CN108690814A - A kind of method and its application of Lipid-producing bacterial strain rapidly and efficiently selection and breeding - Google Patents

A kind of method and its application of Lipid-producing bacterial strain rapidly and efficiently selection and breeding Download PDF

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CN108690814A
CN108690814A CN201810507747.4A CN201810507747A CN108690814A CN 108690814 A CN108690814 A CN 108690814A CN 201810507747 A CN201810507747 A CN 201810507747A CN 108690814 A CN108690814 A CN 108690814A
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strain
mutagenesis
lipid
trichosporon
mutagenic
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龚大春
许鹏飞
刘云
郭金玲
吕育财
雷生娇
田毅红
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China Three Gorges University CTGU
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China Three Gorges University CTGU
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/02—Pretreatment
    • C11B1/025—Pretreatment by enzymes or microorganisms, living or dead
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor

Abstract

The present invention provides a kind of method of rapidly and efficiently selection and breeding Lipid-producing bacterial strain.Mutagenic treatment is carried out to trichosporon cutaneum first with atmospheric pressure at room plasma mutagenesis instrument, it is then seeded on the plating medium containing sesamol, it selects in eugonic inoculation to 48 boreliquid culture mediums, and high flux screening is carried out to culture strain using 96 orifice plate Nile red fluorescent stainings and obtains the mutant strain that proliferation is fast and grease yield is high, finally utilize gas chromatographic detection lubricant component.By mutagenesis and screening, a plant height Lipid-producing strains A 1 is selected, 41.6%, 120.3%, 58.8% has been respectively increased compared with the biomass of original strain, grease yield, lipid-producing;It is identical as original strain by gas-chromatography constituent analysis its aliphatic acid composition, it is palmitic acid, oleic acid, linoleic acid, leukotrienes etc..ARTP plasmas induced-mutation technique and malate dehydrogenase inhibitor, Nile red fluorescent staining are organically combined, realize high flux screening, and successfully select one plant of grease superior strain.

Description

A kind of method and its application of Lipid-producing bacterial strain rapidly and efficiently selection and breeding
Technical field
The invention belongs to biotechnologies, and in particular to a kind of Lipid-producing bacterial strain, the method for rapidly and efficiently selection and breeding and its Using.
Background technology
Biodiesel is a kind of clean reproducible energy that can directly replace fossil fuel.Biodiesel is with vegetable oil It is raw material, the fatty acid methyl ester produced after esterification with animal tallow.But at present because its raw material sources is unstable, at The development in this high market is extremely restricted.Microbial grease refers to utilizing many microorganisms, such as saccharomycete, algae, mould It is by microbial metabolism a large amount of greases that productive accumulation generates in vivo, grease main component Deng under the conditions of certain growth Triglycerides (TAG), it is similar to vegetable oil such as palm oil, rapeseed oil in composition, based on C16 and C18.Realize high yield The key of microbial grease is the selection and breeding of superior strain.
Mutagenesis is to improve a kind of common technology of strain, and current method of mutagenesis has physics and chemical method, but this method Complicated for operation, harmfulness is larger.Atmospheric pressure at room plasma mutagenesis instrument (ARTP) is a kind of novel induced-mutation technique, can quickly be lured Become the mutation of microbial genome.Its by helium atmospheric pressure radio frequency glow discharge, generate a series of action of plasma in Microorganism makes microorganism generate mutation effect.The equipment temperature in use is low, active particle wide variety;Simple in structure, cost It is low;It is safe, pollution-free using simplicity;The spectrum of mutation is wide, and mutation rate is high, is a new breakthrough of Biology Breeding.
The final purpose of mutagenesis is the bacterial strain that grease yield is high in order to obtain, but traditional dyeing and extracting method is compared It is cumbersome.Kimura and Lin Yi etc. measures grease using the method for Nile red fluorescent staining, and the detection method of grease is made to become letter Just, efficiently, high-throughput screening is carried out using fluorescence microplate reader.
Invention content
Based on above-mentioned technical problem, the present invention provides a kind of ARTP induced-mutation techniques trichosporon cutaneum (Trichosporon Cutaneum it) is handled, primary dcreening operation is carried out by malate dehydrogenase inhibitor resistant panel, obtained mutagenic strain is inoculated into 48 It is cultivated in orifice plate, was taken every 24 hours and carry out that bacterium is dense and Nile red fluoroscopic examination in 100uL culture mediums to 96 orifice plates, filter out increasing Bacterial strain rapid, that grease yield is high is grown, and is subsequent hair to its genetic stability and bacterial strain lubricant component into conducting a research Ferment culture optimization provides strain excellent basis.
Technical scheme of the present invention provides a kind of Lipid-producing mutagenic strain, which is with trichosporon cutaneum The bacterial strain of (Trichosporon cutaneum) mutagenesis, the lubricant component of the mutagenic strain includes mainly methyl palmitate, hard Resin acid methyl esters, methyl oleate, methyl linoleate.
The mutagenic strain include Trichosporon cutaneumA1, Trichosporon cutaneumB2, Trichosporon cutaneumC1,Trichosporon cutaneumD4,Trichosporon cutaneumE8。A1, B2,C1,D4,E8.It is Trichosporon cutaneumA1 that further preferred Lipid-producing mutagenic strain, which is mutagenic strain,.
Another technical solution of the present invention provides a kind of rapidly and efficiently selection of Lipid-producing mutagenic strain, skin shape silk spore Yeast specifically comprises the following steps through the mutagenesis of ARTP plasmas, the bacterial strain selected after sesamol sieves again:
ARTP mutagenesis:Trichosporon cutaneum seed bacteria suspension is selected on metal slide glass, metal slide glass is put into mutagenesis room Interior carry out mutagenic treatment, obtains the thalline of mutagenesis;
In the ARTP mutagenesis steps, a concentration of OD of trichosporon cutaneum seed bacteria suspension600=1.0;The temperature of ARTP Degree is maintained at 18-25 DEG C, power regulation 100-140W, helium gas flow 8-12L/min;The bacteria suspension point is carried to metal Piece is gone forward first, and to metal slide glass calcination at least 25-35s, (temperature of further preferably ARTP is maintained at 20 DEG C, and power regulation is 120W, helium gas flow 10L/min;The bacteria suspension selects metal slide glass and goes forward first to metal slide glass calcination at least 30s); Slide glass is put into mutagenesis room, and emitter is apart from bacterium solution 1.5-3nm, preferably 2nm;ARTP mutation times are 10-40s;Mutagenesis After the completion, it is applied on plating medium 28 DEG C of culture 1-3, preferably 2 days.
Sesamol screens:The thalline of mutagenesis is applied in the resistant panel culture medium containing sesamol, obtains luring on tablet The strain of change;
In the sesamol screening step, the thalline of mutagenesis is applied in the resistant panel culture medium containing sesamol, 28 DEG C are cultivated 2 days.
Steps are as follows for the high strain isolation of activity:Using dilution spread flat band method by dilution 10-6~10-8Supernatant It is forwarded to PDA plate, each dilution is made 3 repetitions, cultivated in 29 DEG C of incubators.After flora is grown, in PDA plate On repeatedly scribing line separation, under the microscope observation be determined as pure single bacterium colony after, be inoculated on storage medium and preserved.
High flux screening:The strain of mutagenesis on tablet is inoculated into bamboo stick in culture medium, is cultivated in constant-temperature table, is taken It cultivates in bacterium solution to 96 orifice plates, every 20-28 hours, takes in bacterial strain to orifice plate, while adding Nile red dye liquor into orifice plate, mix It closes and is uniformly protected from light dyeing 5min, with microplate reader with the launch wavelength of 485nm, 595 absorbing wavelength detection cell grease fluorescence is strong Degree, filters out the high strain of fat content;
In the high flux screening step, the strain of mutagenesis is inoculated into the culture medium of 48 hole 1ml, 25-32 DEG C, 110-160h is cultivated in the microwell plate constant-temperature table of 150-220r/min (further preferably in the microwell plate of 30 DEG C of 200r/min 144h is cultivated in constant-temperature table) it takes in the bacterium solution to 96 orifice plates of 150ul, each orifice plate adds 7.5ul Nile red dye liquors, mixing It is uniformly protected from light dyeing 3-5min, OD is detected with microplate reader600The absorbance of nm.
Fermented and cultured:15-25h will be cultivated in the colony inoculation to seed liquor for the high fat content that screening obtains, takes seed Liquid is inoculated into fermentation medium and cultivates, and obtains the bacterium solution of fermented and cultured, as mutagenic strain;
In the fermented and cultured step, it will be cultivated in the colony inoculation to seed liquor for the high fat content that screening obtains 15-20h, take volumetric concentration be 8-15% seed liquors be inoculated into fermentation medium 28 DEG C, 150-220r/min cultivate 3-4 days (further preferred scheme is that will cultivate 18h in the colony inoculation to seed liquor for the high fat content that screening obtains, take volumetric concentration For 10% seed liquor be inoculated into fermentation medium 28 DEG C, 200r/min cultivate 3 days, obtain the bacterium solution of fermented and cultured.)
In above-mentioned technical proposal, used resistant panel medium component is to add the sesamol mother liquor of 60-70mg/mL It is added in plating medium and obtains;The ingredient of the plating medium includes:Glucose 15-20g/L, peptone 15-20g/ L, yeast extract 5-10g/L, agar powder 15-20g/L.Further preferably by resistant panel culture medium, by 62.5mg/mL's Sesamol mother liquor is added in plating medium, obtains a concentration of 0.15mg/mL of sesamol mother liquor.
The ingredient of seed liquid culture medium includes:Sucrose 20-25g/L, yeast extract 4-6g/L, fructus hordei germinatus leaching powder 3-5g/L, Na2HPO42-4g/L, KH2PO40.5-1g/L, MgSO40.3-0.5g/L;
The ingredient of fermentation medium includes:Glucose 60g/L, yeast extract 6g/L, fructus hordei germinatus leaching powder 5g/L, Na2HPO4 4g/ L, KH2PO41g/L, MgSO4 0.5g/L.
Application of the mutagenic strain obtained based on above-mentioned selection and breeding at least one of following (1)-(5):
(1) as the raw material of biodiesel;
(2) grease in the raw material of biodiesel is improved;
(3) methyl palmitate in the raw material of biodiesel is improved;
(4) methyl stearate in the raw material of biodiesel is improved;
(5) methyl linoleate in the raw material of biodiesel is improved.
The method that the mutagenic strain that technical scheme of the present invention obtains selection and breeding extracts grease is as follows:By the bacterium of fermented and cultured Hydrochloric acid is added after liquid centrifugation, drying, is stored at room temperature rear boiling water bath processing 5-15min, it is rapid cooling, 1- is added after being restored to room temperature Chloroform-methanol (the V of 3 times of volumes:V=2:1) solution is stored at room temperature after mixing, is centrifuged, and extraction, rotary evaporation removes dechlorination Imitate to obtain grease.
Technical scheme of the present invention is by ARTP technology combination sesamols and Nile red screening strategy, mutagenesis rapidly and efficiently Trichosporon cutaneum, during being somebody's turn to do, sesamol is the inhibitor of malate dehydrogenase, and malate dehydrogenase is produced in the application Key enzyme in oily metabolic pathway, it can be catalyzed malic acid and generate pyruvic acid and NADPH, for oil and fat accumulation provide raw material with Power, therefore be added into culture medium and obtain the superior strain of one plant of grease for screening, the yield of grease reaches 5.8g/L, 123% is improved than original strain.Show that the grease high yield characteristics of mutagenic strain can keep steady by genetic stability experiment Fixed hereditary capacity.It can be seen that coming, which is simple and efficient, stability is good, strain suitable for saccharomyces oleaginosus Improvement.
Technical scheme of the present invention inhibits ARTP random mutagenesis and sesamol orientation in microorganism oil-producing metabolic pathway Key enzyme is combined, and increases the probability that mutagenic strain is mutated to oil and fat accumulation direction, and the probability for screening strain excellent is made to increase, The workload for reducing screening provides beneficial method for the screening of microorganism dominant strain.
Description of the drawings
Fig. 1 is plasma mutagenic and breeding flow.
Fig. 2 is trichosporon cutaneum ARTP lethality curves.
Fig. 3 is the influence that various concentration sesamol grows trichosporon cutaneum.
Fig. 4 is the opposite fat content of mutant strain.
Fig. 5 is the fluorescence intensity of mutant strain and original strain.
Fig. 6 is the genetic stability of strains A 1.
Specific implementation mode
Embodiment 1
ARTP mutagenesis
OD first is made in seed liquor600The temperature of ARTP is maintained at 20 DEG C by=1.0 bacteria suspension, and regulation power is 120W, helium gas flow 10L/min.It draws 10ul bacteria suspensions to select on the metal slide glass for having used alcohol calcination 30S, slide glass is put Enter into mutagenesis room, emitter apart from bacterium solution 2nm, using different time (0,10,20,30,40,50,60S) carry out at mutagenesis Reason, the thalline of mutagenesis is put into the physiological saline of 1mL and shakes 1min, and 100uL is taken to be applied to 28 DEG C of trainings on plating medium It supports 2 days, calculates lethality.
Sesamol screens
The sesamol mother liquor of 62.5mg/mL is prepared, adds the tablet culture of 0,40,80,120,160, uL to 50mL respectively In base, ultimate density 0,0.05,0.1,0.15,0.2mg/mL.By OD600=1.0 seed bacteria suspension takes 100uL to be applied to In resistant panel containing sesamol, 28 DEG C are cultivated 2 days, and lethality is calculated.
High flux screening
Since the mutant that mutagenesis generates is more, so establishing a kind of quick screening mode of high throughput.It will first be lured on tablet The strain of change is inoculated into bamboo stick in the culture medium of 48 hole 1ml, is cultivated in the microwell plate constant-temperature table of 30 DEG C of 200r/min, often Every 24 hours, takes in the bacterium solution to 96 orifice plates of 150ul and detect OD with microplate reader600The absorbance of nm, with incubation time for horizontal seat Mark, absorbance are that ordinate makees growth curve.
Each orifice plate adds 7.5ul Nile red dye liquors, is uniformly mixed and is protected from light dyeing 5min, with microplate reader with the hair of 485nm Ejected wave is long, and 595 absorbing wavelength detects cell grease fluorescence intensity, using the fluorescence intensity of no addition Nile red dye liquor as blank Control, filters out the high strain of fat content.
Fermented and cultured
The bacterium colony of high fat content and the bacterial strain of low grease content that screening obtains are inoculated into seed liquor cultivate respectively 18h takes 10ml (10%) seed liquor to be inoculated into 28 DEG C of 200r/min cultures in fermentation medium, and detection thalline is dense within each 24 hours Degree and grease fluorescence content, continuous detection 10 days.
Grease extracts
The bacterium solution 8500r/min of fermented and cultured 90h is centrifuged into 10min, thalline is put into the test tube dried and weighed, one It rises and is put into 80 DEG C of baking ovens that drying to constant weight, calculate thalline yield.The hydrochloric acid of 5ml8mol/L is added in every gram of dry mycelium, and mixing is equal It is stored at room temperature after even 1 hour, then boiling water bath processing 10min, rapid cooling, is restored to the chloroform-of 2 times of volumes of addition after room temperature Methanol (V:V=2:1) solution, is stored at room temperature 30min after mixing, and 4500r/min centrifuges 10min.Chloroform layer is extracted to obtain, on Layer solution adds 10ml chloroforms, shaken well, centrifugation to carry out reextraction and obtain chloroform layer, merge chloroform again.Rotary evaporation removes dechlorination Grease is imitated to obtain, is weighed.
Grease yield=oil quality/dry mycelium amount X100%.
Genetic stability
By mutagenic strain, continuous passage 10 times on plating medium, then fermented and cultured is carried out, the heredity for detecting bacterial strain is steady It is qualitative.
Lubricant component measures
It takes 1mL greases to be put into 15mL centrifuge tubes, 3mL n-hexanes is added and add 0.4mol/ after 15min is extracted in infiltration The KOH-CH3OH solution 1mL of L, concussion are put in 50 DEG C of water-bath 15min or so after shaking up, and 5% is added along tube wall after taking-up NaCl solution makes organic layer rise, and after stratification, takes its supernatant liquor with syringe, is added to the gas of 1.5mL after filtering In phase chromatographic sample bottle, gas phase analysis is carried out.The methods of gas phase analysis conditioned reference Zhu Qi think ofs are detected.
As a result with analysis
ARTP lethalities and mutation time measure
Scientific investigations showed that the active particle in plasma acts on microorganism, microorganism wall/film can be made Structure and permeability changes, and cause gene damage, and then make microbial gene sequences and its metabolism network significant changes, finally Microorganism is caused to generate mutation.
Mutation time is respectively 0,10,20,30,40,50,60S, under different induction times, the cause of trichosporon cutaneum Dead rate and time relationship are as shown in Figure 2.
As can be seen from Figure 2 ARTP to the lethality of Trichosporon cutaneum than stronger, when 40s Lethality has just reached 90% or more, when treated between when reach 60s, thalline is substantially all to survive.Due to luring It is random to become the positive and negative mutation generated, depends primarily on the characteristic of method of mutagenesis and bacterial strain itself, to improve bacterial strain existence energy Power selects the 40s that lethality can be made to reach 90% or so for best mutation time
The determination of sesamol additive amount
Sesamol is by inhibiting malate dehydrogenase to generate pyruvic acid and NADPH to control malic acid, to inhibit lipid Synthesis.The sesame phenol solution of high concentration will also inhibit the growth of cell, 0,0.05,0.1,0.15, the sesamol of 0.2mg/mL it is flat Influence of the plate to Trichosporon cutaneum is as shown in Figure 3:
From figure 3, it can be seen that sesamol can effectively inhibit the growth of thalline, at low concentration (0.05mg/mL), inhibit Effect is smaller, and when sesamol concentration reaches 0.1mg/mL, thalli morphology obviously becomes smaller, and growth is suppressed effect, works as sesame When phenol concentration reaches 0.15mg/mL, inhibit to greatly enhance, thalline quantity and form all substantially reduce, and it is dense to continue increasing sesamol Degree, thalline will be no longer grown.In view of the growing state of thalline, the sesamol additive amount conduct for choosing 0.15mg/mL most preferably adds Dosage.
ARTP combination sesamol mutagenesis high flux screenings
According to the inhibition of ARTP lethalities situation and sesamol, by OD600=1.0 seed bacteria suspension, with ARTP instrument With the Power Processing 40s of 120W, then the thalline of mutagenesis is applied on the plating medium containing 0.15mg/mL sesamols 28 DEG C Culture 2 days.It selects eugonic bacterium to be inoculated into 48 hole culture mediums with sterile bamboo stick, every 24 hours, detects cell concentration And fluorescence intensity.
Fig. 4 shows relationship of the 6th day lipid content of mutagenic strain relative to original strain, as seen from the figure, most of The lipid content of mutagenic strain is less than original strain, but have the lipid contents of a small number of mutagenic strains higher than original strain 20% with On.The initial strain of superior strain A1, E8 and low yield bacterial strain C2 as secondary screening is chosen, and examines the feasibility of fluorescence measurement.
The fermented and cultured of mutagenic strain
Mutant strain A1, C2, E8 and original strain are subjected to seed liquor and shake flask fermentation culture, fluorescence is detected daily and contains Amount, testing result are as shown in Figure 5:
As seen from Figure 5, the dominant strain A1 and E8 that primary dcreening operation obtains show to be apparently higher than original strain in shake flask fermentation Lipid accumulation amount and lipid accumulation it is quick, fluorescence intensity reaches maximum when the 9th day, and maximum fluorescence has reached original Three times of bacterial strain or so;The lipid accumulation of disadvantage bacterial strain C2 is always below original strain and accumulation is slow.
By (screening 3-4 days) after these four bacterial strain shaking flask cultures 3-4 days, intracellular grease is extracted, the results are shown in Table 1.
The secondary screening result of 1 trichosporon cutaneum mutagenic strain of table
As shown in Table 1, the mutant strain A1 through mutagenesis screening has large increase, biomass, grease production compared with original strain Amount, fat content have been respectively increased 41.5%, 123%, 58.8%.In shaking flask culture, the grease yield of bacterial strain reaches 5.8g/L is far above original strain.It can be seen that the yield of mutant strain is improved.
Genetic stability
In order to detect the hereditary capacity that can mutagenic strain keep stable, by continuous 10 secondary cultures of mutagenic strain A1, And it is carried out at the same time fermented and cultured, detect the fluorescence intensity of bacterial strain.The results are shown in Figure 6, from 1st generation to the fluorescence of the 10th generation bacterial strain Intensity all keeps stable, it is seen that mutagenic strain possesses good genetic stability, the characteristic for the high Lipid-producing of heredity that can stablize.
Lubricant component is analyzed
The lubricant component of mutagenic strain is measured with gas-chromatography, methyl palmitate, methyl stearate, oil is used in combination Sour methyl esters, methyl linoleate standard items carry out qualitative analysis, and the results are shown in Table 2:
The aliphatic acid table of 2 original strain of table and mutant A1
According to 2 constituent analysis of table, the main component of yeast grease is:Methyl palmitate, methyl stearate, oleic acid first Ester, methyl linoleate are similar to the ingredient of vegetable oil, therefore can be used as the raw material of biodiesel.The fatty acid composition of mutagenic strain Compared with original strain, the relative amount slightly raising of methyl palmitate, methyl stearate, methyl linoleate, and oleic acid The relative amount of methyl esters declines 4% or so, and the fatty acid composition of mutagenic strain and original strain are essentially identical on the whole.

Claims (10)

1. a kind of Lipid-producing mutagenic strain, which is characterized in that the mutagenic strain is with trichosporon cutaneum (Trichosporon Cutaneum the lubricant component of) bacterial strain of mutagenesis, the mutagenic strain includes mainly methyl palmitate, methyl stearate, oleic acid Methyl esters, methyl linoleate.
2. Lipid-producing mutagenic strain described in claim 1, which is characterized in that the mutagenic strain includes Trichosporon Cutaneum mutant strains, including Trichosporon cutaneumA1, Trichosporon cutaneumB2, Trichosporon cutaneumC1,Trichosporon cutaneumD4,Trichosporon cutaneumE8。
3. the Lipid-producing mutagenic strain described in claim 2, which is characterized in that the mutagenic strain is Trichosporon cutaneumA1。
4. the rapidly and efficiently selection of claim 1-3 any one of them Lipid-producing mutagenic strains, which is characterized in that skin shape Trichosporon cutaneum specifically comprises the following steps through the mutagenesis of ARTP plasmas, the bacterial strain selected after sesamol sieves again:
ARTP mutagenesis:Trichosporon cutaneum seed bacteria suspension is selected on metal slide glass, by metal slide glass be put into mutagenesis room into Row mutagenic treatment obtains the thalline of mutagenesis;
Sesamol screens:The thalline of mutagenesis is applied in the resistant panel culture medium containing sesamol, 28 DEG C are cultivated 2 days, are obtained The strain of mutagenesis on tablet;
High flux screening:The strain of mutagenesis on tablet is inoculated into bamboo stick in culture medium, is cultivated in constant-temperature table, bacterium solution is taken It to being cultivated in 96 orifice plates, every 20-28 hours, takes in bacterial strain to orifice plate, while adding Nile red dye liquor into orifice plate, mixing is equal Even to be protected from light dyeing 5min, with microplate reader with the launch wavelength of 485nm, 595 absorbing wavelength detects cell grease fluorescence intensity, sieve Select the high strain of fat content;
Fermented and cultured:15-25h will be cultivated in the colony inoculation to seed liquor for the high fat content that screening obtains, seed liquor is taken to connect It is cultivated in kind to fermentation medium, obtains the bacterium solution of fermented and cultured, as mutagenic strain.
5. the rapidly and efficiently selection of the Lipid-producing mutagenic strain described in claim 4, which is characterized in that
The resistant panel medium component is that the sesamol mother liquor of 60-70mg/mL is added in plating medium to obtain; The ingredient of the plating medium includes:Glucose 15-20g/L, peptone 15-20g/L, yeast extract 5-10g/L, agar Powder 15-20g/L;
The ingredient of the seed liquid culture medium includes:Sucrose 20-25g/L, yeast extract 4-6g/L, fructus hordei germinatus leaching powder 3-5g/L, Na2HPO42-4g/L, KH2PO40.5-1g/L, MgSO40.3-0.5g/L;
The ingredient of the fermentation medium includes:Glucose 60g/L, yeast extract 6g/L, fructus hordei germinatus leaching powder 5g/L, Na2HPO4 4g/L, KH2PO41g/L, MgSO4 0.5g/L.
6. the rapidly and efficiently selection of the Lipid-producing mutagenic strain described in claim 4, which is characterized in that ARTP mutagenesis steps In, a concentration of OD of trichosporon cutaneum seed bacteria suspension600=1.0;The temperature of ARTP is maintained at 18-25 DEG C, and power regulation is 100-140W, helium gas flow 8-12L/min;The bacteria suspension select metal slide glass go forward first to the calcination of metal slide glass at least 25-35s;Slide glass is put into mutagenesis room, and emitter is apart from bacterium solution 1.5-3nm, preferably 2nm;ARTP mutation times are 10- 40s;After the completion of mutagenesis, it is applied on plating medium and cultivates 1-3 days for 28 DEG C.
7. the rapidly and efficiently selection of the Lipid-producing mutagenic strain described in claim 4, which is characterized in that the strain of mutagenesis connects Kind in the culture medium of 48 hole 1ml, 110-160h is cultivated in 25-32 DEG C, the microwell plate constant-temperature table of 150-220r/min, is taken In the bacterium solution of 150ul to 96 orifice plates, each orifice plate adds 7.5ul Nile red dye liquors, is uniformly mixed and is protected from light dyeing 3-5min, uses enzyme It marks instrument and detects OD600The absorbance of nm.
8. the rapidly and efficiently selection of the Lipid-producing mutagenic strain described in claim 4, which is characterized in that fermented and cultured step In, 15-20h will be cultivated in the colony inoculation to seed liquor for the high fat content that screening obtains, it is 8-15% kinds to take volumetric concentration Sub- liquid be inoculated into fermentation medium 28 DEG C, 150-220r/min cultivate 3-4 days.
9. application of the mutagenic strain that any one of the claim 4-8 selection and breeding obtain at least one of following (1)-(5):
(1) as the raw material of biodiesel;
(2) grease in the raw material of biodiesel is improved;
(3) methyl palmitate in the raw material of biodiesel is improved;
(4) methyl stearate in the raw material of biodiesel is improved;
(5) methyl linoleate in the raw material of biodiesel is improved.
10. the application described in claim 9, which is characterized in that the method for the mutagenic strain extraction grease that selection and breeding obtain is as follows:It will Hydrochloric acid is added after drying in the bacterium solution centrifugation of fermented and cultured, is stored at room temperature rear boiling water bath processing 5-15min, rapid cooling, is restored to Chloroform-methanol (the V of 1-3 times of volume is added after room temperature:V=2:1) solution is stored at room temperature after mixing, is centrifuged, and is extracted, rotation Turn evaporation removing chloroform and obtains grease.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109504673A (en) * 2018-12-12 2019-03-22 河西学院 A kind of method for mutation breeding of high Lipid-producing Chlamydomonas reinhardtii
CN111560367A (en) * 2020-05-25 2020-08-21 华东理工大学 Method for high-throughput screening of high-yield sophorolipid strains
CN111690587A (en) * 2019-03-13 2020-09-22 华东理工大学 Method for centrifugally screening grease yeast strains with high oil content and application thereof

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CN101955888A (en) * 2010-06-25 2011-01-26 朱笃 Mutant strain of trichosporon cutaneum B3 for producing grease at high yield, EMS thereof and ultraviolet ray compound mutagenesis breeding method

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CN101955888A (en) * 2010-06-25 2011-01-26 朱笃 Mutant strain of trichosporon cutaneum B3 for producing grease at high yield, EMS thereof and ultraviolet ray compound mutagenesis breeding method

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109504673A (en) * 2018-12-12 2019-03-22 河西学院 A kind of method for mutation breeding of high Lipid-producing Chlamydomonas reinhardtii
CN111690587A (en) * 2019-03-13 2020-09-22 华东理工大学 Method for centrifugally screening grease yeast strains with high oil content and application thereof
CN111690587B (en) * 2019-03-13 2022-10-25 上海凯赛生物技术股份有限公司 Method for centrifugally screening grease yeast strains with high oil content and application thereof
CN111560367A (en) * 2020-05-25 2020-08-21 华东理工大学 Method for high-throughput screening of high-yield sophorolipid strains

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