CN105063012B - A kind of preparation of yeasty fusant and rapid screening method - Google Patents

Abstract

The present invention provides preparation and the rapid screening method of a kind of yeasty fusant, in order to realize ubiquinone₁₀Mass production, the method that the present invention utilizes protoplast fusion, by carrying out fusion treatment to schizosaccharomyces pombe and multiple-shaped nuohan inferior yeast, in conjunction with a kind of method of high frequency zone target yeasty fusant, quickly and efficiently obtain while having both subject fusion of two primary yeast bacterial strain good characteristics, subject fusion has that the speed of growth is fast, high temperature resistant simultaneously, and can efficiently produce ubiquinone₁₀The advantages that.The method of screening yeasty fusant of the present invention, be using the growth performance of thallus includes the speed of growth and heat-resisting ability and target metabolic product as selection markers, specifically with temperature tolerance and contains ubiquinone₁₀Analogue vitamin K₃Culture medium as screening strategy, directly can go out subject fusion by high flux screening.The method of the present invention is a kind of simple, quick, efficient, economic, practical safe improvement yeast method.

Description

A kind of preparation of yeasty fusant and rapid screening method

Technical field

The present invention relates to a kind of preparation of yeasty fusant and its methods of high-flux fast screening.

Background technique

Ubiquinone₁₀For the important electronics hydrogen donor of one in respiratory chain, redox reaction is reversibly carried out, extensively It is present in people, animal, plant and most microorganisms.Currently with microbial method fermentation preparation of cozymase Q₁₀Strain be mainly grain Wine fission yeast (Schizosaccharomyce spombe, S.sprombe), but schizosaccharomyces pombe slow growth, growth item Part requires strictly, to drastically influence ubiquinone₁₀Production efficiency.In recent years, researcher passes through ultraviolet mutagenesis, ion beam mutagenesis The methods of further genetic modification is carried out to it, become ubiquinone₁₀Production carrier.

As the multiple-shaped nuohan inferior yeast (Hansenula polymorpha, H.polymorpha) of food-grade, because of its safety Property it is high, advantage is obvious, be concerned in recent years.It is clear that its advantage is embodied in genetic background, high temperature resistant, be easy to high density fermentation, Breeding is fast, genetic manipulation is simple, can carry out post translational processing and modification and non-toxic metabolic product etc. to foreign product；The multiform Chinese Inferior yeast is a kind of heat-resistant yeast, and optimum growth temperature is 37 DEG C, but maximum growth temperature is up to 49 DEG C；In addition, it can be honest and clean It is grown in the Nonsele ctive culture media of valence, pH wide adaptation range (2.5~8.0), it is easy to accomplish high density fermentation, cell concentration can Up to 120g/L may generally serve as the natural carrier of metabolite production fermentation.Therefore, if by the excellent spy of multiple-shaped nuohan inferior yeast Property (such as the speed of growth fast, high temperature resistant) be efficiently transplanted to ubiquinone₁₀It produces in bacterial strain schizosaccharomyces pombe, must can improve grain wine and split Grow the ubiquinone of yeast₁₀Production efficiency.

Currently, there is not yet about schizosaccharomyces pombe and the preparation of multiple-shaped nuohan inferior yeast Protoplast Fusant and quickly The report of screening technique.

Summary of the invention

The purpose of the present invention is to provide a kind of preparation of yeasty fusant and rapid screening methods.

In order to achieve the above objectives, the invention adopts the following technical scheme:

1) using multiple-shaped nuohan inferior yeast and schizosaccharomyces pombe as starting strain, protoplast is prepared respectively；

2) with multiple-shaped nuohan inferior yeast and schizosaccharomyces pombe for two parents, by amphiphilic this protoplast (multiple-shaped nuohan inferior yeast Protoplast and schizosaccharomyces pombe protoplast) it is merged, obtain fusion treatment cell；

3) fusion treatment cell carry out with 48 DEG C of high-temperature cultivations to obtained bacterial strain is tentatively cultivated after tentatively cultivating Primary dcreening operation, in conjunction with vitamin K₃Selectivity culture carries out secondary screening to the bacterial strain retained after primary dcreening operation, obtains subject fusion bacterial strain.

It is described prepare protoplast the following steps are included:

1.1) it collects thallus: taking corresponding starting strain logarithmic phase bacterium solution and by centrifugation (4000~8000r/min centrifugation 5 ~10min) thallus is collected, with PBS buffer solution washing thalline 2~3 times；

1.2) it cell pretreatment: is suspended using pretreatment fluid (dosage as logarithmic phase bacterium solution volume 0.5~1 times) and passes through PBS The thallus of buffer washing passes through centrifugation (4000~8000r/ then in 25~35 DEG C of 5~10min of water bath processing after processing Min thallus) is collected；The pretreatment fluid the preparation method comprises the following steps: by 15~25 μ L 0.05mol/L EDTA aqueous solutions and 15~ 25 μ L beta -mercaptoethanols are dissolved in 20mL PB buffer；

1.3) it broken wall: is suspended and is walked using 2.0~2.5% glusulase enzyme solutions (dosage as logarithmic phase bacterium solution volume 0.5~1 times) The rapid thallus 1.2) collected makes somatic cells be changed into protoplast, so then in 25~40 DEG C of 30~60min of water bath processing Protoplast is collected by centrifugation (3000~6000r/min is centrifuged 5~10min) afterwards, is then washed with KCl high osmotic buffer former Raw plastid 2~3 times, the KCl high osmotic buffer the preparation method comprises the following steps: 4~5g KCl is dissolved in 100mL PB buffer.

It is described fusion the following steps are included:

2.1) inactivate: utilizing step 1.1)~1.3) this protoplast of amphiphilic is prepared respectively, respectively by this plasm of amphiphilic Body irradiates 1~5min in the UV lamp, and 15~20min is then saved in dark, primary after respectively obtaining two parents inactivation Plastid；

2.2) suspended respectively with KCl high osmotic buffer the protoplast after two parents inactivation, and it is thin to obtain amphiphilic this protoplast Born of the same parents' suspension, the KCl high osmotic buffer the preparation method comprises the following steps: 4~5g KCl is dissolved in 100mL PB buffer；

2.3) (3000~6000r/min is centrifuged 5~10min) is centrifuged after mixing amphiphilic this protoplast cell suspension simultaneously Cell is collected, with PEG-CaCl₂Suspend buffer 1 times of logarithmic phase bacterium solution volume (dosage be) cell, then in 25~35 Then DEG C 15~20min of water bath processing collects cell by centrifugation (3000~6000r/min is centrifuged 5~10min), with KCl high Oxidation buffer washs the cell 2~3 times, obtains fusion treatment cell, the PEG-CaCl₂Buffer the preparation method comprises the following steps: by 6~ 8g PEG₆₀₀₀, 0.8~1.0g CaCl₂And 30~50 μ L beta -mercaptoethanol be dissolved in 20mL PB buffer.

The irradiation power of the ultraviolet lamp is 15~30W, and irradiation distance is 30~50cm.

The step 3) specifically includes the following steps:

3.1) solid medium culture (preliminary culture, long cell wall): by fusion treatment cell KCl high osmotic buffer 0.3~0.5 times of logarithmic phase bacterium solution volume (dosage be) is spread evenly across on the hypertonic culture medium of KCl after suspending, then in 28~ 37 DEG C of 36~60h of constant temperature incubation, then the preferable bacterial strain of 80~120 plants of upgrowth situations of picking is used as fusion yeast candidate strain, The KCl high osmotic buffer the preparation method comprises the following steps: 4~5g KCl is dissolved in 100mL PB buffer；

3.2) high temperature screens: fusion yeast candidate strain is inoculated in respectively on solid YPD culture medium, then in 48 DEG C of perseverances Temperature 36~60h of culture, screens high temperature resistant fusant bacterial strain；

3.3) vitamin K₃Resistance secondary screening: high temperature resistant fusant bacterial strain is respectively coated on solid vitamin K₃Selective agar medium On, then in 28~37 DEG C of 36~60h of constant temperature incubation, the vitamin K filtered out₃Resistant strain, that is, subject fusion bacterial strain.

The PB buffer is by 87.7mL 0.2mol/L NaH₂PO₄Aqueous solution and 12.3mL 0.2mol/L Na₂HPO₄ Aqueous solution is formulated.

The composition of the hypertonic culture medium of KCl are as follows: yeast extract 10g/L, peptone 20g/L, glucose 20g/L, agar Powder 20g/L and KCl 40g/L.

The solid vitamin K₃It is 0.16mg/mL vitamin K that Selective agar medium, which is added with concentration,₃Solid YPD culture Base.

The beneficial effects of the present invention are embodied in:

In order to realize ubiquinone₁₀Mass production, the present invention utilize protoplast fusion method, by grain wine fragmentation Yeast and multiple-shaped nuohan inferior yeast carry out fusion treatment, quickly high in conjunction with a kind of method of high frequency zone target yeasty fusant Effect ground obtains while having both subject fusion of the good characteristic of two primary yeast bacterial strains, and subject fusion has growth simultaneously Speed is fast, high temperature resistant, and can efficiently produce ubiquinone₁₀The advantages that.The method of screening yeasty fusant of the invention, be with The high temperature resistant (i.e. 48 DEG C of high temperature, too high or too low to cannot achieve screening) and target metabolic product (ubiquinone of thallus₁₀) as sieve Choosing label, specifically with high-temperature stability and addition ubiquinone₁₀Analogue vitamin K₃Culture medium as screening means, can Directly to filter out subject fusion.The method of the present invention is that simple, quick, efficient, economic, practical, the safe yeast of one kind melts Zygote construction method.

Detailed description of the invention

Fig. 1 is ubiquinone₁₀High performance liquid chromatography detection map, in which: A is ubiquinone₁₀Standard items, B are subject fusion bacterial strain Fermentation broth extract.

Fig. 2 is that No. 5 subject fusion bacterial strains continuous eight are commissioned to train feeding ubiquinone₁₀Yield mapping.

Specific embodiment

It elaborates with reference to the accompanying drawings and examples to the present invention.

Strain of the present invention: multiple-shaped nuohan inferior yeast (Hansenula polymorpha) DL-1 is derived from Chinese industrial Organism Depositary, deposit number are ATCC No.26012；Schizosaccharomyces pombe (Schizosaccharomyce spombe) (S.promb 2.1794) is purchased from Institute of Microorganism, Academia Sinica.

Culture medium of the present invention:

1) YPD culture medium: yeast extract 10g/L, peptone 20g/L, glucose 20g/L, PH are naturally, (prepare solid training Agar powder 20g/L need to be separately added in feeding base).

2) the hypertonic culture medium of KCl: yeast extract 10g/L, peptone 20g/L, glucose 20g/L, agar powder 20g/L, KCl 40g/L, PH are natural.

3) vitamin K₃Selective agar medium: a certain amount of vitamin K is added in YPD culture medium₃。

4) malt extract medium: yeast extract 3g/L, peptone 4g/L, glucose 10g/L, malt flour 10g/L, PH are natural (agar powder 13g/L need to be separately added by preparing solid medium).

Reagent of the present invention:

1) PB buffer: 0.2mol/L NaH₂PO₄Aqueous solution 87.7mL and 0.2mol/L Na₂HPO₄Aqueous solution 12.3mL is formulated；

2) PBS buffer solution: 14.6g mannitol is dissolved in PB buffer, is settled to 100mL, spare；

3) pretreatment fluid: with 20 μ L of 20 μ L of 0.05mol/L EDTA aqueous solution and beta -mercaptoethanol, it is dissolved in 20mL PB It is spare in buffer；

4) 2.5% glusulase enzyme solution: 2.5mg glusulase (sigma) is dissolved in 100mL PBS buffer solution, spare；

5) KCl high osmotic buffer: 5g KCl is dissolved in 100mL PB buffer, spare；

6)PEG-CaCl₂Buffer: PEG₆₀₀₀8g, CaCl₂It is slow to be dissolved in 20mL PB by 40 μ L of 0.9g and beta -mercaptoethanol Fliud flushing, it is spare.

The preparation of yeasty fusant of the present invention and rapid screening method, comprising the following steps:

One, vitamin K₃The determination of minimum inhibitory concentration

Schizosaccharomyces pombe preservation of bacteria strain is taken, is inoculated in malt extract medium with 5% (v/v) inoculum concentration, 28 DEG C of constant temperature Culture carries out actication of culture, until OD₅₀₀It is 1；Bacterium solution is taken to be inoculated in malt extract medium again, inoculum concentration is 10% (v/v), 28 DEG C of constant temperature incubation 16h obtain schizosaccharomyces pombe logarithmic phase bacterium solution.Logarithmic phase bacterium solution is taken, being diluted to concentration is 1 × 10⁶A/ ML is respectively coated on solid vitamin K₃Selective agar medium, vitamin K₃Concentration from small to large, respectively 0.00,0.04,0.08, 0.12,0.16,0.20,0.24mg/mL.Bacterium colony growing state is observed after 28 DEG C of constant temperature incubation 48h, to determine vitamin K₃Most Small Mlc, it is final to determine that minimal inhibitory concentration is 0.16mg/mL.

Two, the preparation of fusant

1, the preparation of protoplast (this method for preparing protoplast of amphiphilic is consistent)

1) it collects thallus: taking logarithmic phase bacterium solution 1mL, 6000r/min to be centrifuged 10min, supernatant is abandoned, with 1mL PBS buffer solution Washing 2 times, thalline were collected by centrifugation by 6000r/min；The logarithmic phase bacterium solution of multiple-shaped nuohan inferior yeast and schizosaccharomyces pombe logarithmic phase bacterium Liquid preparation method is identical；

2) cell pretreatment: with 1mL pretreatment fluid resuspending step 1) gained thallus, 30 DEG C of water bath processings 10min, 6000r/ Min centrifugation, abandons supernatant, collects thallus；

3) broken wall: with 2.5% glusulase enzyme solution resuspending step 2 of 1mL) gained thallus, 35 DEG C of water bath processing 60min, microscopy (90% or more somatic cells are changed into protoplast), 4000r/min are centrifuged 10min, collect protoplast, then with 1mL KCl high osmotic buffer washs 2 times, and the protoplast after washing is spare.

2, protoplast fusion

1) by this protoplasm somatocyte of amphiphilic, (power 20W, distance 45cm) irradiates under aseptic operating platform ultraviolet lamp respectively 5min, dark in save 20min, with this protoplasm somatocyte of 0.5mL KCl high osmotic buffer suspension amphiphilic；

2) this protoplast of amphiphilic cell suspension is mixed, 4000r/min centrifugation collects cell, with 1mL PEG-CaCl₂It is slow Fliud flushing suspension cell, 30 DEG C of water bath processing 20min；4000r/min is centrifuged 10min, cell is collected, with the hypertonic buffering of 1mL KCl Liquid washs cell 2 times, obtains fusion treatment cell；

3) will gained fusion treatment cell suspended with 0.3mL KCl high osmotic buffer after be coated on the hypertonic culture medium of KCl, 37 DEG C culture 48h.

Three, the screening of subject fusion bacterial strain

1, primary dcreening operation under 48 DEG C of hot conditions

Pick out that 100 plants of speeds of growth are very fast, the good single colonie of growth conditions at random from the hypertonic culture medium of above-mentioned KCl Bacterial strain, as fusion yeast candidate strain.

Aseptically by above-mentioned 100 plants of fusion yeast candidate strain, inoculation (scribing line) divides in solid YPD culture medium Not using multiple-shaped nuohan inferior yeast and schizosaccharomyces pombe as positive and negative control, constant temperature incubation 48h at 48 DEG C, filtering out has The bacterium colony of high-temperature stability.As a result, have in 100 plants of fusion yeast candidate strains 58 plants can smooth continued growth, show high temperature resistant Characteristic, same to multiple-shaped nuohan inferior yeast；And schizosaccharomyces pombe and other 42 plants of fusions yeast candidate strains cannot be grown.Show This 58 plants fusion yeast candidate strains have multiple-shaped nuohan inferior yeast growth characteristics resistant to high temperature, may for subject fusion bacterial strain or Multiple-shaped nuohan inferior yeast.

2, vitamin K₃Resistance screening

Using above-mentioned 58 plants of fusions yeast candidate strain as research object, aseptically, it is inoculated with (scribing line) Yu Gu Body vitamin K₃Selective agar medium, respectively using schizosaccharomyces pombe and multiple-shaped nuohan inferior yeast as positive and negative control, 37 DEG C Constant temperature incubation 48h, screening have vitamin K₃Resistant strain.As a result, have in 58 plants of fusion yeast candidate strains 7 plants can smoothly after Continuous growth, shows vitamin K₃Resistance trait, same to schizosaccharomyces pombe；And multiple-shaped nuohan inferior yeast and other 51 plants of fusion yeast are waited Select bacterial strain that cannot grow.Show that this 7 plants fusion yeast candidate strains are while having high temperature resistant and vitamin K₃Resistance Subject fusion bacterial strain, number is 1~No. 7 respectively.

Four, final product detects

Above-mentioned 7 plants of subject fusion bacterial strains are inoculated in liquid YPD medium respectively, 37 DEG C of fermented and cultured 72h are right respectively Its fermentation liquid extracts separating treatment with Pyrogallil acid, is combined high performance liquid chromatography (HPLC) and mentions to fermentation liquid Object is taken to carry out ubiquinone₁₀Content detection.

Chromatographic condition are as follows: C18 chromatographic column；Methanol/dehydrated alcohol (volume ratio 1:1) is mobile phase；35 DEG C of column temperature；Flow velocity is 1mL/min；Detection wavelength is 275nm；Sample volume is 20 μ L.

By to the resulting ubiquinone that ferments₁₀Yield is detected, and finds the yield of 1~No. 7 subject fusion bacterial strain compared with grain wine Fission yeast starting strain all increases.Schizosaccharomyces pombe starting strain ubiquinone₁₀Yield is 9.24mg/L, subject fusion 1~No. 7 ubiquinone of bacterial strain₁₀Yield be respectively 16.31mg/L, 18.78mg/L, 12.47mg/L, 15.82mg/L, 20.03mg/L, 15.08mg/L, 17.56mg/L, yield increases separately about 76.5%, 103.2%, 35.0%, 71.2%, 116.8%, 90.0%, HPLC map are shown in Fig. 1.

Five, subject fusion bacterial strain inheritance stability Journal of Sex Research

Using the highest No. 5 subject fusion bacterial strains of yield as research object, inheritance stability Journal of Sex Research is carried out to it.By No. 5 mesh Mark fusant bacterial strain is inoculated in liquid YPD medium, and 37 DEG C, shaking flask culture is carried out under 125r/min, 48h passes a generation, passage training After supporting for 8 generations, separation is extracted to the fermentation liquid of every generation respectively, detects ubiquinone₁₀Yield.As a result see Fig. 2, ubiquinone₁₀It produces Amount is respectively 20.03mg/L, 19.87mg/L, 19.23mg/L, 19.52mg/L, 18.63mg/L, 18.94mg/L, 18.81mg/ L, 18.79mg/L declines by a small margin and fluctuates although having, and whole to tend towards stability, yield maintains 18.63~20.03mg/L； Show the multiple-shaped nuohan inferior yeast and schizosaccharomyces pombe recombinant bacterial strain constructed based on protoplast fusion, although in secondary culture Middle ubiquinone₁₀Yield has to be declined by a small margin, but to entire effect and little, so having genetic stability.

By multiple repetition experiment (carrying out fusant preparation and screening since starting strain), can obtain every time To 5~20 plants of subject fusion bacterial strains, ubiquinone₁₀Yield is improved, and has genetic stability.

Claims (5)

1. preparation and the rapid screening method of a kind of yeasty fusant, it is characterised in that: the following steps are included:

1) it is with multiple-shaped nuohan inferior yeast (Hansenula polymorpha) DL-1 and schizosaccharomyces pombe (S.promb) 2.1794 Starting strain prepares protoplast respectively；

2) with multiple-shaped nuohan inferior yeast and schizosaccharomyces pombe for two parents, by amphiphilic, this protoplast is merged, and is obtained at fusion Manage cell；

3) fusion treatment cell is carried out carrying out primary dcreening operation to obtained bacterial strain is tentatively cultivated with 48 DEG C of high-temperature cultivations after tentatively cultivating, In conjunction with vitamin K₃Selective medium carries out secondary screening to the bacterial strain retained after primary dcreening operation, obtains subject fusion bacterial strain, vitamin K₃Choosing Culture medium is selected to be 0.16mg/mL vitamin K added with concentration₃Solid YPD culture medium；

It is described prepare protoplast the following steps are included:

1.1) it collects thallus: taking corresponding starting strain logarithmic phase bacterium solution and by the way that thalline were collected by centrifugation, bacterium is washed with PBS buffer solution Body 2~3 times；

1.2) cell pretreatment: with pretreatment fluid suspend by washing thallus, then in 25~35 DEG C of water bath processings 5~ 10min, by the way that thalline were collected by centrifugation after processing；The pretreatment fluid the preparation method comprises the following steps: by 15~25 μ L 0.05mol/L EDTA aqueous solution and 15~25 μ L beta -mercaptoethanols are dissolved in 20mL PB buffer；

1.3) broken wall: with 2.0~2.5% glusulase enzyme solution resuspending steps 1.2) collect thallus, then in 25~40 DEG C of water-baths 30~60min is handled, somatic cells is made to be changed into protoplast, it is then hypertonic with KCl then by the way that protoplast is collected by centrifugation Buffer wash protoplast 2~3 times, the KCl high osmotic buffer the preparation method comprises the following steps: 4~5g KCl is dissolved in 100mL PB buffer；

It is described fusion the following steps are included:

2.1) inactivate: by amphiphilic, this protoplast irradiates 1~5min in the UV lamp respectively, then in dark save 15~ 20min, the protoplast after respectively obtaining two parents inactivation；

2.2) it is outstanding to be obtained with the protoplast after the suspension two parents inactivation of KCl high osmotic buffer for this protoplasm somatocyte of amphiphilic respectively Liquid, the KCl high osmotic buffer the preparation method comprises the following steps: 4~5g KCl is dissolved in 100mL PB buffer；

2.3) it is centrifuged and collects cell after mixing amphiphilic this protoplast cell suspension, with PEG-CaCl₂Buffer suspends, and this is thin Born of the same parents, then by the way that cell is collected by centrifugation, wash this with KCl high osmotic buffer then in 25~35 DEG C of 15~20min of water bath processing Cell 2~3 times, obtain fusion treatment cell, the PEG-CaCl₂Buffer the preparation method comprises the following steps: by 6~8g PEG₆₀₀₀, 0.8~ 1.0g CaCl₂And 30~50 μ L beta -mercaptoethanol be dissolved in 20mL PB buffer.

2. a kind of preparation of yeasty fusant and rapid screening method according to claim 1, it is characterised in that: described ultraviolet The irradiation power of lamp is 15~30W, and irradiation distance is 30~50cm.

3. a kind of preparation of yeasty fusant and rapid screening method according to claim 1, it is characterised in that: the step 3) specifically includes the following steps:

3.1) solid medium culture: the hypertonic training of KCl is spread evenly across after fusion treatment cell is suspended with KCl high osmotic buffer It supports on base, then in 28~37 DEG C of 36~60h of constant temperature incubation, the then preferable bacterial strain conduct of 80~120 plants of upgrowth situations of picking Merge yeast candidate strain, the KCl high osmotic buffer the preparation method comprises the following steps: 4~5g KCl is dissolved in 100mL PB buffer；

3.2) high temperature screens: fusion yeast candidate strain being inoculated on solid YPD culture medium, then in 48 DEG C of constant temperature incubations 36 ~60h screens high temperature resistant fusant bacterial strain；

3.3) vitamin K₃Resistance secondary screening: high temperature resistant fusant bacterial strain is coated on solid vitamin K₃On Selective agar medium, then in 28~37 DEG C of 36~60h of constant temperature incubation, the vitamin K filtered out₃Resistant strain, that is, subject fusion bacterial strain.

4. according to claim 1 or a kind of preparation of 3 yeasty fusants and rapid screening method, it is characterised in that: described PB buffer is by 87.7mL 0.2mol/L NaH₂PO₄Aqueous solution and 12.3mL 0.2mol/L Na₂HPO₄Aqueous solution prepare and At.

5. a kind of preparation of yeasty fusant and rapid screening method according to claim 3, it is characterised in that: the KCl The composition of hypertonic culture medium are as follows: yeast extract 10g/L, peptone 20g/L, glucose 20g/L, agar powder 20g/L and KCl 40g/L。