054 inoculum Substances 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims description 4
- 238000011218 seed culture Methods 0.000 claims description 4
- 238000010186 staining Methods 0.000 claims description 4
- BSYNRYMUTXBXSQ-FOQJRBATSA-N 59096-14-9 Chemical compound CC(=O)OC1=CC=CC=C1[14C](O)=O BSYNRYMUTXBXSQ-FOQJRBATSA-N 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 238000002203 pretreatment Methods 0.000 claims description 3
- 238000012807 shake-flask culturing Methods 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 229920000742 Cotton Polymers 0.000 claims description 2
- -1 compound lithium chloride Chemical class 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 230000035040 seed growth Effects 0.000 claims description 2
- 230000008569 process Effects 0.000 abstract description 9
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 abstract description 7
- 229960001138 acetylsalicylic acid Drugs 0.000 abstract description 7
- 230000034659 glycolysis Effects 0.000 abstract description 3
- 150000003628 tricarboxylic acids Chemical class 0.000 abstract description 3
- 238000012258 culturing Methods 0.000 abstract description 2
- 230000003213 activating effect Effects 0.000 abstract 1
- 230000004136 fatty acid synthesis Effects 0.000 abstract 1
- 230000000813 microbial effect Effects 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- 238000012262 fermentative production Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 238000013459 approach Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 2
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 238000005468 ion implantation Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 231100000219 mutagenic Toxicity 0.000 description 2
- 230000003505 mutagenic effect Effects 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- 150000003180 prostaglandins Chemical class 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- KIWBPDUYBMNFTB-UHFFFAOYSA-N Ethyl hydrogen sulfate Chemical compound CCOS(O)(=O)=O KIWBPDUYBMNFTB-UHFFFAOYSA-N 0.000 description 1
- 241000235575 Mortierella Species 0.000 description 1
- 241001219224 Mortierella elongata Species 0.000 description 1
- VZUNGTLZRAYYDE-UHFFFAOYSA-N N-methyl-N'-nitro-N-nitrosoguanidine Chemical compound O=NN(C)C(=N)N[N+]([O-])=O VZUNGTLZRAYYDE-UHFFFAOYSA-N 0.000 description 1
- 102000004005 Prostaglandin-endoperoxide synthases Human genes 0.000 description 1
- 108090000459 Prostaglandin-endoperoxide synthases Proteins 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000003570 biosynthesizing effect Effects 0.000 description 1
- 230000001851 biosynthetic effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 235000021323 fish oil Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 238000003012 network analysis Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 235000020777 polyunsaturated fatty acids Nutrition 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000002787 reinforcement Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000008467 tissue growth Effects 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a mutagenesis screening method of mortierella alpina of arachidonic acid producing bacteria. After the microbial glycolysis pathway, a tricarboxylic acid transport system, a fatty acid synthesis pathway and other related pathways in the process of producing AA by fermenting Mortierella alpina are analyzed, iodoacetic acid and acetylsalicylic acid are selected as screening agents, and strains with high growth rate of arachidonic acid, short fermentation period and high yield of arachidonic acid can be obtained by the steps of activating seeds, culturing the seeds, pretreating seed liquid, mutagenizing seed treatment liquid, primary screening, secondary screening, rescreening and the like.
Description
Technical field
The invention belongs to technical field of bioengineering, the arachidonic field of Mortierella alpina fermentative Production especially is specifically related to obtain the mutagenesis screening method of a plant height output Mortierella alpina.
Background technology
(Arachidonic acid is the important unsaturated fatty acids that belongs to ω-6 series AA) to arachidonic acid, is human body prostaglandin(PG) synthetic important precursor, also is that content is the highest in the human body, and the widest a kind of polyunsaturated fatty acid distributes.It in reducing blood-fat, anticoagulant, anti-inflammatory, anticancer, lipotropism matter oxidation, promote aspect such as cerebral tissue growth to have the unique biological activity, so Application Areas is very extensive, has caused the great interest of people.Arachidonic acid extensively and slightly is present in animal body and the microorganism, and at present, the commercialization arachidonic acid is mainly derived from animal lipid or fish oil.But because resource-constrained, and arachidonic acid content is very low in the product that is extracted, and not only quality product can not satisfy people's consumers demand, and cost is high always, be not suitable for scale operation, thereby its research for a long time and use is restricted always.Utilize Production by Microorganism Fermentation arachidonic acid owing to have not to be subjected to starting material and weather restriction, with short production cycle, advantage such as cost is lower, product content height, separation and Extraction are easy, become current research focus.
Arachidonic production bacterial classification mainly is a mould, with mortierella for.A good bacterial classification is the core of fermentation industry, and in recent years, countries in the world are studied strain improvement in succession.Yamada in 1987 etc. separate from soil and obtain many strains AA and produce bacterium, obtain a plant height through natural seed selection and produce bacterium Mortierella elongata IS-5, utilize glucose to carry out fermentative production as carbon source, and AA output is 4.3g/L.2000, Yao Jianming etc. utilize ionic fluid injection biotechnology that Mortierella alpine is carried out mutagenesis, and screening obtains a plant height and produces bacterium, and every liter of nutrient solution can get biomass 30.80g, the dry mycelium fat content is 25.8%, and arachidonic content accounts for 45.37% of total fat.2004, MNNG mutagenesis M.alpina 1S-4 such as Eiji obtained the low mutant strain Y11 of ω-3 dehydrogenase activity, and its AA output can reach 4.97g/L.2008, Zhao Mo etc. are starting strain with the mould ultraviolet mutagenesis mutant strain A1.13 of wheel stalk, prepare its protoplastis and adopt ultraviolet ray, microwave and ethyl sulfate to carry out the mutagenesis of many wheels continuously as mutagenic compound, sieve again by 15 ℃ of low temperature and 0.12g/mL acetylsalicylic acid primary dcreening operation and shake flask fermentation, obtain the mutant strain MW40+DES20 II-4 that the arachidonic acid yield improves, its arachidonic acid accounts for the content of total fatty acids and brings up to 15.098%.Sum up to find that from domestic and foreign literature purpose all is in order to improve the arachidonic acid ultimate capacity, but a lot of strain improvement means are more loaded down with trivial details, process is tediously long, and blindly, randomness is bigger, has expended great amount of manpower and material resources.
Before the present patent application, some are also arranged, and (publication number has CN1362522A, CN1587378A) about the report of the relevant patent of arachidonic acid strain improvement.Wherein in CN1362522A, with the Mortierella alpina is starting strain, by the N+ ion implantation mutagenesis, obtain strain activity mutagenesis bacterium preferably, by fermentation, biomass can reach 30g/L, grease 10.8g/L, arachidonic acid productive rate 4.65g/L, this patent shortcoming is ion implantation perfect inadequately with the interactional mechanism research of mould on the one hand, be that the arachidonic acid yield is lower on the other hand, be difficult to resist the pressure that extraneous intratype competition person brings.Among the publication number CN1587378A, disclose a kind of separating screening method of arachidonic acid high yielding strain, effect is obvious, but this only is a kind of process of natural seed selection, and AA output can not be greatly enhanced.
Summary of the invention
Technical problem to be solved by this invention provides the mutagenesis screening method that a kind of arachidonic acid that simply is easy to realize produces the bacterium Mortierella alpina.
For solving the problems of the technologies described above, thinking of the present invention is mainly based on the rational breeding technique of pathways metabolism, by to arachidonic acid biosynthetic metabolism network analysis, selected suitable selective agent, selecting the starting point of selective agent is by the arachidonic acid metabolic pathway of synthesizing being analyzed, crucial mesostate being regulated and control to obtain the higher bacterial strain of arachidonic acid yield.
Concrete technical scheme is as follows:
A kind of arachidonic acid produces the mutagenesis screening method of bacterium Mortierella alpina, comprises the steps:
(1) activation of seed;
(2) cultivation of seed;
(3) pre-treatment of seed liquor:
(3a) seed liquor obtains finely dispersed bacteria suspension after stroke-physiological saline solution dilution, concussion, filtration;
(3b) will add lithium chloride in the bacteria suspension, 1～6h is handled in concussion;
(4) the seed treatment liquid that step (3b) is obtained carries out mutagenesis;
(5) screening:
(5a) primary dcreening operation: the bacteria suspension after the mutagenesis that step (4) is obtained is coated on the flat board that contains EMP Embden Meyerbof Parnas pathway inhibitor and PGSI, chooses to grow at first and single bacterium colony that diameter is bigger;
(5b) multiple sieve: the single bacterium colony switching inclined-plane that will choose, treat that the inclined-plane covers with after, carry out shake-flask culture again, in seed liquor, add the TCC staining agent, by observing, pick out the bacterium colony that the variable color time is the shortest, color is the darkest;
(5c) rescreen: will sieve the seed liquor of choosing again and carry out the shake flask fermentation cultivation, be final screening index with biomass, grease yield, arachidonic acid yield, chooses the strongest bacterial strain of arachidonic acid energy for growth.
In the step (1), the activation method of seed is: 25～28 ℃ on the PDA inclined-plane that mycelium is inoculated in the PDA inclined-plane or has added nutritional factor was cultivated 5～7 days, wherein, described nutritional factor is any one or a few the mixture in yeast extract paste, peptone and the extractum carnis, and the addition of nutritional factor is 1～5g/L.
In the step (2), the cultural method of seed is: activation mycelium inclined-plane well washes mycelium come with the sterilized water of 5～10mL, then mycelium is inserted in the shake-flask seed substratum inoculum size 10～20% (v/v), cultivate 48～72h, treat that seed growth is to logarithmic phase for 20～28 ℃.
In the step (3a), seed liquor inserts in the groove triangular flask that is covered with granulated glass sphere that sterilising treatment has been crossed, with volume is that 8～10 times of seed liquor, concentration are the stroke-physiological saline solution dilution of 0.9g/100mL, 25 ℃, 25～30min is handled in the 250rpm concussion, filter with aseptic 2～8 layers of gauze or absorbent cotton, obtain finely dispersed bacteria suspension;
In the step (3b), bacteria suspension adds in the triangular flask of the physiological salt liquid that the lithium chloride that volume is identical with bacteria suspension, concentration is 0.6～2.0g/100mL is housed that (concentration is that the compound method of physiological salt liquid of the lithium chloride of 0.6～2.0g/100mL is: the stroke-physiological saline solution of preparing 0.9g/100mL earlier, dissolve in the lithium chloride of 0.6～2.0g/100mL again), triangular flask is placed 20～28 ℃ of concussion incubators, and rotating speed 100～150r/m handles 1～6h.
In the step (4), described mutafacient system is the compound lithium chloride mutagenesis of ultraviolet, be about to adding that step (3b) obtains the bacteria suspension of lithium chloride place under 15～30W ultraviolet lamp, irradiation distance 20～35cm, irradiation time is 60～80s.
In the step (5a), described EMP Embden Meyerbof Parnas pathway inhibitor is an iodoacetic acid, and add-on is 0.03～0.075g/L; Described PGSI is an acetylsalicylic acid, and add-on is 0.1～0.4g/L.The present invention is by analyzing microorganism glycolytic pathway, tricarboxylic acid delivery system and lipid acid route of synthesis and other relational approach in the Mortierella alpina fermentative production arachidonic acid process, find: a, iodoacetic acid is by reducing the activity of glyceraldehyde 3-phosphate dehydro-genase, suppressed EMP Embden Meyerbof Parnas pathway, corresponding reinforcement HMP approach, thereby produce more NADPH, and a large amount of supplies of NADPH arachidonic acid synthetic key point just can promote effectively arachidonic synthetic; B; acetylsalicylic acid can suppress the oxidizing reaction in the prostaglandin(PG) building-up process by the aminoterminal acetylize to prostaglandin synthetase; thereby acetylsalicylic acid can suppress arachidonic biosynthesizing; the bacterial strain that screening has resistance to acetylsalicylic acid also can improve arachidonic generation.Therefore, the present invention has selected for use iodoacetic acid and acetylsalicylic acid as " sieve ".
In the step (5b), described TCC staining agent is that concentration is the TCC aqueous solution of 2～4g/L, and the adding volume of the TCC aqueous solution and seed liquor volume ratio are 1: 0.25～2.
Wherein, described seed culture based formulas is: glucose 20～60g/L, and yeast extract paste 3～6g/L, potassium primary phosphate 2～5g/L, SODIUMNITRATE 2～5g/L, magnesium sulfate heptahydrate 0.3～1g/L, all the other are water, pH6.0～8.5.
Wherein, described fermentative medium formula is: glucose 60～150g/L, and yeast extract paste 5～12g/L, potassium primary phosphate 2～6g/L, SODIUMNITRATE 2～6g/L, magnesium sulfate heptahydrate 0.5～1g/L, all the other are water.
Wherein, the pretreatment process of iodoacetic acid is to utilize 0.22 μ m or 0.44 μ m sterilized water membrane filtration behind the deionized water dissolving again, keeps in Dark Place; Acetysalicylic pretreatment process is to utilize 0.22 μ m or the aseptic organic membrane filter of 0.44 μ m behind the anhydrous alcohol solution again, keeps in Dark Place.
Beneficial effect: the present invention is to the microorganism glycolytic pathway in the Mortierella alpina fermentative production AA process, after tricarboxylic acid delivery system and lipid acid route of synthesis and other relational approach are analyzed, selected iodoacetic acid for use, acetylsalicylic acid is as selective agent, through primary dcreening operation, multiple sieve, several steps such as rescreen, it is fast to obtain the arachidonic acid growth velocity, fermentation period is short, the very high bacterial strain of arachidonic acid yield increase rate, it is the rational breeding technique of a kind of very ideal bacterial classification, can realize that arachidonic acid produces the high flux screening of bacterium Mortierella alpina, have very high economic benefit and social benefit, industrial prospect is good.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, processing condition and result thereof only are used to illustrate the present invention, and should also can not limit the present invention described in detail in claims.
The used first-selected culture medium prescription of following examples is as follows:
Seed culture medium: glucose 30g/L, yeast extract paste 5g/L, potassium primary phosphate 3g/L, SODIUMNITRATE 3g/L, magnesium sulfate heptahydrate 0.6g/L, all the other are water.
Solid medium: PDA+PE+BE: murphy juice 200g/L, glucose 20g/L, peptone 1.5g/L, extractum carnis 1.5g/L, agar 20g/L (, being PDA) as undeclared.
Fermention medium: glucose 60g/L, yeast extract paste 5g/L, potassium primary phosphate 3g/L, SODIUMNITRATE 6g/L, magnesium sulfate heptahydrate 1g/L, all the other are water.
Embodiment 1:
1. the activation of seed:
Mortierella alpine mould species ATCC 16266 is inoculated in the PDA+PE+BE inclined-plane to be cultivated 5 days for 28 ℃.
2. the cultivation of seed:
After treating that 1. the inclined-plane covers with, wash mycelia with the 10mL sterilized water, be inoculated in the seed culture medium, inoculum size 20% (v/v) was cultivated 60 hours for 25 ℃, and this moment, seed grew to logarithmic phase.
3. the pre-treatment of seed liquor:
Pretreatment process: the 2. seed liquor 5mL of gained, insert in the 250mL groove triangular flask that is covered with granulated glass sphere that sterilising treatment has been crossed, the stroke-physiological saline solution 45mL that adds 0.9g/100mL, 25 ℃, 30min is handled in the 250r/m concussion, with 4 layers of filtered through gauze of the bacterium of going out, resulting bacteria suspension is got 5mL and is added and to be equipped with in the aseptic triangular flask of physiological salt liquid of LiCl that 5mL concentration is 0.6～2.0g/100mL, places 25 ℃ of incubators of concussion, rotating speed 120r/m to handle 2h triangular flask.
4. the mutagenesis of seed liquor:
3. gained seed treatment liquid is injected the aseptic blank culture dish that is equipped with magnetic stirring bar, and bacterium liquid thickness 2mm carries out ultraviolet mutagenesis under the dark condition.Mutagenic condition: ultraviolet lamp 30W, irradiation distance 30cm, irradiation time is 75s.Under the red light condition, take out culture dish, carry out the flat board coating after the physiological saline dilution with 0.9g/100mL.
5. screening:
A, with the seed liquor that 4. obtains under the red light condition with 10 times of physiological saline dilutions, be coated on and contain on 0.075g/L iodoacetic acid and the acetysalicylic solid plate of 0.2g/L, dark culturing 2 days is chosen and is grown bacterial strain 30 strains that colony diameter is big at first.
B, the 30 strain bacterial strains that will choose switching PDA inclined-plane carries out the seed shake-flask culture again.Get seed liquor 2mL in test tube, after 28 ℃ of insulation balances, add 2 3g/L TCC aqueous solution (about 1mL), shake up, timing, it is darker to choose color, bacterial strain 10 strains that the variable color time is the shortest.
C, the 10 strain bacterial strain seed liquor that will choose are seeded in the fermention medium, and inoculum size is 10% (v/v), fermentation to sugar consumption is zero, time 6.5-7 days, measure biomass, grease yield and arachidonic acid yield respectively, compare, obtain all much higher bacterial strain of 5 strain biomasss, grease yield, AA output than original strain, difference called after HM-1, HM-2, HM-3, HM-4, HM-5.
Embodiment 2: genetic stability is investigated.
The 5 plant heights product bacterial strain that embodiment 1 is obtained carries out the genetic stability investigation, goes down to posterity 10 times, whenever goes down to posterity and all carries out the shake flask fermentation experiment for 1 time, measures its biomass, grease yield and AA output.Experimental results show that, through repeatedly going down to posterity and fermenting checking, biomass, grease yield and AA output are all suitable with activation fermentation level once, that wherein the highest the and growth vigor of AA output is the strongest is HM-5, in first sugared concentration is under the 60g/L condition, biomass is stabilized in about 35g/L, and grease yield maintains about 18g/L, and AA output maintains about 9g/L.
Embodiment 3: leavening property relatively before and after the bacterial strain mutagenesis.
Experimentize chamber 50mL shake flask fermentation of bacterial strain HM-5 is cultivated, 25 ℃ of temperature, rotating speed 120rpm, initial sugared concentration 60g/L fermented 7 days.The fermentation result sees Table 1 before and after the contrast mutagenesis.
Fermentation result contrast before and after the mutagenesis of table 1 bacterial strain
| Biomass (g/L) | Total grease (g/L) | AA output (g/L) | Fermentation time (h) | AA productive rate (g/Lh) | |
| Original strain | ??25.0 | ??13.0 | ??5.1 | ??168 | ??0.03 |
| ??HM-5 | ??35 | ??18 | ??9 | ??156 | ??0.058 |
By on show, the strain growth activity increases after the mutagenesis, fermentation time obviously shortens, the AA productive rate rises to 0.058g/Lh by 0.03g/Lh.
Embodiment 4:
With the method for embodiment 1, step that different is 5. in, the iodoacetic acid add-on is 0.03g/L; Described PGSI is an acetylsalicylic acid, and add-on is 0.4g/L.
Claims (10)
1. the mutagenesis screening method of an arachidonic acid generation bacterium Mortierella alpina is characterized in that this method comprises the steps:
(1) activation of seed;
(2) cultivation of seed;
(3) pre-treatment of seed liquor:
(3a) seed liquor obtains finely dispersed bacteria suspension after stroke-physiological saline solution dilution, concussion, filtration;
(3b) will add lithium chloride in the bacteria suspension, 1～6h is handled in concussion;
(4) the seed treatment liquid that step (3b) is obtained carries out mutagenesis;
(5) screening:
(5a) primary dcreening operation: the bacteria suspension after the mutagenesis that step (4) is obtained is coated on the flat board that contains EMP Embden Meyerbof Parnas pathway inhibitor and PGSI, chooses to grow at first and single bacterium colony that diameter is bigger;
(5b) multiple sieve: the single bacterium colony switching inclined-plane that will choose, treat that the inclined-plane covers with after, carry out shake-flask culture again, in seed liquor, add the TCC staining agent, by observing, pick out the bacterium colony that the variable color time is the shortest, color is the darkest;
(5c) rescreen: will sieve the seed liquor of choosing again and carry out the shake flask fermentation cultivation, be final screening index with biomass, grease yield, arachidonic acid yield, chooses the strongest bacterial strain of arachidonic acid energy for growth.
2. arachidonic acid according to claim 1 produces the mutagenesis screening method of bacterium Mortierella alpina, it is characterized in that in the step (1), the activation method of seed is: 25～28 ℃ on the PDA inclined-plane that mycelium is inoculated in the PDA inclined-plane or has added nutritional factor was cultivated 5～7 days, wherein, described nutritional factor is any one or a few the mixture in yeast extract paste, peptone and the extractum carnis, and the addition of nutritional factor is 1～5g/L.
3. arachidonic acid according to claim 1 produces the mutagenesis screening method of bacterium Mortierella alpina, it is characterized in that in the step (2), the cultural method of seed is: activation mycelium inclined-plane well washes mycelium come with the sterilized water of 5～10mL, mycelium is inserted in the shake-flask seed substratum then, inoculum size 10～20% (v/v), cultivate 48～72h, treat that seed growth is to logarithmic phase for 20～28 ℃.
4. arachidonic acid according to claim 3 produces the mutagenesis screening method of bacterium Mortierella alpina, it is characterized in that described seed culture based formulas is: glucose 20～60g/L, yeast extract paste 3～6g/L, potassium primary phosphate 2～5g/L, SODIUMNITRATE 2～5g/L, magnesium sulfate heptahydrate 0.3～1g/L, all the other are water, pH6.0～8.5.
5. arachidonic acid according to claim 1 produces the mutagenesis screening method of bacterium Mortierella alpina, it is characterized in that in the step (3a), seed liquor inserts in the groove triangular flask that is covered with granulated glass sphere that sterilising treatment has been crossed, with volume is that 8～10 times of seed liquor, concentration are the stroke-physiological saline solution dilution of 0.9g/100mL, 25 ℃, 25～30min is handled in 250rpm concussion, with aseptic 2～8 layers of gauze or absorbent cotton filtration, obtains finely dispersed bacteria suspension.
6. arachidonic acid according to claim 1 produces the mutagenesis screening method of bacterium Mortierella alpina, it is characterized in that in the step (3b), bacteria suspension adds in the triangular flask of the physiological salt liquid that the lithium chloride that volume is identical with bacteria suspension, concentration is 0.6～2.0g/100mL is housed, and places 20～28 ℃ of incubators of concussion, rotating speed 100～150r/m to handle 1～6h triangular flask.
7. arachidonic acid according to claim 1 produces the mutagenesis screening method of bacterium Mortierella alpina, it is characterized in that in the step (4), described mutafacient system is the compound lithium chloride mutagenesis of ultraviolet, be about to adding that step (3b) obtains the bacteria suspension of lithium chloride place under 15～30W ultraviolet lamp, irradiation distance 20～35cm, irradiation time is 60～80s.
8. arachidonic acid according to claim 1 produces the mutagenesis screening method of bacterium Mortierella alpina, it is characterized in that in the step (5a), described EMP Embden Meyerbof Parnas pathway inhibitor is an iodoacetic acid, and add-on is 0.03～0.075g/L; Described PGSI is an acetylsalicylic acid, and add-on is 0.1～0.4g/L.
9. arachidonic acid according to claim 1 produces the mutagenesis screening method of bacterium Mortierella alpina, it is characterized in that in the step (5b), described TCC staining agent is that concentration is the TCC aqueous solution of 2～4g/L, and the adding volume of the TCC aqueous solution and seed liquor volume ratio are 1: 0.25～2.
10. arachidonic acid according to claim 1 produces the mutagenesis screening method of bacterium Mortierella alpina, it is characterized in that in the step (5c), described fermentation culture, its culture medium prescription is: glucose 60～150g/L, yeast extract paste 5～12g/L, potassium primary phosphate 2～6g/L, SODIUMNITRATE 2～6g/L, magnesium sulfate heptahydrate 0.5～1g/L, all the other are water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910234375A CN101709297A (en) | 2009-11-25 | 2009-11-25 | Mutagenesis screening method of arachidonic acid producing strain mortierella alpina |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910234375A CN101709297A (en) | 2009-11-25 | 2009-11-25 | Mutagenesis screening method of arachidonic acid producing strain mortierella alpina |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101709297A true CN101709297A (en) | 2010-05-19 |
Family
ID=42402102
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910234375A Pending CN101709297A (en) | 2009-11-25 | 2009-11-25 | Mutagenesis screening method of arachidonic acid producing strain mortierella alpina |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101709297A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703332A (en) * | 2012-06-19 | 2012-10-03 | 南京工业大学 | Bacterial strain for producing arachidonic acid grease and application thereof |
| CN103484385A (en) * | 2013-10-11 | 2014-01-01 | 武汉轻工大学 | Mortierella alpina mutant strain capable of producing high-yield arachidonic acid, and fermentation method and application thereof |
| CN105861339A (en) * | 2016-06-16 | 2016-08-17 | 江南大学 | Recombination mortierella alpine of overexpression GTP ring type hydrolytic enzyme gene and construction method and application of recombination mortierella alpine |
| CN106834125A (en) * | 2017-01-24 | 2017-06-13 | 江南大学 | It is a kind of to produce culture medium of polyunsaturated fatty acid filamentous fungi and application thereof for quick screening |
| CN108410916A (en) * | 2018-05-30 | 2018-08-17 | 湖北福星生物科技有限公司 | The production method of arachidonic acid oil |
| CN110331099A (en) * | 2019-07-31 | 2019-10-15 | 江南大学 | A kind of rapid screening method of oil-producing filamentous fungi genetic modification bacterial strain |
-
2009
- 2009-11-25 CN CN200910234375A patent/CN101709297A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703332A (en) * | 2012-06-19 | 2012-10-03 | 南京工业大学 | Bacterial strain for producing arachidonic acid grease and application thereof |
| CN103484385A (en) * | 2013-10-11 | 2014-01-01 | 武汉轻工大学 | Mortierella alpina mutant strain capable of producing high-yield arachidonic acid, and fermentation method and application thereof |
| CN105861339A (en) * | 2016-06-16 | 2016-08-17 | 江南大学 | Recombination mortierella alpine of overexpression GTP ring type hydrolytic enzyme gene and construction method and application of recombination mortierella alpine |
| CN105861339B (en) * | 2016-06-16 | 2019-10-18 | 江南大学 | Recombination Mortierella alpina, its construction method and the application of one plant of overexpression GTP cyclohydrolase gene |
| CN106834125A (en) * | 2017-01-24 | 2017-06-13 | 江南大学 | It is a kind of to produce culture medium of polyunsaturated fatty acid filamentous fungi and application thereof for quick screening |
| CN108410916A (en) * | 2018-05-30 | 2018-08-17 | 湖北福星生物科技有限公司 | The production method of arachidonic acid oil |
| CN110331099A (en) * | 2019-07-31 | 2019-10-15 | 江南大学 | A kind of rapid screening method of oil-producing filamentous fungi genetic modification bacterial strain |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101109015B (en) | Preparation method of arachidonic acid grease | |
| CN102174449B (en) | Method for producing high-yield gamma-propalanine and application thereof | |
| CN102864111B (en) | Schizochytrium limacinum strain for producing docosahexaenoic acid | |
| JP2014509188A (en) | Process for the production of microalgae, cyanobacteria and their metabolites | |
| CN101709297A (en) | Mutagenesis screening method of arachidonic acid producing strain mortierella alpina | |
| CN112239728B (en) | Synthetic medium containing reduced glutathione and suitable for cordyceps militaris culture, preparation method and application | |
| CN107164238B (en) | Schizochytrium limacinum strain and mutagenesis method and application thereof | |
| CN113308387B (en) | Bacterial strain for co-production of unsaturated fatty acid and carotenoid and application thereof | |
| CN103571896A (en) | Method for producing arachidonic acid grease by utilizing mortierella alpina mutant strain, and arachidonic acid grease produced by method | |
| CN101445815A (en) | Microbial synthesis method of gamma-linolenic acid oil | |
| CN108004277B (en) | Method for preparing linoleic acid by using bacillus megatherium | |
| CN102703339A (en) | High-yield arginine deiminase bacterial strain and method for producing L-citrulline by same | |
| CN101113410B (en) | Mortierella alpina and application thereof | |
| CN109429911A (en) | A kind of fermentation cordyceps sinensis culture solution and preparation method thereof prepared with jerusalem artichoke | |
| CN101591617B (en) | Docosahexaenoic acid producing strain, mutation screening method and application thereof | |
| CN105969702B (en) | Serratia marcescens RZ 21-C6 and its application | |
| CN103451247B (en) | A kind of arachidonic preparation method | |
| CN101861794A (en) | Method for producing liquid strain of cordyceps militaris | |
| CN105420143A (en) | Acetobacter orientalis and method for producing astragalus polysaccharide through same | |
| CN102219567B (en) | Method for producing biological organic fertilizer by using methane liquid as basic culture medium through fermentation | |
| CN103642698B (en) | Mortierella alpina mutant strain and application thereof | |
| CN102827780B (en) | Mortierella alpine strain for producing arachidonic acid | |
| CN105349588A (en) | Method for preparing docosahexenoic acid from schizochytrium sp. | |
| CN102766663A (en) | Preparation method of active polysaccharides from phellinus linteus | |
| CN105483171B (en) | A kind of raising ubiquinone10The production method of industrial output |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20100519 |