CN101434910A - Microbial bacterial strain for producing sphingolipid alkali - Google Patents
Microbial bacterial strain for producing sphingolipid alkali Download PDFInfo
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- CN101434910A CN101434910A CNA2007101681679A CN200710168167A CN101434910A CN 101434910 A CN101434910 A CN 101434910A CN A2007101681679 A CNA2007101681679 A CN A2007101681679A CN 200710168167 A CN200710168167 A CN 200710168167A CN 101434910 A CN101434910 A CN 101434910A
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Abstract
The invention relates to a microbial strain which can produce sphingosine, dihydrosphingosine, phytosphingosine and/or the derivatives thereof with improved level. In addition, the invention further discloses a method which is based on induced mutation or other selection technologies, and the strain is prepared accordingly. As an example, a mutant strain of pichia pastris is provided; and compared with the wild-type strain, compounds produced by the mutant strain are higher by about 50 percent.
Description
The present invention relates to utilize mutagenesis and select technique improvement to produce the method for the microorganism strains of sphingolipid alkali.
Background of invention
Term " sphingolipid " is meant one group of lipid derived from sphingosine.Sphingolipid occurs in animal, plant and microbial cell film usually.The actual functional capability of sphingolipid in human body is also unknown, but known this group compound participates in the electric signal transmission in neural system and the cell membrane stabilityization.Also pointed out sugar sphingosine to play a role in immunity system: the specificity sugar sphingosine plays the bacteriotoxin acceptor, also can be used as bacterium and Virus receptors.
Sphingolipid contains sphingosine, D-sphinganine or phytosphingosine, and it is as the amido linkage of alkali and lipid acid formation.Sphingosine or phytosphingosine alkali are can be used as parent material in synthesizing of ceramide at one group of concrete sphingolipid.Ceramide is upper layers of skin-cuticular main lipid composition.Stratum corneum has important barrier function, usually the externalizing compound is blocked in the forfeiture of its barrier outside and restriction moisture.In skin cosmetics, add sphingolipid such as ceramide and can improve the barrier function of skin and preserve moisture character (Curatolo, 1987; Kerscher etal., 1991).
At present, the allos sheath grease article that is used for makeup mainly extracts from animal.Obviously, this is the very high method of a kind of cost on technical scale.In addition, have been found that these starting material are owing to for example existing ox statural spongiosus myelitis (BSE) to have the potential insecurity in the ox tissue.Therefore, shown that cosmetic industry is more and more interested for the new source of sphingolipid pure, that character is determined, described new source can derive from other sources except animal tissues.
Have been found that such as before being called Hansenula and Endomycopsisciferrii (Barnett et al., 1990; Stodola and Wickerham, 1960; Wickerham andStodola, 1960; Wickerham et al., 1954; Wickerham, 1951) west not pichia spp microorganisms such as (Pichia ciferrii) can produce sphingolipid, and sphingosine, phytosphingosine and/or its derivative.This discovery provides the source of sphingolipid self and has been used to produce the source of the parent material of other compound that commercial value is arranged, and it can be the feasible alternative thing of the animal-origin of these compounds.
For example; the acetyl derivatives of sphingosine, D-sphinganine and phytosphingosine can deacetylation; thus obtained sphingosine, D-sphinganine or phytosphingosine can be chemically converted to relevant compound such as ceramide, false manifestation of vitality through acid amides and/or sugar (glyco) ceramide; can be used for (Smeets and Weber, 1993) in makeup and the therapeutic product thereupon.
In yeasts candida utilis (Candida utills) and yeast saccharomyces cerevisiae (Saccharomycescerevisiae) (Wagner and Zofcsik, 1966; Oda and Kamiya; 1958), Hansenlaspora valbyensis (Braun and Snell; 1967) and also demonstrated the generation of phytosphingosine and/or its acetyl derivatives among the Torulopsisutills (Kulmacz and Schroepfer Jr., 1978).The generation of phytosphingosine is also reported in fungi aspergillus sydowi (Aspergillus sydowi) and some mould (Penicillium notatum) (Stodola andWickerham, 1960).
In addition, be selected from sugar yeast (Saccharomyces) in analysis, kluyveromyces (Kluyvermoyces), Debaromyces, pichia spp (Pichia), debaryomyces hansenii (Hansenula), saccharomyces oleaginosus (Lipomyces), shadow yeast (Sporobolomyces), cryptococcus (Cryptococcus), torulopsis (Torulopsis), candiyeast (Candida), in the research of 30 yeast kinds that trichosporon (Trichosporon) and rhodotorula (Rhodotorula) belong to, find that all yeast kinds of being analyzed all contain the sphingolipid (ceramide monohexoside) of at least a form, therefore can be used for sphingolipid and produce (Kaneko et al., 1977).
Produce in the mutant of thanomin yeast saccharomyces cerevisiae a kind of, demonstrate phytosphingosine and gather, so this primary yeast can be used as the source (Ishida-Schick andAtkinson, 1983) of thanomin.
The generation of sphingolipid is also as Sphingobacterium (Sphingobacterium) (Yano etal., 1983), confirm in the bacterial strain that acetic bacteria (Acetobacter), bacterioide (Bacteroides), Bdellovibrio (Bdellovibrio), Xanthomonas campestris (Xanthomonas) and Flavobacterium (Flavobacterium) (Tahara et al., 1986) belong to.
Stoffel etc. (1968) find that yeast Hansenula (Pichia) clferrii makes all long-chain alkali acetylizes that are used as precursor in the research.Sphingosine be converted into triacetyl sphingosine and D-sphinganine be converted into triacetyl-, diacetyl-and N-acetyl-D-sphinganine.In addition, separated three kinds of acetyl derivatives of phytosphingosine, promptly the tetrem acyl-, triacetyl-and N-acetyl phytosphingosine.Except these acetyl derivatives, west not debaryomyces hansenii (Hansenula ciferrii) produces the long-chain ceramide in the substratum that contains long-chain alkali.
The biosynthetic pathway of the tetrem acyl phytosphingosine (TAPS) in the western not pichia spp is described by (1973) such as Barenholz.The biosynthetic pathway of sphingosine and D-sphinganine is described by (1971) such as Dimari.
(1971﹠amp such as Barenholz; 1973) studied the metabolism background that in Xi Fuhanxun (finishing red) four bacterial strains of zymic, produces TAPS and other sphingolipid alkali.In research after a while; (west is debaryomyces hansenii NRRL Y-1031 not, E-11, sex b to have contrasted the low yield bacterium; 8-205) (west is debaryomyces hansenii NRRL Y-1031 not, the pattern of the biosynthetic four kinds of microsomal enzymes that are specific to acetylize sphingosine F-60-10) with the high yield bacterium.The specific activity of discovery 3-ketone D-sphinganine synthetic enzyme and long-chain alkali acetyl-CoA Transacetylase is compared with the low yield bacterium and is increased by 5-10 times and 30 times respectively, and palmityl thiokinase and 3-ketone D-sphinganine reductase enzyme is active similar.This shows in the low yield bacterium, and the activity of synthetic enzyme and long-chain alkali acetyl-CoA Transacetylase is limited in acetylize sphingosine synthetic.Under specified growth conditions, discovery west not debaryomyces hansenii NRRLY-1031 F-60-10 produces 300 μ mol/l sphingosines (approximately 0.15g/l) alkali, and wherein at least 250 μ mol/l are that born of the same parents are outer.Under the culture condition that is optimized at TAPS production, only obtain 0.485g/l TAPS (0.024g TAPS/g dry yeast) (Maister et al., 1962).
Yet, up to now Yan Jiu yeast strains even west not pichia spp NRRL Y-1031F-60-10 none can produce the sphingolipid alkali of q.s such as sphingosine, phytosphingosine or its derivative as interested source on the effective economics of this compounds.For example, the availability that can produce the yeast strains of higher level TAPS will significantly be improved the economics feasibility and the magnetism of producing this important parent material, and described parent material can be converted to end product with commercial value such as ceramide, false manifestation of vitality subsequently through acid amides and glycosyl ceramide.
Summary of the invention
The invention provides the method that produces the Pichi strain of energy production higher level TAPS by mutagenesis and selection technology as claimed in claim 1.Consequent mutant strain is compared with its parental strain and produces the higher levels of product of wishing under the same conditions.The production level of these increases provides attractive source on a kind of economics for thereby this compound is converted into the end product with commercial value as parent material.
Detailed Description Of The Invention
The present invention confirms that the output of TAPS can increase by the circulation of the microorganism strains of determining this compound of generation being carried out successive mutagenesis and selection.By this way, biosynthetic weak link can progressively be eliminated, and makes the output of these products increase.
The invention provides generation as claimed in claim 1 and its parental strain mutually specific energy produce the method for the Pichi strain of higher level TAPS, wherein described parental strain is carried out mutagenic treatment at least one time, comprise described parental strain is handled with the suitable mutagen of q.s, so that such mutant strain to be provided, when cultivating under the same conditions its with parental strain mutually specific energy produce higher level and wish product; Increase and the selection mutant strain based on the output of wishing product subsequently.
That produce by the inventive method and as the mutant strain of cultivation as described in the embodiment hereinafter can produce 0.030g TAPS/g yeast dry weight, preferably at least 0.050,0.060,0.075,0.10,0.15g TAPS/g yeast dry weight and 0.20g TAPS/g yeast dry weight at least most preferably.
The yeast strains that is used for the present invention can be selected from not pichia spp kind (before being called not debaryomyces hansenii of west) of Pichia bacterial classification, particularly west.Most preferably belong to Pichia and the west yeast strains of pichia spp kind (particularly west not pichia spp NRRL Y-1031F-60-10) not most preferably.
According to the present invention, can carry out mutagenesis by using suitable mutagen, make sphingosine, D-sphinganine, phytosphingosine and/or its derivative output increase.Preferred mutagen for example is UV irradiation, ethyl methane sulfonates (ethyl methane sulphonate) and N-methyl-N '-nitro-N-nitrosoguanidine (Stanbury, 1988; Shay, 1987; Masanari er al., 1985).
Mutagenesis can be carried out single mutagenesis, but find to carry out two or repeatedly mutagenesis be favourable.After each mutagenesis step, bacterial strain produces the hope product of increasing amount such as the ability of TAPS is improved.
After finishing mutagenesis reaction, can select the gained mutant, the mutant of selecting is when cultivating under the condition that the sphingosine that is being of value to hope, D-sphinganine, phytosphingosine and/or its derivative produce, and shows the mutant of the hope product of production peak level.
Those skilled in the art are known to separating the mutant of wishing (inferior) mutant of very many poor-performings for producing the bacterial strain that for example amino acid whose bacterial strain of primary metabolite produces the secondary metabolism thing easier (Stanbury, 1988).
On agar plate, carry out the preselected of high yield mutant by the zone of a crystal of estimating single bacterium colony, subsequently it is separated.Selection illustrates maximum diameter crystalline bacterium colony with respect to the bacterium colony size.Also can use other preselected method, for example to the resistance of amino acid analogue or protein synthesis inhibitor.
Isolated cells can be cultivated in shaking bottle or culture test tube by this way, determines that by the suitable dilution of analyzing fermenting broth the production level of wishing product selects suitable mutant.Provide the growth conditions described in detail as embodiment as a reference with the production level of the hope product of determining bacterial strain of the present invention.Yet, in the scope that does not depart from spirit of the present invention, also can use other culture condition of wishing that product produces that is of value to well known by persons skilled in the art.
Quantitative analysis can be undertaken by the whole bag of tricks, for example by with periodate continuous deacetylation being carried out in the oxidation of TAPS and related compound.Form the aldehyde of stoichiometric amount, it is the degradation production of the oxidation of TAPS and related compound.
Based on the selection of TAPS generation level can be further or shaking in the bottle separately and using CDCl
3Extraction is also analyzed by NMR and to be carried out.TAPS is preferably used as the standard substance that NMR analyzes.
As object of reference, can quantitatively determine other hope product such as sphingosine, phytosphingosine and D-sphinganine with the TAPS standard substance in the same manner; The triacetyl of sphingosine, phytosphingosine and D-sphinganine, diacetyl and N-acetyl derivative; 3-ketone D-sphinganine and/or 3-ketone sphingosine.
In case obtain the product of hope, they can directly be used or the further processing for last application.For example; the acetyl derivatives of sphingosine, D-sphinganine and phytosphingosine can be through enzyme or chemical treatment and deacetylation; in acid amides synthetic, be used as parent material (Smeetsand Weber, 1993) the sphingolipid product with commercial value such as ceramide, glycosyl ceramide and false manifestation of vitality subsequently.
In following embodiment, the contriver makes great efforts to guarantee that the numerical value (for example quantity, temperature, pH etc.) that is adopted is accurate, but should be taken into account some experimental errors and deviation.Unless otherwise indicated, then temperature is degree centigrade, and pressure is in or near normal atmosphere.
Preparation embodiment A: the preparation of tetrem acyl phytosphingosine standard substance
With the mixture reflux of 1.0g (2.8mmol) phytosphingosine hydrochloride (Sigma), 2.7ml (28mmol) diacetyl oxide, 2.1ml (15mmol) triethylamine and the pure chloroform of 10ml and stirred 8 hours.After being cooled to room temperature, be 7 with the washing of the saturated aqueous solution of sodium bicarbonate until reaching pH with this mixture.Subsequently with organic layer at 50 ℃ at MgSO
4Last dry, filter and vacuum-evaporation.Use Merck:Lobor Fertich Saule GroBec (440-37) Lichroprep Si60 (40-60 μ m) post to pass through column chromatography purification resistates.The mixture of methylene dichloride and methyl alcohol (25:1) is as elutriant (pumping velocity is 10ml/ minute).The product that obtains is a white solid, and yield is 80%, and fusing point is 41-43 ℃.The purity of the TAPS of Xing Chenging is at CDCl thus
3In determine that by NMR (estimation purity is 96% for proton N MR, 360MHz) (ρ-nitrotoluene is as internal standard).
15mg TAPS/ml CDCl as above-mentioned generation
3In all embodiment of the present invention all as standard substance.
Preparation Embodiment B: TAPS nutrient agar inclined-plane
With 122g Nemoutex (Diastatische Produkten B.V.; Leiden TheNetherlands) is dissolved in 1 premium on currency and 110 ℃ of sterilizations 60 minutes.With this suspension in the room temperature incubated overnight, the solids removed by filtration particle.PH is equilibrated at 6.4.Add 10g/l agar (Bacto), solution was sterilized 30 minutes at 120 ℃.
Preparation Embodiment C: TAPS substratum (cultivating and shake-flask culture) for test tube
| Compound | Quantity (g/l) |
| KH-phthalic acid NaClMgSO 4·7H 2OCaCl 2·2H 2ONH 4ClKH 2PO 4(NH 4) 2Fe(SO 4) 2ZnSO 4CuSO 4MnSO 4H 3BO 3The amino benzoate vitamin B6 vitamin H yeast extract (Difco) of Sodium orthomolybdate K1 inositol nicotinic acid Ca-D-panthotenate VITMAIN B1 p- | 200.060.880.204.831.00.0270.0050.00750.00060.00060.00060.000150.0590.0030.0030.0030.0020.00030.000011.0 |
In shaking bottle and culture test tube, adding glucose to final concentration is respectively 33 and 7g/l.After described composition dissolving, be 5.4 with pH regulator.
In 100ml Erlenmeyer flask (no baffle plate), add the 30ml substratum, 110 ℃ of sterilizations 30 minutes.For agar plate, in substratum, add 20g agar (Bacto), use identical sterilizing program.
Preparation embodiment D:YEP-D substratum
| Bactopeptone (Bactopeptne) yeast extract dextrose bacteria (Bacto)-agar | 20g10g20g20g |
Add 1000ml and remove mineral water, pH is equilibrated at 7.0.Substratum was sterilized 30 minutes at 110 ℃.
Preparation embodiment E: physiological saline substratum
Remove dissolving 8.5g NaCl in the mineral water at 1 liter, 120 ℃ of sterilizations 20 minutes.
Preparation embodiment F: the growth conditions of cell cultures
With the west not pichia spp NRRL Y-1031 F-60-10 and mutant thereof in incubator on inclined-plane or agar plate 24 ℃ of growths.
In containing the 40ml glass culture test tube (diameter 2.5cm) of 3.0ml TAPS substratum, carry out the growth in the culture test tube.With culture test tube 3 days (300rpm, 24 ℃) of vertical insulation on the test-tube stand of Gallenkamp orbital incubator.After finishing fermentation, consider the steam output between soak and the concentration of proofreading and correct thus the TAPS that forms.Mutant is with quadruplicate test.
Shake the growth in the bottle (no baffle plate) in 30ml TAPS substratum (seeing above-mentioned) in the 100ml Erlenmeyer flask or the 100ml substratum in the 500ml Erlenmeyer flask.3 days (250rpm, 24 ℃) of insulation in the Gallenkamporbital incubator.After finishing fermentation, consider the steam output between soak and the concentration of proofreading and correct thus the TAPS that forms.Mutant is with quadruplicate test.
Embodiment 1: with UV-light (UV) mutagenesis
With the west not the semilog culture (100ml) of pichia spp NRRL Y-1031 F-60-10 or its deutero-mutant on YEP-D or TAPS substratum, grow (24 hours, 24 ℃, 250rpm).With culture centrifugal (Heraeus sepatech minifuge RF whizzer, 5000 * g, 5 minutes) and with throw out 25ml physiological saline substratum washed twice.The cell suspending liquid dilution is final concentration 10
8Individual cell/ml, and this suspension of 10ml moved to (diameter 9cm) in the sterile petri dish with suction pipe.Then this flat board is carried out the UV irradiation,, shone respectively 0,15,30,60 and 90 second apart from irradiating source (Osram lamp HQV, 125 watts) 23cm.To place dark (30 minutes, 20 ℃) through the cell of irradiation then.Be to determine survival rate, the diluent of the suspension of mutant cell is plated on the YEP-D nutrient agar.Remaining cell suspending liquid is grow overnight on the TAPS substratum.If desired, can add glycerine to final concentration is 10%, with this suspension-20 ℃ of storages.After 3 days, the counting bacterium colony also calculates survival per-cent 24 ℃ of insulations.To cause survival rate through radiation treatment is that about 10% cell suspending liquid is used to select TAPS high yield mutant.
Embodiment 2: use ethyl methane sulfonate mutagenesis
As make as described in the embodiment 1 west not the semilog culture (100ml) of pichia spp NRRL Y-1031 F-60-10 or its deutero-mutant grow and wash, difference is that throw out is washed with 50ml 1M Tris-HCl pH7.5 replacement physiological saline substratum.Afterwards, cell being diluted to final concentration in the 1.0M of 25ml Tris-HCl pH of buffer 7.5 is 10
8Individual cell/ml.
In the cell suspending liquid of 6ml, add 300 μ l EMS and be incubated in room temperature.At the 0th, 1,3,6 and 12 minute, get the 1ml sample.Subsequently cell is used 9ml physiological saline substratum washed twice, suitable diluent is plated on the YEP-D nutrient agar.As described in embodiment 1, make remaining mutant cell grow overnight, calculate survival per-cent.Selection illustrates the sample of 10% survival rate to separate TAPS high yield mutant.
Embodiment 3: with N-methyl-N '-nitro-N-nitrosoguanidine mutagenesis
As described in the embodiment 1 with the west not the semilog culture (100ml) of pichia spp NRRL Y-1031 F-60-10 or its deutero-mutant grow and wash, difference is to replace the physiological saline substratum to wash with throw out with 50ml 1M Tris-HCl pH7.5.Afterwards, cell being diluted to final concentration in the 1.0M of 25ml Tris-HCl pH of buffer 7.5 is 10
8Individual cell/ml.
In the cell suspending liquid of 6ml, add the NTG stoste of 600 μ l prepared fresh and be incubated in room temperature.At the 0th, 1,3,6 and 12 minute, get the 1ml sample.Subsequently cell is used 9ml physiological saline substratum washed twice, suitable diluent is plated on the YEP-D nutrient agar.As described in embodiment 1, make remaining mutant cell grow overnight, calculate survival per-cent.Selection illustrates the sample of 10% survival rate to separate TAPS high yield mutant.
Embodiment 4: mutant preselected
On the TAPS-nutrient agar, carry out preselected.Suitable cell diluent (25-50 bacterium colony/flat boards) is as acquisition as described in embodiment 1,2 and 3, with its in incubator 24 ℃ of insulations 4-7 days.In order to stimulate crystallization, should flat board 4 ℃ of incubated overnight.Afterwards, select to have the bacterium colony (diameter (the mm)/colony diameter of percent crystallization in massecuite=periphery of bacterial colonies zone of a crystal) of high percent crystallization in massecuite.
Separate these bacterium colonies and move in the agar plate that contains the TAPS nutrient agar.These flat boards are known as " main dull and stereotyped ", and these bacterium colonies are known as " primary colony ".Cell-starting material on these main flat boards are used for the highest TAPS producer who is chosen in culture test tube, shakes bottle and are used to store purpose.
Embodiment 5: the TAPS at the culture of culture test tube growth is analyzed (method 1)
Further be chosen in the cell that obtains among the embodiment 4 at culture test tube in the bottle with shaking subsequently, as described in the preparation embodiment F, carry out, analyze as following.TAPS determines that program is divided into three parts (paragraph 5a-c).
5a: the deacetylation of tetrem acyl phytosphingosine
With the TAPS stoste of 0.05-0.3g/l with for referencial use.In the 1ml sample, add sodium hydroxide to 8 times that exceed the TAPS desired amount, this solution is incubated 2 hours at 50 ℃.
5b: the oxidation of phytosphingosine
Carry out the oxidation of phytosphingosine by adding sodium periodate.
CH
3-(CH
2)
13-CHOH-CHOH-CHNH
2-CH
2-OH→
CH
3-(CH
2)
13-CHO+2HCOOH+NH
3+HCHO
In the 1ml30mM potassium periodate among the deacetylation blend sample adding 0.1M acetate buffer pH5.4 that will in above-mentioned steps a, obtain.Subsequently, room temperature in the dark oxidation spend the night, during reaction mixture is shaken (125rpm) gently on IKA Vibrax VXR electricity shaking table.Reaction tube is carefully sealed aldehyde evaporation to produce in preventing to react.
5c: the determining of aldehyde
The amount of the aldehyde that produces in step b can be by determining as the described colorimetric method of Avigad (1983).
After the oxidation program of step b, the 0.3ml among the 1N NaOH 1% (w/v) Purpald (Aldrich) is added in the 0.2ml sample.Described Purpald reagent should be prepared fresh (being no more than 2 hours).(180-200rpm) go up insulation 2 hours at IKA Vibrax VXR Electric cradle with this mixture.Subsequently by adding 0.5ml 0.2% NaBH among the 1N NaOH (w/v)
4Termination reaction.The absorbancy of sample is measured at the 548nm wavelength.
Embodiment 6: analyze TAPS (method 2) by NMR
Further be chosen in the cell that obtains among the embodiment 4 at culture test tube in the bottle with shaking subsequently, as described in the preparation embodiment F, carry out, analyze as following.
6a: extract
Extracting TAPS from shake bottle growth medium can use various hydrophobic organic compounds to carry out.The organic extraction compound that is used for present embodiment is tritiate chloroform (CDCl
3).(25ml) uses 2ml CDCl with substratum
3Extract, mixed 5 minutes in room temperature powerful (at full speed) level on IKA Vibrax VXR electricity shaking table.Then with this mixture centrifugal (Heraeus sepatechminifuge RF whizzer, 10,000 * g, 5 minutes) and remove CDCl
3, analyze by NMR.
6b: nucleus magnetic resonance (NMR) is analyzed
CDCl
3The TAPS content of layer is measured in the program identical with the TAPS standard substance, uses AMR360 NMR instrument (Bruker-Carisruhe, Germany) for this reason.Following adjusting NMR instrument:
Receiver gain value: fixed value
The Relaxation time: 5 seconds
Number of scans: fixing
Pulse: 45 °
The Fourier transform of the FID (free induction decay) that obtains carries out with absolute strength.The quantity of TAPS (representing with the mg/g culture) is as shown in the formula calculating:
The weight of standard substance and purity are represented with g and per-cent respectively.Described standard substance contains 15mg TAPS/ml CDCl
3
Embodiment 7: dry weight is measured
In the dry centrifuge tube of known weight (A), add about 10ml cell suspending liquid, weigh once more (B) and centrifugal.Subsequently with throw out dry (24 ℃, 105 ℃), gravimetry (C) once more in incubator.The following calculating of dry weight (A, B, C represent with g):
Dry weight (g/kg)=[(C-A)/(B-A)] * 1000g dry-matter/kg cell suspending liquid
For shake-flask culture, the following calculating of TAPS content (g TAPS/g dry weight):
Therefore, the production level of TAPS is represented with g TAPS/g yeast dry weight.
Embodiment 8: by cultivate not the determining of amount of the TAPS that forms of the mutant of pichia spp NRRL Y-1031F-6010 and acquisition of west on the TAPS substratum
In shaking bottle on the TAPS substratum as described in the preparation embodiment F under the condition, not pichia spp NRRL Y-1031 F-6010 is with 40 times of growths in the west, CBS111, CBS1710 and CBS1910 are with ten times of growths, mutants which had is with five times of growths.TAPS extracts and the NMR analysis is carried out as described in embodiment 6.Dry weight is measured and is carried out as described in embodiment 7.
The CDCl that obtains by the culture that extracts TAPS production yeast strains
3The position of the NMR collection of illustrative plates resonance peak of layer with to analyzing identical among the gained result as preparing the TAPS standard substance of preparation as described in the embodiment A.
The mean yield (productivity) of 40 shake-flask culture things of F-60-10 is a 0.025gTAPS/g yeast dry weight.The output of CBS111, CBS1710 and CBS1910 is the mean value of 10 independent measured values, and the output of mutants which had is the mean value of 5 independent measured values.The output of 5 mutants which hads (5F11,14D11,15G8,20A11 and 27D10) is shown in the following table.Shown in table, mutants which had has higher production level, than the output high about 40-60% of parental strain.
Described 5 mutants which hads and parental strain west not pichia spp NRRL-Y1031F-60-10 are deposited in the microbial strains preservation center (Centraal Bureau voor Schimmelcultures) of Dutch Baarn on July 6th, 1994 (CBS), preserving number are: CBS403.94, CBS404.94, CBS405.94, CBS406.94, CBS407.94 (mutants which had) and CBS408.94 (parental strain).
TAPS output in the shake-flask culture of western not Pichi strain CBS111, CBS1710 and CBS1910 and NRRL-Y1031 F-60-10 and mutant thereof is as follows:
*ND=can not detect
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Claims (8)
1. method that produces the mutant Pichi strain, described mutant Pichi strain can produce 0.030g tetrem acyl phytosphingosine (TAPS)/g pichia spp dry weight at least, it is characterized in that the parental generation Pichi strain of the natural generation TAPS of energy is carried out mutagenic treatment at least one time, comprise with the suitable mutagen of capacity and handle described parental strain, compare the mutants which had that can produce higher levels of TAPS when cultivating under the same conditions with described parental strain to provide; Be chosen in subsequently in the liquid TAPS substratum in the Erlenmeyer flask and can produce the mutants which had of 0.030g TAPS/g pichia spp dry weight at least in 3 days in 24 ℃ of cultivations.
2. the method for claim 1 is characterised in that described mutant Pichi strain is selected from the not bacterial strain of pichia spp kind of west.
3. claim 1 or 2 method are characterised in that described mutagen is selected from UV irradiation, ethyl methane sulfonate or N-methyl-N '-nitro-N-nitrosoguanidine.
4. a mutant Pichi strain can produce 0.030g TAPS/g pichia spp dry weight at least when cultivating 3 days for 24 ℃ in its liquid TAPS substratum in Erlenmeyer flask.
5. the mutant Pichi strain of claim 4 is characterised in that it is selected from the not bacterial strain of pichia spp kind of west.
6. the mutant Pichi strain of claim 5, wherein said bacterial strain are selected from the group that CBS403.94, CBS404.94, CBS405.94, CBS406.94 and CBS407.94 form.
7. a generation is selected from the method for the hope compound of phytosphingosine or its acetyl derivatives, it is characterized in that the bacterial strain of one of claim 4-6 is cultivated under the condition that TAPS produces helping, and randomly with consequent TAPS deacetylation.
8. the method for claim 7 is characterised in that and describedly wishes that compound is a phytosphingosine.
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